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Responses of cultured cerebellar neurons to iontophoretically applied amino acids
Brain Research, 74 (1974) 67-80
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
RESPONSES OF C U L T U R E D C E R E B E L L A R P H O R E T I C A L L Y APPLIED A M I N O ACIDS
NEURONS
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HERBERT M. GELLER* AND DONALD J. WOODWARD Department of Physiology, University of Rochester School of Medicine and Dentistry, Rochester, N. Y. 14642 (U.S.A.)
(Accepted January 9th, 1974)
SUMMARY Electrical activity of spontaneously firing neurons in explant cultures of neonatal rat cerebellum was recorded with extracellularly placed microelectrodes. Several different normal patterns o f firing were noted, common ones being single spikes at regular intervals, irregularly spaced single spikes and rapid bursts o f up to l0 spikes separated by periods o f silence. The firing rate could be modified by the local application o f drugs through iontophoresis onto the soma and adjacent processes. Firing rates were increased by glutamate and homocysteate, and decreased by gamma-aminobutyric acid (GABA) and glycine. The action o f GABA was blocked by bicuculline and picrotoxin. GABA was relatively more effective than glycine. Acetylcholine was generally ineffective, but rarely had weak excitatory actions. We conclude that the sensitivities of the cultured Purkinje cell with respect to these drugs is the same as the adult Purkinje cell in vivo and similar to the immature cell at the time o f explantation. We suggest that pharmacological and physiological criteria be used to identify cell types and evaluate the extent of differentiation of neurons in culture.
I NTRODUCTION It has long been recognized that organotypic explant cultures of central nervous system have considerable potential for gaining insight into basic physiological and pharmacological properties of neurons. To exploit such potential we have investigated * Present address: C.M.D.N.J.-Rutgers Medical School, Department of Pharmacology, P.O Box 101, Piscataway, N.J. 08854, U.S.A.
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the physiology and pharmacology of neurons in roller tube cultures of rat cerebellum by extracellular unit recording in conjunction with iontophoretic methods of local drug application. Earlier studies6,17 have demonstrated that some neurons in culture generate continuous spiking activity. Recent interest in this phenomenon has led to work showing that sustained activity can exist in culture in the presence of high magnesium and low calcium, suggesting an independence from synaptic input of at least some components of this activity 1~. In preliminary investigations we also found unit activity which was maintained for a long period of time (more than 1 h). We were therefore encouraged in this study to examine the possibility that this steady activity of neurons in explant cultures could be employed as a basis for examining pharmacological properties of the membranes of these neurons. Earlier studies indicated that addition of pharmacologic agents to the bathing medium can have an influence on aggregate synaptic and spiking activity of cultured neurons7, lz. Recent work investigating the site of action of such drugs has shown cultured dissociated neurons to be individually sensitive to drugs applied to the surface membrane~9, ~3. In studies reported here we have examined the pharmacological sensitivity of neurons in the more normal neuroglial frameworks of the organ explant grown and bathed in an artificial medium. It is shown that many characteristic actions of pharmacologically active agents on sustained spiking are similar in culture and in vivo. A preliminary report o f this work has appeared a~. MATERIALS AND METHODS
The techniques used to prepare newborn rat cerebellar cultures have been reported in detail 5. Cultures of 0-day postpartum animals were prepared weekly. Feeding medium consisted of Eagle's basal medium (GIBCO), 83 ~ ; fetal calf serum (GIBCO), 14 ~ ; chick embryo extract (GIBCO), 1 ~ ; and 2.7 M glucose, 1 ~ , to bring the final glucose concentration in the feeding medium to 33 raM. The medium was exchanged at weekly intervals. Cultures between 2-6 weeks of age were selected on the basis of neuronal outgrowth and thinning out of the original explant in order to provide a closely matched sample space for electrophysiological and neuropharmacological studies. They were placed in a temperature-regulated chamber at 35-36 °C, perfused with Gey's balanced salt solution 15, and inspected with a 40 × objective to locate areas suitable for recording. Such areas showed clearly visible neuronal perikarya with prominent round nuclei and nucleoli, and almost always exhibited electrical activity. In particularly thin cultures, the proximal processes could also be visualized, but they were usually obscured by more highly refractile somata. A single-barreled NaC1 filled recording electrode, 1-3 # m in diameter, with a resistance of 4-10 M ~ was then introduced into the culture and placed near the outline of a neuron soma until spikes could be easily discriminated. Standard recording and monitoring electronics were used. The initial several minutes of spike record were
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recorded on audiotape for later processing by the PDP-12 computer 8°. Integrated activity was displayed on a strip chart recorder to indicate effect o f drug application. After a stable record of several minutes was obtained, a 5-barreled (or at times 3barreled) drug micropipette o f 3-8 # m tip diameter observed directly was maneuvered near to the neuron, until drug effects were observed. Spiking remained stable in this situation for several hours. Drugs were applied through the various barrels through the use of a specially constructed microiontophoresis unit as described by Geller and Woodward 14. The center barrel o f the array was used as a balance barrel, to eliminate the possibility of current-induced artifacts. Drugs used in the various barrels were: 1 M GABA, pH 4; 1 M sodium glutamate, pH 8; 1 M glycine, pH 4; 1 M DL-homocysteic acid (DLH), pH 7.5; saturated picrotoxin; 5 m M bicuculline (in 165 m M NaC1), p H 3.5; 1 M acetylcholine chloride, p H 4.0. Drugs were dissolved in distilled H20 and pH was adjusted with N a O H or HCI. At the conclusion o f the experiment, cultures were placed in Bodian's fixative, and stained according to a modification o f the Bodian silver methodL RESULTS
Morphology of organotypic explants Bodian silver staining techniques were used to characterize the cultures typically employed for the physiological studies (Fig. 1). These studies demonstrated that the morphology was generally similar to that o f neonatal cerebellum described in both Maximov 3,24 and roller tube 16 cultures. The most consistent feature revealed by the Bodian stains was large numbers o f long neural fiber processes. These processes are not detected by the stain in immature cultures, at a time when neurons clearly exist. Presumably, neurofibrillary material accumulates in time inside the fibers which allow their detection by the Bodian stain. Such fiber processes are typically several hundred microns in length and in many cases can be seen extending several millimeters through the area in and around the explant. Usually fibers appear to course in groups as though guided non-randomly in their growth by some aspect o f the glial-fibroblast network. Most fibers could not be traced microscopically back to their cells of origin, either being lost in the complicated network, or becoming small at both ends and impossible to follow. In some cases, fibers end in ring-like structures which have been called 'synapses' elsewhere 1. Fibers were never found in a cell-free area, and are always embedded in a non-neural cell matrix, presumably o f fibroblasts and glial cells. Large neurons, presumably Purkinje cells, were routinely observed in both living and stained cultures (Fig. 1). Multiple fibers containing dense fibrillary material could be seen branching in many directions from the soma which itself was sparsely stained. Cell bodies were sometimes distinguishable within the central explant but much more clearly in the thinner region at the periphery of the original edge of the explant. In contrast to fibers, cell bodies were never seen more than a few hundred microns away from the explants and appeared to have limited mobility. Many small
Fig. 1. Organization a n d structures revealed in silver-stained preparations of rat cerebellar cultures. Cultures stained with a modification of Bodian's silver stain. A: low power p h o t o m i c r o g r a p h of a developed cerebellar explant. Presumptive cortical tissue is located on either side of a fissural r e m n a n t at lower middle portion of explant (20 days in vitro (DIV), x 22). B: row of aligned Purkinje cells. This culture displayed an extremely high degree of organization, illustrated here a n d in C (25 DIV, x 350). C: high power view o f several Purkinje cell s o m a t a with proximal dendrites. T h e dendrites are aligned in a pattern reminiscent of the #t vivo situation (25 DIV, .-: 625). D: typical high magnification view of a silver-stained culture. Several weakly stained neuron s o m a t a (arrow) s u r r o u n d one well-impregnated n e u r o n (N). Note the large a m o u n t of neurofibrillary material which s u r r o u n d s the s o m a t a (44 DIV, x 475). E: ring form ending (long arrow) in close proximity to a lightly stained neuron s o m a (N). T h e neuronal nucleus is clearly visible, a n d the s o m a , out of focus in this micrograph, has been outlined with arrows (44 D1V, ;~ 1150). F: small, bipolar n e u r o n s whose processes are not visible due to the short depth of field of the objective. M a n y of these cells p r e s u m e d to be granule n e u r o n s can be seen in cerebellar cultures (25 D1V, x 825).
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oblate cells (Fig. IF) were visible in each culture in close proximity to the large neurons and fiber tracts. A striking feature occurring only occasionally consisted of a line o f many somata of presumed Purkinje cells, each with its primary dendrite oriented in the same direction stimulating the orientation of the apical dendrite system of the molecular layer in vivo (Fig. 1B, C). More typically, development in culture evidently leads to the multipolar configuration which would be the form most often encountered by microelectrodes. Most cell bodies did not have visible stained endings terminating on them; a few, however, have shown boutons on the soma or nearby processes (Fig. IE). Little definitive information is provided by the Bodian technique on the prevalence of synaptic connections. Electron microscopic studies were carried out on several typical explants (West, M. J., unpublished observations). Synaptic structures and myelinated axons were readily observed in many areas. Many round cells of 6-8/~m diameter with scant cytoplasm, closely resembling granule cells 21, were observed. Multicontact synaptic structures were often observed resembling glomeruli in vivo and similar to those observed in culture by others zg. The phase contrast optics used during electrophysiological studies usually allowed only a view limited to the neuronal soma. Typically, large, round cell bodies could be distinguished among the network of cells and processes, with round, clearly visible nuclei and nucleoli. Occasionally in thinner regions of the culture under the best conditions the thick adjacent parts o f the neural processes could be followed 100/~m or more into the nearby regions. Finer neural elements, however, could not be resolved optically, nor could axonal processes be followed for appreciable distances. During the typical recording situation, the tip of a recording electrode could be located visually within a few microns of the soma. A multi-barrel array of drug-containing electrodes was positioned on the opposite side of the soma. Thus, it appears from the visualization under phase contrast that recording and drug application are both restricted to the soma and a small adjacent portion of the long fibrous processes. Normal spontaneous activity Spontaneous neural activity was detected in these cultures by advancing a singlebarrel electrode through the multi-layered structure of the explant toward the outline o f a cell until detection of activity proved it to be a neuron. Activity appeared to be a stable property and ceils routinely displayed the initial pattern observed until the recording was terminated after from several minutes to more than an hour. Mean firing rates were calculated for 46 cells recorded in culture. The range o f firing rates extended from 1/sec to a maximum o f 35.6/sec, with a mean rate of 9.23 8.22 (S.D.). Fig. 4 shows representative interspike interval histograms of several such units illustrating the range o f variability in the patterning o f firing observed. The most typical unit is shown in Fig. 2B having a mean firing rate o f 17.7/sec and for which the intervals were distributed exponentially in an approximate unimodal distribution. This apparent randomness o f long and short intervals is contrasted with another unit as shown in Fig. 2A displaying an extremely high regularity o f firing as revealed in the sharp peak o f the interspike histogram. Unit activity could also be found which was
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Fig. 2. Interspike interval histograms and sample spike records constructed from taped records of activity from cultured Purkinje cells. See text for interpretation. A : very regular firing pattern from a neuron in a 16-day-old culture. Firing rate of 9.5 spikes/sec, with a modal interspike interval of 105 msec. B: interspike interval histogram of irregularly firing neuron from a 15-day-old culture. The distribution has a mean rate of 17.7 spikes/sec and is coupled with a modal interval of 5 msec. The long tail represents the low, but finite, probability of long intervals. C: interval histogram of irregularly firing cell, 15 days in culture. The mean rate of this cell of 8.1 spikes/sec was coupled with a modal interspike interval of 192 msec. D: interspike interval and interburst interval histograms recorded from a Purkinje neuron in an 18-day-old culture. The mean firing rate for this cell was 5.0 spikes/sec. DI: the interspike interval histogram shows the most probable interval within bursts was 71 msec, with the long tail representing longer intervals between bursts. Substantial time is represented by intervals longer than the 500 msec scale. Dm: the interburst interval histogram constructed on-line through a special burst detection subroutine29. The burst detection of cycling is most accurately described by points between 0.75 and 2 sec. Burst intervals less than 0.75 sec represent phases of slow irregular firing.
extremely irregular as in Fig. 2C. F u r t h e r m o r e , other units displayed consistent bursting forms o f activity, pauses o f slow activity alternating with periods of fast, rapid activity (Fig. 2D). U n i t s employed in drug studies mostly exhibited the slightly irregular p a t t e r n as in Fig. 2B.
E~'ects of pharmacologic agents After a cell was located by positioning a single-barrel electrode o n one side o f the soma, a second multi-barrel electrode was placed o n the other side o f the soma a n d drugs applied by iontophoretic currents t h r o u g h the various barrels. N o effects were observed unless the drug electrode was visibly close to the soma. S p o n t a n e o u s activity in this preparation proved to be extremely sensitive to
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GABA. Forty-six cells were examined, all o f which were inhibited by GABA ejected with currents between 0 and 125 nA. Inhibition o f spontaneous activity could often be elicited simply by turning offthe holding current used to prevent diffusion from the electrode tip. In our experiments we were able to put this observation to use by allowing GABA to diffuse from the barrel during the initial placement of the drug micropipette. The micropipette was slowly advanced towards the cell until the GABA leak noticeably affected firing rate. We then stopped movement, and the drug micropipette was left in place for the duration of the experiment. Firing returned to control levels upon restoring holding current for GABA. Whereas clear effects on neural activity were easy to determine by inspection of the effect on firing rate, the certainty of a negative effect was more difficult to determine. We labeled a test as having no effect in those instances where at least one other drug, in most cases GABA, was effective and where no clear sign o f drug electrode block or other evidence could be found o f failure of drugs to be released. Our denotation o f ' n o effect' is conservative in that more instances can be attributed to a simple technical failure to release sufficient drug near the cell. Excitatory amino acids active in vivo had similar actions in culture. Glutamate, ejected with currents from 0 to 100 nA, excited 7 cells with latencies between 0 and 16 sec and had no effect on two cells. DL-Homocysteic acid, an amino acid not occurring in nature, excited 4 of 7 cells. Neither o f these agents at any time showed an inhibitory action upon spontaneous activity when applied in low doses. In some instances 10-8 M glutamate was injected in the bathing medium. In such cases cells first became excited and discharged at progressively faster rates. Spike size then became smaller and the cell went into a phase o f inactivation. Changing back to the original bathing medium caused reversal to normal firing. Such inactivation can be accounted for by excessive depolarization of the cell membrane. Glycine was found in 3 cells to have an inhibitory action on spontaneous activity (Fig. 5). No effects were observed on 8 cells. In 4 additional instances, cells exhibited uncertain or ambivalent effects in which inhibition appeared on a single trial application o f iontophoretic currents, but not on subsequent trials. The iontophoretic currents required for the successful glycine effects were greater in every instance than those required for GABA inhibitions, with 100-2t30 nA required in most cases. The ambivalent or negative effects can be likely accounted for here by the requirement o f relatively large amounts o f drug close to the membrane to evoke effective inhibition. Acetylcholine was tested on 15 cells. There was virtually no effect of spontaneous activity (Fig. 5) in 9, whereas in 6 somewhat ambivalent excitatory effects were found. The excitation was gradual and had a long latency of onset (5-20 sec) (Fig. 5C) compared with the actions of glutamate and GABA, and the firing rate slowly returned to control levels (with delays of up to 1 min) following termination of ejection. One m M atropine was placed in the bath 3 times and found to have no effect on spontaneous activity. Similarly, 1 m M D-tubocurarine was put in the bathing media and twice not found to have an effect on the spontaneous activity. Strychnine was placed in the bathing media 3 times at 1 m M concentration without influence on spontaneous activity.
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Fig. 3. Some effects of GABA on spontaneously firing cerebellar neurons in culture. In this figure and those following, the ordinate represents neuronal discharges in successive epochs, and the abscissa represents time, with the length of drug application indicated by bars with arrows. The drug being applied is indicated above the bar, with the application current in nA. A: the effect of the competitive antagonist alkaloid bicuculline on GABA inhibition of cultured Purkinje cells. GABA was allowed to diffuse out of the pipette for periods of 5 sec at a supramaximal dose and was retained for 5 sec. The application of bicuculline for a period of 1 min significantly reduced the effectiveness of GABA in shutting off the neuron. B: the cell in this figure was quiescent. Two short pulses of GABA were applied to the cell. Upon cessation of drug application, the cell responded with a burst of 15-20 spikes over the next 2 sec. See text for discussion. C: a pulse of GABA sufficient to shut off neuronal firing was applied for 20 sec. When the drug was stopped, the firing resumed at approximately double the control rate and did not return to baseline for 2 rain. D: GABA retaining current was reduced here from 40 nA to 10 nA. The cell continued to fire spontaneously for 3-4 sec, emitted a burst of 7-10 spikes, and then, in the next 4 sec, stopped firing.
P i c r o t o x i n a n d b i c u c u l l i n e , k n o w n to i n h i b i t the a c t i o n o f G A B A 9, w e r e tested by a p p l y i n g t h e m f r o m a n o t h e r b a r r e l o f t h e p i p e t t e . P i c r o t o x i n b l o c k e d t h e a c t i o n o f G A B A in 2 i n s t a n c e s a n d h a d n o effect in 3 instances. B i c u c u l l i n e b l o c k e d t h e a c t i o n o f G A B A o n 2 cells in w h i c h it d i d n o t alter the p a t t e r n o f firing. O n 7 o t h e r cells, b i c u c u l l i n e e v o k e d cyclic bursts o f firing, a n d m a y h a v e o b s c u r e d a b l o c k i n g o f G A B A action. M a n y f e a t u r e s o f d r u g effects o n l o n g - t e r m a c t i v i t y o f n e u r o n s in c u l t u r e s r e s e m -
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Fig. 4. Effects of iontophoretically applied excitatory agents on quiescent and steadily firing neurons. A : cell which was firing at 0-2 spikes/sec was speeded up to 7 spikes/sec by application of 75 nA of sodium glutamate. The effect of GABA at 25 nA was to reduce the firing of the cell, while at 50 nA the cell was shut off completely. B: the effect of DL-homocysteate upon spontaneous firing of a cerebellar Purkinje cell. Initially a dose of 125 nA provokes a slight increase in firing rate. As the drug is repeatedly applied, the efficiency is increased, until 75 nA causes the same rise as the initial 125 nA.
ble actions on cells in vivo. Depending on the position of the drug electrode effects of drugs on firing rates could be shown in some cells to vary up or down as more iontophoretic current was applied, indicating a rough dose response relation (Fig. 4A). Also, a repeated series o f several seconds o f iontophoretic current from individual drug barrels gave successively greater effects (Fig. 4B). This observation is probably due to an effect of prolonged retaining currents loading the end of the drug releasing pipette with inactive ions. With repeated drug application, a higher amount of active ions are brought to the tip and ejected. Fig. 3 shows some interesting features on the inhibition of spontaneous activity. At times, firing rates did not gradually slow down when GABA was applied at effective levels, but instead there often occurred a phase of excitation before the total inhibition of activity (Fig. 3D). Also, application of drugs causing total inhibition for a few seconds was often followed by a rebound phase of a much more rapid activity lasting 10-20 sec (Fig. 3C). The onset and release of the GABA effect involved several seconds o f delayed onset so the more immediate direct current effects are likely not involved. Excitatory and inhibitory drugs exhibited clear interactions, as in Fig. 4A. Glutamate could overcome the action o f GABA. This indicated that GABA was not causing a blockade o f the spike generation mechanism, but an inhibition which could be overcome by a depolarizing substance. In addition, cells which were stimulated to fire more rapidly by glutamate could also be depressed with GABA. DISCUSSION
The present results show that drug-induced changes in the spontaneous activity
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Fig. 5. The effects of microiontophoretically applied drugs on cerebellar Purkinje neurons. A: GABA at 10 nA consistently reduces the firing rate of tke neuron, while applied glycine at a dose of 250 nA does not affect the cell. B: GABA is efficient at reducing the firing of this cell at a dose of 25 hA, while glycine has the same effect only at the larger current of 100 nA. Both drugs provoke a rebound of the firing of the cell to higher than control levels. Acetylcholine (ACh) at a current of 100 nA was ineffective in influencing the cell firing. C: acetylcholine was effective in increasing the discharge rate of this neuron at a dose of 80 nA. The latency of onset was over 10 sec, and the time to peak response was almost 1 rain. After cessation of drug application, the firing rate decreased, but did not return to control level. In this same cell, glutamate, 2 hA, caused an increase in the discharge rate with a latency of less than 1 sec, which returned to baseline levels upon stoppage of the ejection current (not illustrated).
o f n e u r o n s in c u l t u r e c a n be used as a m e a s u r e o f t h e sensitivity o f t h e m e m b r a n e to a p p l i e d p h a r m a c o l o g i c a l agents. T h e effects o f the a m i n o a c i d p u t a t i v e n e u r o t r a n s m i t t e r s o n c u l t u r e d c e r e b e l l a r P u r k i n j e cells are c o m p a r a b l e to effects in vivo a n d a l l o w us to c o n c l u d e t h a t t h e s u r f a c e m e m b r a n e retains its sensitivities to t h e s e agents. The morphological investigations confirm the observations of many investigat o r s t h a t c u l t u r e d c e r e b e l l a r n e u r o n s w i t h i n e x p l a n t s will d e v e l o p in this in vitro system. T h e r o l l e r t u b e m e t h o d , s i m i l a r to t h a t e m p l o y e d b y H i l d 16, is s i m p l e to use and promotes rapid thinning out of the culture, favoring extended survival through
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adequate nourishment. The degree of neuronal organization seen in these roller tube cultures can be comparable to that reported by other investigators using Maximov cultures 3,21,26 where less flattening occurs. It has long been presumed in previous morphological studies5,12,17 that the large neurons of the type we observed here are Purkinje cells, because this is the main cell type which would grow a large soma with long axonal processes. To these criteria we can now add the identification of some pharmacological sensitivities to amino acids characteristic of the Purkinje cell in vivo. Our interest is to eventually consider similar criteria for other cells in explant cultures. We propose that it is now feasible to employ pharmacological investigations generally in the identification of cell type, or in determining whether basic cell properties have deviated in vitro. The wide variation of mean firing rates reported here correlates with previous observations in cultureS, 11. A similar wide range of activity has been reported for adult Purkinje cells in vivo is. Sustained activity has been reported in all studies involving absence of innervation of Purkinje cells: immature cerebellum before synaptogenesisa2, pedunculotomized cerebellum1°, and undercut cerebellar cortex 2s. Such phenomena suggest a dominant autonomous component in the generation of activity. If this is the case, the wide range of mean rates may not be due to variation in tonic synaptic inputs as much as to variation in individual neuronal metabolism or resting conductance properties of cell membrane. The different patterns of firing in culture seem likely due to excitatory and inhibitory synaptic influences superposed upon the autonomous activity. An irregularization of firing as shown in Fig. 2D could be due to excitatory input reducing interspike intervals as suggested in the theoretical and experimental studies on spinal motoneurons 4. Longer pauses or bursts could be generated by excitation or by inhibition in more complex interactions. The presence of synaptic activity has been postulated to account for similar interval distributions observed in cerebellar cultures by others z~. The effect of the applied pharmacological agents is consistent with the hypothesis that Purkinje cells have retained their sensitivities to these drugs during maturation in culture. The developing Purkinje cell is rather unique in the extent of detailed information available concerning functional properties at the time of explantation. At this time, the Purkinje cell of the rat is observed as a small neuron with many fine fibrous processes extending from the soma and a fine axonal process leading down to the cerebellar nuclei. The large dendritic tree which characterizes the mature cell develops from days 7 to 21 postnatally. The Purkinje cell at birth, though not yet innervated, has been demonstrated to be capable of generating action potentials and to display well-characterized responses to the pharmacological agents used in this studya2. The physiological and pharmacological properties displayed by these cells in culture, which have matured in the sense of growing a large soma and long axonal processes, are thus not ones which they have acquired, but ones which they have evidently retained as part of previously expressed genetic information. GABA consistently decreased the firing rate of cultured Purkinje cells presumably by hyperpolarizing the neuronal membrane, as has been shown in previous studies~7,aa. The specificity of the action of GABA was supported by the blockade of
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these effects with picrotoxin and bicuculline. Other responses with GABA parallel results from in vivo investigations. Depending on the position of the drug barrel with respect to the cell, weak dose-response relations could be demonstrated. The latency to onset and threshold current level also could be shown to vary much as in vivo. Furthermore, long-lasting rebound increases of the firing rate to supra-control levels were often observed following GABA application, as in Fig. 3C. Such an effect could result from a loss ofintracellular K + during the period of presumed drug-induced high K + conductance after which the membrane would tend to be depolarized towards threshold causing the sustained increase in activity. The implication of this rebound effect is that mean activity of a neuron may be influenced over long periods of time by ionic imbalances induced by relatively short-term alterations of membrane conductances. As a consequence, substantial alterations in firing may be induced during periods when alteration by active synaptic input is not present. Glycine generally had an inhibitory effect on the cells on which it was tested, but at drug application currents much higher than necessary for GABA. This agrees with the ratio of 3.6 calculated to be the equipotent glycine current to GABA current in cerebellar Purkinje cells in cats 2°. This relationship of effectiveness provides further evidence for the similarity of in vivo and in vitro neurons. Experiments in vitro have directly demonstrated an hyperpolarization caused by glycine application on motoneurons 19, The excitatory actions of the glutamic and homocysteic amino acids on most cells studied were consistent with similar actions reported for in vivo investigations 9. In experiments in which glutamate was in the bathing medium, neurons reached instantaneous rates of over 100 spikes/sec. Firing then stopped abruptly, presumably due to excessive depolarization of the membrane, then returned to normal after a delay of several seconds following exchange of the salt solution. Such effects are readily explained by the depolarizing action of these amino acids. The ambivalent excitatory or nonexistent effects of acetylcholine contrasted with the potent effect of other drugs. The excitatory responses observed were of long latency, about 20 sec, and gradual in onset, compared with a few seconds for GABA or glutamate. Large doses (100-200 nA) of acetylcholine were required to elicit effects on those neurons which responded, while doses of up to 500 nA were often without effect. This pattern of responsiveness is similar to the pattern found in immature rat cerebellum a2. Other work, in the intact adult rat (Hoffer, personal communication) and cat s, suggests that some form of sensitivity to acetylcholine exists within the cerebellar cortex of several species in vivo. Acetylcholine sensitivity also survives intact in the rat when interneurons have been eliminated by postnatal X-irradiation al. Our results from culture suggest that intact afferent systems or normal neuroglial morphological relationships may be necessary for these effects. In general, our studies clearly indicate that cultured Purkinje neurons possess chemosensitivity to excitatory and inhibitory putative neurotransmitters. This is a critical piece of information necessary for establishing a potential for synaptic interaction. It now appears feasible to analyze the synaptic connections in culture z2 employing pharmacological interaction with suspected transmitter systems. Also, it
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should now be possible to employ the procedures developed in this study to characterize and manipulate the unique pharmacological membrane properties expected to be encountered in cultured neuronal cell types from many different brain regions. ACKNOWLEDGEMENTS
The technical assistance of Ms. Debra Bickett is gratefully acknowledged. This research was jointly supported by the National Institutes of Health (Grant MH 18367-01), the National Science Foundation (Grant GB 28873-X), and by a General Research Support Grant from the University of Rochester. Dr. Geller held an N.I.H. Postdoctoral Fellowship (GM 50876) and Dr. Woodward an N.I.H.T.I.S.T. Award (NS 11030) during the course of this work.
REFERENCES 1 ALLERAND, C. D., Regeneration of synapses in vitro, Exp. Neurol., 25 (1969) 482-493. 2 BODIAN, D. B., The staining of paraitin sections of nervous tissue with activated Protargol - - the role of fixative, Anat. Rec., 69 (1937) 153-162. 3 BORNSTE1N,M. B., AND MURRAY, M. R., Serial observations on patterns of growth, myelin formation, maintenance, and degeneration in cultures of new-born rat and kitten cerebellum, J. biophys. biochem. Cytol., 4 (1958) 499-504. 4 CALVIN,W., AND STEVENS,C. F., Synaptic noise and other sources of randomness in motoneuron interspike intervals, J. Neurophysiol., 31 (1968) 574-587. 5 CECHNER,R. L., FLEMING,D. G., AND GELLER, H. M., Neurons in vitro: a tool for basic and applied research in neural electrodynamics. In M. CLYNES AND J. MILSUM (Eds.), Biomedical Engineering Systems, McGraw-Hill, New York, 1970, Ch. 15. 6 CRAIN, S. M., Development of 'organotypic' bioelectric activities in central nervous tissues during maturation in culture, Int. Rev. Neurobiol., 9 (1966) 1-43. 7 CRAIN, S. M., ALFEI, L., AND PETERSON,E. R., Neuromuscular transmission in cultures of adult human and rodent skeletal muscle after innervation in vitro by fetal rodent spinal cord, J. Neurobiol., 1 (1970)471-489. 8 CRAWFORD,J. M., CURTIS,D. R., VOORHOEVE,P. E., AND WILSON, V. J., Acetylcholine sensitivity of cerebellar neurons in the cat, J. Physiol. (Lond.), 186 (1966) 139-165. 9 CURTIS, D. R., AND FELIX, D., The effect of bicuculline upon synaptic inhibition in the cerebral and cerebellar cortices of the cat, Brain Research, 34 (1971) 301-321. 10 ECCLES,J. C., ITO, M., AND SZENT,~GOTHAI,J., The Cerebellum as a Neuronal Machine, Springer, New York, 1967. 11 G.~HWILER,B. H., MAMOON,A., SCHLAPFER,W. Z., AND TOBIAS, C. A., Effects of temperature on spontaneous bioelectric activity of cultured nerve cells, Brain Research, 40 (1972) 527-533. 12 G.~HWILER, B. H., MAMOON,A. M., AND TOBIAS, C. H., Spontaneous bioelectric activity of cultured Purkinje ceils during exposure to agents which prevent synaptic transmission, Brain Research, 53 0973) 71-79. 13 GELLER, H. M., AND WOODWARD, D. J., Modification by iontophoretic drug application of spontaneous activity of single neurons in long term tissue cultures of rat cerebellum, Fed. Proc., 31 (1972) 231A. 14 GELLER, H. M., AND WOODWARD, O. J., An improved constant current source for micro-iontophoretic drug application studies, Electroenceph. clin. Neurophysiol., 33 (1972) 430--432. 15 GEY, G. O., AND GEY, M. K., The maintenance of h u m a n normal cells and tumor cells in continuous culture. I. Preliminary report: cultivation of mesoblastic tumors and normal tissue and notes on methods of cultivation, Amer. J. Cancer, 27 (1936) 45-76. 16 MILD, W., Cell types and neuronal connections in cultures of mammalian central nervous system, Z. Zellforsch., 69 (1966) 155-188.
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17 HILD, W., AND TASAKI,I., Morphological and physiological properties of neurons and glial cells in tissue culture, J. Neurophysiol., 25 (1962) 277-304. 18 HOFFER,B. J., SIGGINS,G. R., AND BLOOM, F. E., Studies on norepinephrine containing afferents to Purkinje cells of rat cerebellum. 11. Sensitivity of Purkinje cells to norepinephrine and related substances administered by microiontophoresis, Brain Research, 25 (1971) 523-534. 19 HOSLI, L., ANDRES, P. F., AND HOSLI, E., Effects of glycine on spinal neurons grown in tissue culture, Brain Research, 34 0971) 399-402. 20 KAWAMURA,H., AND PROVINI, L., Depression of cerebellar Purkinje cells by microiontophoretic application of GABA and related amino acids, Brain Research, 24 0970) 293-304. 21 K~M, S. U., Observations on cerebellar granule cells in tissue culture. A silver and electron microscope study, Z. Zellforsch., 107 (1970) 454-465. 22 LEIMAN,A. L., AND SERE,F. J., Spontaneous and evoked bioelectric activity in organized cerebellar tissues, Exp. Neurol., 40 (1973) 748-758. 23 OBATA, K., Acetylcholine and gamma-aminobutyric acid action on tissue cultured cells from sympathetic ganglion, dorsal root ganglion and diaphragm muscle, Fed. Proc., 31 (1972) 231A. 24 Ross, L. L., BORNSTEIN,M. B., AND LEHRER,G. M., Electron microscopic observations of rat and mouse cerebellum in tissue culture, J. Cell Biol., 14 (1962) 19-30. 25 SCHLAPFER,W. T., MAMOON, A., AND TOBIAS,C. A., Spontaneous bioelectric activity of neurons in cerebellar cultures: evidence for synaptic interactions, Brain Research, 45 (1972) 345-363. 26 SEIL, F. J., Neuronal groups and fiber patterns in cerebellar tissue cultures, Brain Research, 42 (1972) 33-51. 27 SIGGINS, G. R., HOFFER, B. J., AND BLOOM, F. E., Cyclic adenosine monophosphate: possible mediator for norepinephrine effects on cerebellar Purkinje cells, Science, 165 (1969) 1018-1020. 28 SNIDER, R. S., TERAMOTO,S., AND BAN, J. T., Activity of Purkinje and basket cells in isolated cerebellar folia, Exp. Neurol., 19 (1967) 445454. 29 WOLr, M. K., AND DUBOIS-DALCQ,M., Anatomy of cultured mouse cerebellum. I. Golgi and electron microscope demonstrations of granule cell, their afferent and efferent synapses, J. comp. Neurol., 140 (1970) 261-280. 30 WOODWARD, D. J., Poststimulus and Interspike Interval Histogram Program, Digital Equipment Corporation Users Society, Digital Equipment Corporation, Maynard, Mass., 1971, Decus 12-65. 31 WOODWARD,D. J., HOFr'ER,B. J., AND ALTMAN,J., Physiological and pharmacological properties of Purkinje cells in rats degranulated by postnatal X-irradiation, J. Neurobiol., in press. 32 WOODWARD, D. J., HOFFER, B. J., SI~INS, G. R., AND BLOOM, F. E., The ontogenetic development of synaptic junctions. Synaptic activation and responsiveness to neurotransmitter substances in rat cerebellar Purkinje cells, Brain Research, 34 (1971) 73-97. 33 WOODWARD,D. J., HOFFER, B. J., SIQCINS, 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) 91-100.
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
RESPONSES OF C U L T U R E D C E R E B E L L A R P H O R E T I C A L L Y APPLIED A M I N O ACIDS
NEURONS
TO
67
IONTO-
HERBERT M. GELLER* AND DONALD J. WOODWARD Department of Physiology, University of Rochester School of Medicine and Dentistry, Rochester, N. Y. 14642 (U.S.A.)
(Accepted January 9th, 1974)
SUMMARY Electrical activity of spontaneously firing neurons in explant cultures of neonatal rat cerebellum was recorded with extracellularly placed microelectrodes. Several different normal patterns o f firing were noted, common ones being single spikes at regular intervals, irregularly spaced single spikes and rapid bursts o f up to l0 spikes separated by periods o f silence. The firing rate could be modified by the local application o f drugs through iontophoresis onto the soma and adjacent processes. Firing rates were increased by glutamate and homocysteate, and decreased by gamma-aminobutyric acid (GABA) and glycine. The action o f GABA was blocked by bicuculline and picrotoxin. GABA was relatively more effective than glycine. Acetylcholine was generally ineffective, but rarely had weak excitatory actions. We conclude that the sensitivities of the cultured Purkinje cell with respect to these drugs is the same as the adult Purkinje cell in vivo and similar to the immature cell at the time o f explantation. We suggest that pharmacological and physiological criteria be used to identify cell types and evaluate the extent of differentiation of neurons in culture.
I NTRODUCTION It has long been recognized that organotypic explant cultures of central nervous system have considerable potential for gaining insight into basic physiological and pharmacological properties of neurons. To exploit such potential we have investigated * Present address: C.M.D.N.J.-Rutgers Medical School, Department of Pharmacology, P.O Box 101, Piscataway, N.J. 08854, U.S.A.
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the physiology and pharmacology of neurons in roller tube cultures of rat cerebellum by extracellular unit recording in conjunction with iontophoretic methods of local drug application. Earlier studies6,17 have demonstrated that some neurons in culture generate continuous spiking activity. Recent interest in this phenomenon has led to work showing that sustained activity can exist in culture in the presence of high magnesium and low calcium, suggesting an independence from synaptic input of at least some components of this activity 1~. In preliminary investigations we also found unit activity which was maintained for a long period of time (more than 1 h). We were therefore encouraged in this study to examine the possibility that this steady activity of neurons in explant cultures could be employed as a basis for examining pharmacological properties of the membranes of these neurons. Earlier studies indicated that addition of pharmacologic agents to the bathing medium can have an influence on aggregate synaptic and spiking activity of cultured neurons7, lz. Recent work investigating the site of action of such drugs has shown cultured dissociated neurons to be individually sensitive to drugs applied to the surface membrane~9, ~3. In studies reported here we have examined the pharmacological sensitivity of neurons in the more normal neuroglial frameworks of the organ explant grown and bathed in an artificial medium. It is shown that many characteristic actions of pharmacologically active agents on sustained spiking are similar in culture and in vivo. A preliminary report o f this work has appeared a~. MATERIALS AND METHODS
The techniques used to prepare newborn rat cerebellar cultures have been reported in detail 5. Cultures of 0-day postpartum animals were prepared weekly. Feeding medium consisted of Eagle's basal medium (GIBCO), 83 ~ ; fetal calf serum (GIBCO), 14 ~ ; chick embryo extract (GIBCO), 1 ~ ; and 2.7 M glucose, 1 ~ , to bring the final glucose concentration in the feeding medium to 33 raM. The medium was exchanged at weekly intervals. Cultures between 2-6 weeks of age were selected on the basis of neuronal outgrowth and thinning out of the original explant in order to provide a closely matched sample space for electrophysiological and neuropharmacological studies. They were placed in a temperature-regulated chamber at 35-36 °C, perfused with Gey's balanced salt solution 15, and inspected with a 40 × objective to locate areas suitable for recording. Such areas showed clearly visible neuronal perikarya with prominent round nuclei and nucleoli, and almost always exhibited electrical activity. In particularly thin cultures, the proximal processes could also be visualized, but they were usually obscured by more highly refractile somata. A single-barreled NaC1 filled recording electrode, 1-3 # m in diameter, with a resistance of 4-10 M ~ was then introduced into the culture and placed near the outline of a neuron soma until spikes could be easily discriminated. Standard recording and monitoring electronics were used. The initial several minutes of spike record were
CEREBELLAR PHARMACOLOGY ill vitro
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recorded on audiotape for later processing by the PDP-12 computer 8°. Integrated activity was displayed on a strip chart recorder to indicate effect o f drug application. After a stable record of several minutes was obtained, a 5-barreled (or at times 3barreled) drug micropipette o f 3-8 # m tip diameter observed directly was maneuvered near to the neuron, until drug effects were observed. Spiking remained stable in this situation for several hours. Drugs were applied through the various barrels through the use of a specially constructed microiontophoresis unit as described by Geller and Woodward 14. The center barrel o f the array was used as a balance barrel, to eliminate the possibility of current-induced artifacts. Drugs used in the various barrels were: 1 M GABA, pH 4; 1 M sodium glutamate, pH 8; 1 M glycine, pH 4; 1 M DL-homocysteic acid (DLH), pH 7.5; saturated picrotoxin; 5 m M bicuculline (in 165 m M NaC1), p H 3.5; 1 M acetylcholine chloride, p H 4.0. Drugs were dissolved in distilled H20 and pH was adjusted with N a O H or HCI. At the conclusion o f the experiment, cultures were placed in Bodian's fixative, and stained according to a modification o f the Bodian silver methodL RESULTS
Morphology of organotypic explants Bodian silver staining techniques were used to characterize the cultures typically employed for the physiological studies (Fig. 1). These studies demonstrated that the morphology was generally similar to that o f neonatal cerebellum described in both Maximov 3,24 and roller tube 16 cultures. The most consistent feature revealed by the Bodian stains was large numbers o f long neural fiber processes. These processes are not detected by the stain in immature cultures, at a time when neurons clearly exist. Presumably, neurofibrillary material accumulates in time inside the fibers which allow their detection by the Bodian stain. Such fiber processes are typically several hundred microns in length and in many cases can be seen extending several millimeters through the area in and around the explant. Usually fibers appear to course in groups as though guided non-randomly in their growth by some aspect o f the glial-fibroblast network. Most fibers could not be traced microscopically back to their cells of origin, either being lost in the complicated network, or becoming small at both ends and impossible to follow. In some cases, fibers end in ring-like structures which have been called 'synapses' elsewhere 1. Fibers were never found in a cell-free area, and are always embedded in a non-neural cell matrix, presumably o f fibroblasts and glial cells. Large neurons, presumably Purkinje cells, were routinely observed in both living and stained cultures (Fig. 1). Multiple fibers containing dense fibrillary material could be seen branching in many directions from the soma which itself was sparsely stained. Cell bodies were sometimes distinguishable within the central explant but much more clearly in the thinner region at the periphery of the original edge of the explant. In contrast to fibers, cell bodies were never seen more than a few hundred microns away from the explants and appeared to have limited mobility. Many small
Fig. 1. Organization a n d structures revealed in silver-stained preparations of rat cerebellar cultures. Cultures stained with a modification of Bodian's silver stain. A: low power p h o t o m i c r o g r a p h of a developed cerebellar explant. Presumptive cortical tissue is located on either side of a fissural r e m n a n t at lower middle portion of explant (20 days in vitro (DIV), x 22). B: row of aligned Purkinje cells. This culture displayed an extremely high degree of organization, illustrated here a n d in C (25 DIV, x 350). C: high power view o f several Purkinje cell s o m a t a with proximal dendrites. T h e dendrites are aligned in a pattern reminiscent of the #t vivo situation (25 DIV, .-: 625). D: typical high magnification view of a silver-stained culture. Several weakly stained neuron s o m a t a (arrow) s u r r o u n d one well-impregnated n e u r o n (N). Note the large a m o u n t of neurofibrillary material which s u r r o u n d s the s o m a t a (44 DIV, x 475). E: ring form ending (long arrow) in close proximity to a lightly stained neuron s o m a (N). T h e neuronal nucleus is clearly visible, a n d the s o m a , out of focus in this micrograph, has been outlined with arrows (44 D1V, ;~ 1150). F: small, bipolar n e u r o n s whose processes are not visible due to the short depth of field of the objective. M a n y of these cells p r e s u m e d to be granule n e u r o n s can be seen in cerebellar cultures (25 D1V, x 825).
CEREBELLAR PHARMACOLOGY
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oblate cells (Fig. IF) were visible in each culture in close proximity to the large neurons and fiber tracts. A striking feature occurring only occasionally consisted of a line o f many somata of presumed Purkinje cells, each with its primary dendrite oriented in the same direction stimulating the orientation of the apical dendrite system of the molecular layer in vivo (Fig. 1B, C). More typically, development in culture evidently leads to the multipolar configuration which would be the form most often encountered by microelectrodes. Most cell bodies did not have visible stained endings terminating on them; a few, however, have shown boutons on the soma or nearby processes (Fig. IE). Little definitive information is provided by the Bodian technique on the prevalence of synaptic connections. Electron microscopic studies were carried out on several typical explants (West, M. J., unpublished observations). Synaptic structures and myelinated axons were readily observed in many areas. Many round cells of 6-8/~m diameter with scant cytoplasm, closely resembling granule cells 21, were observed. Multicontact synaptic structures were often observed resembling glomeruli in vivo and similar to those observed in culture by others zg. The phase contrast optics used during electrophysiological studies usually allowed only a view limited to the neuronal soma. Typically, large, round cell bodies could be distinguished among the network of cells and processes, with round, clearly visible nuclei and nucleoli. Occasionally in thinner regions of the culture under the best conditions the thick adjacent parts o f the neural processes could be followed 100/~m or more into the nearby regions. Finer neural elements, however, could not be resolved optically, nor could axonal processes be followed for appreciable distances. During the typical recording situation, the tip of a recording electrode could be located visually within a few microns of the soma. A multi-barrel array of drug-containing electrodes was positioned on the opposite side of the soma. Thus, it appears from the visualization under phase contrast that recording and drug application are both restricted to the soma and a small adjacent portion of the long fibrous processes. Normal spontaneous activity Spontaneous neural activity was detected in these cultures by advancing a singlebarrel electrode through the multi-layered structure of the explant toward the outline o f a cell until detection of activity proved it to be a neuron. Activity appeared to be a stable property and ceils routinely displayed the initial pattern observed until the recording was terminated after from several minutes to more than an hour. Mean firing rates were calculated for 46 cells recorded in culture. The range o f firing rates extended from 1/sec to a maximum o f 35.6/sec, with a mean rate of 9.23 8.22 (S.D.). Fig. 4 shows representative interspike interval histograms of several such units illustrating the range o f variability in the patterning o f firing observed. The most typical unit is shown in Fig. 2B having a mean firing rate o f 17.7/sec and for which the intervals were distributed exponentially in an approximate unimodal distribution. This apparent randomness o f long and short intervals is contrasted with another unit as shown in Fig. 2A displaying an extremely high regularity o f firing as revealed in the sharp peak o f the interspike histogram. Unit activity could also be found which was
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Fig. 2. Interspike interval histograms and sample spike records constructed from taped records of activity from cultured Purkinje cells. See text for interpretation. A : very regular firing pattern from a neuron in a 16-day-old culture. Firing rate of 9.5 spikes/sec, with a modal interspike interval of 105 msec. B: interspike interval histogram of irregularly firing neuron from a 15-day-old culture. The distribution has a mean rate of 17.7 spikes/sec and is coupled with a modal interval of 5 msec. The long tail represents the low, but finite, probability of long intervals. C: interval histogram of irregularly firing cell, 15 days in culture. The mean rate of this cell of 8.1 spikes/sec was coupled with a modal interspike interval of 192 msec. D: interspike interval and interburst interval histograms recorded from a Purkinje neuron in an 18-day-old culture. The mean firing rate for this cell was 5.0 spikes/sec. DI: the interspike interval histogram shows the most probable interval within bursts was 71 msec, with the long tail representing longer intervals between bursts. Substantial time is represented by intervals longer than the 500 msec scale. Dm: the interburst interval histogram constructed on-line through a special burst detection subroutine29. The burst detection of cycling is most accurately described by points between 0.75 and 2 sec. Burst intervals less than 0.75 sec represent phases of slow irregular firing.
extremely irregular as in Fig. 2C. F u r t h e r m o r e , other units displayed consistent bursting forms o f activity, pauses o f slow activity alternating with periods of fast, rapid activity (Fig. 2D). U n i t s employed in drug studies mostly exhibited the slightly irregular p a t t e r n as in Fig. 2B.
E~'ects of pharmacologic agents After a cell was located by positioning a single-barrel electrode o n one side o f the soma, a second multi-barrel electrode was placed o n the other side o f the soma a n d drugs applied by iontophoretic currents t h r o u g h the various barrels. N o effects were observed unless the drug electrode was visibly close to the soma. S p o n t a n e o u s activity in this preparation proved to be extremely sensitive to
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GABA. Forty-six cells were examined, all o f which were inhibited by GABA ejected with currents between 0 and 125 nA. Inhibition o f spontaneous activity could often be elicited simply by turning offthe holding current used to prevent diffusion from the electrode tip. In our experiments we were able to put this observation to use by allowing GABA to diffuse from the barrel during the initial placement of the drug micropipette. The micropipette was slowly advanced towards the cell until the GABA leak noticeably affected firing rate. We then stopped movement, and the drug micropipette was left in place for the duration of the experiment. Firing returned to control levels upon restoring holding current for GABA. Whereas clear effects on neural activity were easy to determine by inspection of the effect on firing rate, the certainty of a negative effect was more difficult to determine. We labeled a test as having no effect in those instances where at least one other drug, in most cases GABA, was effective and where no clear sign o f drug electrode block or other evidence could be found o f failure of drugs to be released. Our denotation o f ' n o effect' is conservative in that more instances can be attributed to a simple technical failure to release sufficient drug near the cell. Excitatory amino acids active in vivo had similar actions in culture. Glutamate, ejected with currents from 0 to 100 nA, excited 7 cells with latencies between 0 and 16 sec and had no effect on two cells. DL-Homocysteic acid, an amino acid not occurring in nature, excited 4 of 7 cells. Neither o f these agents at any time showed an inhibitory action upon spontaneous activity when applied in low doses. In some instances 10-8 M glutamate was injected in the bathing medium. In such cases cells first became excited and discharged at progressively faster rates. Spike size then became smaller and the cell went into a phase o f inactivation. Changing back to the original bathing medium caused reversal to normal firing. Such inactivation can be accounted for by excessive depolarization of the cell membrane. Glycine was found in 3 cells to have an inhibitory action on spontaneous activity (Fig. 5). No effects were observed on 8 cells. In 4 additional instances, cells exhibited uncertain or ambivalent effects in which inhibition appeared on a single trial application o f iontophoretic currents, but not on subsequent trials. The iontophoretic currents required for the successful glycine effects were greater in every instance than those required for GABA inhibitions, with 100-2t30 nA required in most cases. The ambivalent or negative effects can be likely accounted for here by the requirement o f relatively large amounts o f drug close to the membrane to evoke effective inhibition. Acetylcholine was tested on 15 cells. There was virtually no effect of spontaneous activity (Fig. 5) in 9, whereas in 6 somewhat ambivalent excitatory effects were found. The excitation was gradual and had a long latency of onset (5-20 sec) (Fig. 5C) compared with the actions of glutamate and GABA, and the firing rate slowly returned to control levels (with delays of up to 1 min) following termination of ejection. One m M atropine was placed in the bath 3 times and found to have no effect on spontaneous activity. Similarly, 1 m M D-tubocurarine was put in the bathing media and twice not found to have an effect on the spontaneous activity. Strychnine was placed in the bathing media 3 times at 1 m M concentration without influence on spontaneous activity.
74
H. M. G E L L E R A N D D. J. W O O D W A R D
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Fig. 3. Some effects of GABA on spontaneously firing cerebellar neurons in culture. In this figure and those following, the ordinate represents neuronal discharges in successive epochs, and the abscissa represents time, with the length of drug application indicated by bars with arrows. The drug being applied is indicated above the bar, with the application current in nA. A: the effect of the competitive antagonist alkaloid bicuculline on GABA inhibition of cultured Purkinje cells. GABA was allowed to diffuse out of the pipette for periods of 5 sec at a supramaximal dose and was retained for 5 sec. The application of bicuculline for a period of 1 min significantly reduced the effectiveness of GABA in shutting off the neuron. B: the cell in this figure was quiescent. Two short pulses of GABA were applied to the cell. Upon cessation of drug application, the cell responded with a burst of 15-20 spikes over the next 2 sec. See text for discussion. C: a pulse of GABA sufficient to shut off neuronal firing was applied for 20 sec. When the drug was stopped, the firing resumed at approximately double the control rate and did not return to baseline for 2 rain. D: GABA retaining current was reduced here from 40 nA to 10 nA. The cell continued to fire spontaneously for 3-4 sec, emitted a burst of 7-10 spikes, and then, in the next 4 sec, stopped firing.
P i c r o t o x i n a n d b i c u c u l l i n e , k n o w n to i n h i b i t the a c t i o n o f G A B A 9, w e r e tested by a p p l y i n g t h e m f r o m a n o t h e r b a r r e l o f t h e p i p e t t e . P i c r o t o x i n b l o c k e d t h e a c t i o n o f G A B A in 2 i n s t a n c e s a n d h a d n o effect in 3 instances. B i c u c u l l i n e b l o c k e d t h e a c t i o n o f G A B A o n 2 cells in w h i c h it d i d n o t alter the p a t t e r n o f firing. O n 7 o t h e r cells, b i c u c u l l i n e e v o k e d cyclic bursts o f firing, a n d m a y h a v e o b s c u r e d a b l o c k i n g o f G A B A action. M a n y f e a t u r e s o f d r u g effects o n l o n g - t e r m a c t i v i t y o f n e u r o n s in c u l t u r e s r e s e m -
75
CEREBELLAR PHARMACOLOGY #l vitro
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Fig. 4. Effects of iontophoretically applied excitatory agents on quiescent and steadily firing neurons. A : cell which was firing at 0-2 spikes/sec was speeded up to 7 spikes/sec by application of 75 nA of sodium glutamate. The effect of GABA at 25 nA was to reduce the firing of the cell, while at 50 nA the cell was shut off completely. B: the effect of DL-homocysteate upon spontaneous firing of a cerebellar Purkinje cell. Initially a dose of 125 nA provokes a slight increase in firing rate. As the drug is repeatedly applied, the efficiency is increased, until 75 nA causes the same rise as the initial 125 nA.
ble actions on cells in vivo. Depending on the position of the drug electrode effects of drugs on firing rates could be shown in some cells to vary up or down as more iontophoretic current was applied, indicating a rough dose response relation (Fig. 4A). Also, a repeated series o f several seconds o f iontophoretic current from individual drug barrels gave successively greater effects (Fig. 4B). This observation is probably due to an effect of prolonged retaining currents loading the end of the drug releasing pipette with inactive ions. With repeated drug application, a higher amount of active ions are brought to the tip and ejected. Fig. 3 shows some interesting features on the inhibition of spontaneous activity. At times, firing rates did not gradually slow down when GABA was applied at effective levels, but instead there often occurred a phase of excitation before the total inhibition of activity (Fig. 3D). Also, application of drugs causing total inhibition for a few seconds was often followed by a rebound phase of a much more rapid activity lasting 10-20 sec (Fig. 3C). The onset and release of the GABA effect involved several seconds o f delayed onset so the more immediate direct current effects are likely not involved. Excitatory and inhibitory drugs exhibited clear interactions, as in Fig. 4A. Glutamate could overcome the action o f GABA. This indicated that GABA was not causing a blockade o f the spike generation mechanism, but an inhibition which could be overcome by a depolarizing substance. In addition, cells which were stimulated to fire more rapidly by glutamate could also be depressed with GABA. DISCUSSION
The present results show that drug-induced changes in the spontaneous activity
76
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I
Fig. 5. The effects of microiontophoretically applied drugs on cerebellar Purkinje neurons. A: GABA at 10 nA consistently reduces the firing rate of tke neuron, while applied glycine at a dose of 250 nA does not affect the cell. B: GABA is efficient at reducing the firing of this cell at a dose of 25 hA, while glycine has the same effect only at the larger current of 100 nA. Both drugs provoke a rebound of the firing of the cell to higher than control levels. Acetylcholine (ACh) at a current of 100 nA was ineffective in influencing the cell firing. C: acetylcholine was effective in increasing the discharge rate of this neuron at a dose of 80 nA. The latency of onset was over 10 sec, and the time to peak response was almost 1 rain. After cessation of drug application, the firing rate decreased, but did not return to control level. In this same cell, glutamate, 2 hA, caused an increase in the discharge rate with a latency of less than 1 sec, which returned to baseline levels upon stoppage of the ejection current (not illustrated).
o f n e u r o n s in c u l t u r e c a n be used as a m e a s u r e o f t h e sensitivity o f t h e m e m b r a n e to a p p l i e d p h a r m a c o l o g i c a l agents. T h e effects o f the a m i n o a c i d p u t a t i v e n e u r o t r a n s m i t t e r s o n c u l t u r e d c e r e b e l l a r P u r k i n j e cells are c o m p a r a b l e to effects in vivo a n d a l l o w us to c o n c l u d e t h a t t h e s u r f a c e m e m b r a n e retains its sensitivities to t h e s e agents. The morphological investigations confirm the observations of many investigat o r s t h a t c u l t u r e d c e r e b e l l a r n e u r o n s w i t h i n e x p l a n t s will d e v e l o p in this in vitro system. T h e r o l l e r t u b e m e t h o d , s i m i l a r to t h a t e m p l o y e d b y H i l d 16, is s i m p l e to use and promotes rapid thinning out of the culture, favoring extended survival through
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adequate nourishment. The degree of neuronal organization seen in these roller tube cultures can be comparable to that reported by other investigators using Maximov cultures 3,21,26 where less flattening occurs. It has long been presumed in previous morphological studies5,12,17 that the large neurons of the type we observed here are Purkinje cells, because this is the main cell type which would grow a large soma with long axonal processes. To these criteria we can now add the identification of some pharmacological sensitivities to amino acids characteristic of the Purkinje cell in vivo. Our interest is to eventually consider similar criteria for other cells in explant cultures. We propose that it is now feasible to employ pharmacological investigations generally in the identification of cell type, or in determining whether basic cell properties have deviated in vitro. The wide variation of mean firing rates reported here correlates with previous observations in cultureS, 11. A similar wide range of activity has been reported for adult Purkinje cells in vivo is. Sustained activity has been reported in all studies involving absence of innervation of Purkinje cells: immature cerebellum before synaptogenesisa2, pedunculotomized cerebellum1°, and undercut cerebellar cortex 2s. Such phenomena suggest a dominant autonomous component in the generation of activity. If this is the case, the wide range of mean rates may not be due to variation in tonic synaptic inputs as much as to variation in individual neuronal metabolism or resting conductance properties of cell membrane. The different patterns of firing in culture seem likely due to excitatory and inhibitory synaptic influences superposed upon the autonomous activity. An irregularization of firing as shown in Fig. 2D could be due to excitatory input reducing interspike intervals as suggested in the theoretical and experimental studies on spinal motoneurons 4. Longer pauses or bursts could be generated by excitation or by inhibition in more complex interactions. The presence of synaptic activity has been postulated to account for similar interval distributions observed in cerebellar cultures by others z~. The effect of the applied pharmacological agents is consistent with the hypothesis that Purkinje cells have retained their sensitivities to these drugs during maturation in culture. The developing Purkinje cell is rather unique in the extent of detailed information available concerning functional properties at the time of explantation. At this time, the Purkinje cell of the rat is observed as a small neuron with many fine fibrous processes extending from the soma and a fine axonal process leading down to the cerebellar nuclei. The large dendritic tree which characterizes the mature cell develops from days 7 to 21 postnatally. The Purkinje cell at birth, though not yet innervated, has been demonstrated to be capable of generating action potentials and to display well-characterized responses to the pharmacological agents used in this studya2. The physiological and pharmacological properties displayed by these cells in culture, which have matured in the sense of growing a large soma and long axonal processes, are thus not ones which they have acquired, but ones which they have evidently retained as part of previously expressed genetic information. GABA consistently decreased the firing rate of cultured Purkinje cells presumably by hyperpolarizing the neuronal membrane, as has been shown in previous studies~7,aa. The specificity of the action of GABA was supported by the blockade of
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these effects with picrotoxin and bicuculline. Other responses with GABA parallel results from in vivo investigations. Depending on the position of the drug barrel with respect to the cell, weak dose-response relations could be demonstrated. The latency to onset and threshold current level also could be shown to vary much as in vivo. Furthermore, long-lasting rebound increases of the firing rate to supra-control levels were often observed following GABA application, as in Fig. 3C. Such an effect could result from a loss ofintracellular K + during the period of presumed drug-induced high K + conductance after which the membrane would tend to be depolarized towards threshold causing the sustained increase in activity. The implication of this rebound effect is that mean activity of a neuron may be influenced over long periods of time by ionic imbalances induced by relatively short-term alterations of membrane conductances. As a consequence, substantial alterations in firing may be induced during periods when alteration by active synaptic input is not present. Glycine generally had an inhibitory effect on the cells on which it was tested, but at drug application currents much higher than necessary for GABA. This agrees with the ratio of 3.6 calculated to be the equipotent glycine current to GABA current in cerebellar Purkinje cells in cats 2°. This relationship of effectiveness provides further evidence for the similarity of in vivo and in vitro neurons. Experiments in vitro have directly demonstrated an hyperpolarization caused by glycine application on motoneurons 19, The excitatory actions of the glutamic and homocysteic amino acids on most cells studied were consistent with similar actions reported for in vivo investigations 9. In experiments in which glutamate was in the bathing medium, neurons reached instantaneous rates of over 100 spikes/sec. Firing then stopped abruptly, presumably due to excessive depolarization of the membrane, then returned to normal after a delay of several seconds following exchange of the salt solution. Such effects are readily explained by the depolarizing action of these amino acids. The ambivalent excitatory or nonexistent effects of acetylcholine contrasted with the potent effect of other drugs. The excitatory responses observed were of long latency, about 20 sec, and gradual in onset, compared with a few seconds for GABA or glutamate. Large doses (100-200 nA) of acetylcholine were required to elicit effects on those neurons which responded, while doses of up to 500 nA were often without effect. This pattern of responsiveness is similar to the pattern found in immature rat cerebellum a2. Other work, in the intact adult rat (Hoffer, personal communication) and cat s, suggests that some form of sensitivity to acetylcholine exists within the cerebellar cortex of several species in vivo. Acetylcholine sensitivity also survives intact in the rat when interneurons have been eliminated by postnatal X-irradiation al. Our results from culture suggest that intact afferent systems or normal neuroglial morphological relationships may be necessary for these effects. In general, our studies clearly indicate that cultured Purkinje neurons possess chemosensitivity to excitatory and inhibitory putative neurotransmitters. This is a critical piece of information necessary for establishing a potential for synaptic interaction. It now appears feasible to analyze the synaptic connections in culture z2 employing pharmacological interaction with suspected transmitter systems. Also, it
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should now be possible to employ the procedures developed in this study to characterize and manipulate the unique pharmacological membrane properties expected to be encountered in cultured neuronal cell types from many different brain regions. ACKNOWLEDGEMENTS
The technical assistance of Ms. Debra Bickett is gratefully acknowledged. This research was jointly supported by the National Institutes of Health (Grant MH 18367-01), the National Science Foundation (Grant GB 28873-X), and by a General Research Support Grant from the University of Rochester. Dr. Geller held an N.I.H. Postdoctoral Fellowship (GM 50876) and Dr. Woodward an N.I.H.T.I.S.T. Award (NS 11030) during the course of this work.
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