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Effects of ethanol on Purkinje cells recorded from cerebellar slices
Ah'ohol, Vol 1, pp 353-358, 1984 ~ AnkholnternatmnalInc Pnnted m the U S A
0741-8329/84$3 00 + 00
Effects of Ethanol on Purkinje Cells Recorded From Cerebellar Slices FRANK GEORGE AND NAI-SHIN CHU"
D e p a r t m e n t o f Neurology, University o f Cahfornia lrvine M e d w a l Center, Orange, CA 92668 R e c e i v e d 11 M a y 1984 GEORGE, F AND N -S CHU Effects of ethanol on Pur,~tnje tells recordedfrorn cerebellar shces ALCOHOL 1(5) 353-358, 1984 --The effects of ethanol on rat cerebellar Purklnle cells were studied using brain shce preparatmns_ The spontaneous actlwty of Purklnje cells exhibited either steady or cyclic finng patterns The steady units were generally lnh~b~tedby ethanol, but some units were excited by low doses The cychng units showed a dose-related decrease In finng rate and the duratmn of cychc finng_ The data indicate that ethanol in concentrations which produce behaworal effects is primarily inhibitory to cerebellar Purklnle cells. Ethanol
Cerebellar Purklnje cells
Brain slice
ATAXIA is the prominent symptom in ethanol intoxication, suggesting that cerebellum may be one of the major sites of ethanol action Single unit study also indicates that cerebellar cortex is one of the most sensitive brain regions to ethanol [9]. However, the effects of ethanol on cerebellar Purklnje cells in m wvo studies have been variable Ethanol is inhibitory to cerebellar Purkinje cells m some studies [6,9], but excitatory in others [14, 16, 17]. Apparently, the observed effects were complex ones The altered actwlty of cerebellar Purkinje cells might be in part due to dosage, the systemic effects of ethanol and its metabohtes, influences from other parts of the brain, and the use of anesthetics [2, 14, 15, 16, 17]. It is Important that a different approach is used to elucidate the direct actions of ethanol on cerebellar Purkinje cells. In recent years, brain slice methods have become increasingly useful for electrophyslological and pharmacological studies [5] The technique eliminates the blood-brain barrier and the systemic and synaptic interactmns. In many cases, cells and their regional architecture can be identified visually. Properly prepared, it is possible to retain most, if not all, of the local neuronal circuitry. It has advantage over mlcroiontophoresls to allow more precise dose-response study. Thus, this method is ideal for examining drug effects on neuronal actlwty Recently, this technique was utilized to demonstrate the actmns of ethanol in hippocampus slices [1] There are also several electro-physiological studies with cerebellar slice preparations [10, 11, 12, 20] In this study, cerebellar slices were used to examine the dose-response relationship between ethanol and cerebellar Purkmje cell activity. METHOD Male Slmonsen rats between 30 and 50 days old and
weighing 150-180 grams were used. Animals were decapitated between l0 a.m and 12 p.m. Whole brain, including brainstem, was quickly excised and placed in ice-cold artificial CSF which was composed of NaCl 124.0, KCI 3 3, KH2PO4 l 2, MgSO4 2 4, CaCI2 2.5, NaHCO~ 25 7, and D-glucose 10.0 in raM. The brain was cooled for approximately 2 to 4 minutes to reduce the possibility of anoxia before it was removed from solution and grossly dissected to isolate the cerebellum Several 300/zm slices were cut saggitally from the vermis using a mechanical tissue chopper and immediately immersed in the ice-cold artificial CSF. The slices were gently transferred to a nylon mesh platform in a recording chamber which was located in the center of a larger heating chamber filled with normal saline (Fig. 1). The slices were rested on the nylon mesh such that the flow and the level of the solution could be vaned with little effect on the tissue. With the opening of the larger chamber covered, the temperature of the bathing solution was gradually increased from 34°C to and malntamed at 37°C Heated and humidified air consisting of 95% 02 and 5% CO~ was continuously circulated through the recording chamber. Fluid level was maintained at or just above the top of the slices. This method typically provided viable tissues for at least 5 hours. Solutions were introduced into the recording chamber through a valve system. Complete exchange of perfusion solution within the recording chamber was achieved in approximately 2 minutes with a gravity-assisted flow rate at 5 ml/min which was manually controlled with an IV infusion dnp-clamp apparatus The slices were maintained in the recording chamber for 1 hour prior to the recording. A tungsten mlcroelectrode with 1 /zm tip and an impedance of 9-12 MO was lowered through the central opening of the cover into the Purkinje cell layer under direct visual
~Thls research was supported by a grant from Nauonal Instnute on Alcohol Abuse and Alcoholism (K02-AA00049-04)to Dr Chu. -'Requests for reprints should be addressed to N -Shin Chu, Department of Neurology, University of California Irvme Medical Center, 101 City Dnve South, Orange, CA 92668
353
354
GEORGE AND CHU
Oscilloscope Polygraph
Mmroscope I I
Stimulator
I
I
Temperature
Controller
U Outflow
FIG 1 Schemat=c for m
vitro
recording system See text for detads
guidance using a low-power microscope. Extracellular action potentials were amplified through a dual stage amplification system and displayed on an oscilloscope Wellisolated action potentials were converted to pulses of constant voltage via a window dlscrirmnator. The constant pulses were then counted by a ratemeter and summed over 1 sec intervals and displayed on a polygraph. Purkinje cells were verified by climbing fiber responses following white matter stimulatmn with a Grass SD9 stimulator using a bipolar tungsten electrode Baseline spontaneous activity of the Purkmje cell unit was observed for 30 minutes using control CSF medium. One of several ethanol solutions ranging in concentrations from 0 05% v/v (40 mg%), 0 1% (80 mg%), 0.2% (160 mg%) to 0.3% (240 mg%) was then introduced into the recording chamber. After approximately 10 minutes, the solution was again switched to control medium. Continuous recording of the unit activity during this entire period allowed for comparison of neuronal activity between control, ethanol and recovery phases RESULTS
Of a total of 63 Purkinje cell units, 30 displayed a steady firing pattern with a mean discharge rate of 3 9+- 16 per second (mean-+S.D.) (Fig. 2A) and the remmnlng 33 exhibited cychc discharges. The cycling units typically fired for a period which ranged from several seconds to several minutes (Fig. 3C) However, the majority of the units had a firing period of approximately 1 minute The firing period was followed by a brief silence period of approximately 10-20 seconds. This firing pattern was consistent within a given cell, but there was considerable variation between cells. Within these cycling units, there appeared to be two groups of fast fining and slow firing cells. The fast units (N=21) had a mean f-tnng rate of 143-+32 per second during their discharge periods (Fig 2B), whereas the slow units (N= 12) had a mean fining rate of 40 + - 19 per second, a rate similar to the steady units. The recordings were usually maintained for 1 to 4 hours without alterations in the size and shape of the spikes The effects of several ethanol doses on spontaneous Pur-
kinje cell activity were successfully observed in 36 units (Table 1). Ethanol generally produced consistent, doserelated responses The onset of Purkinje cell responses to ethanol became evident 2 to 3 minutes following introduction of the ethanol solution and could be accounted for by the time required for media exchange and tissue diffusion [1]. In steady units, the 0.05% ethanol solution produced either a mild excitation or a mild inhibition (Table l and Fig. 3A and 3B). Higher ethanol concentrations resulted in a dose-related decrease in spontaneous firing All Purklnje cell units were inhibited by high ethanol doses of 0 2% and 0 3% There was also a dose-related progressive increase in the degree of inhibition of finng rates (Table I). In addition to decreased finng rates, some units showed periodic bursting discharges (Fig. 3B). All cycling units showed dose-related decrease in both firing rate and the duration of cyclic finng (Table l and Fig 3C). Even at low ethanol dose of 0.05% the discharge rates of all 5 units studied were decreased to a mean of 39% The cyclic firing was completely inhibited by the high ethanol dose of 0 3%_ An exception was the response of a unit to 0 2% ethanol dose (Table l) The discharge rate was not decreased, but the duration of cychc finng was decreased by 40% Finally, while the finng periods of these units were decreased, their silent periods were almost not changed at all by ethanol. DISCUSSION
Autorhythmicity of cerebellar Purkinje cells has been observed in both acutely and chromcally Isolated cerebellar tissues [10, l l , 12, 18, 20] The studies by Yamamoto [20] and by Llinas and Suglmon [10,11] have shown that the majority of Purkinje cells in guinea-pig cerebellar slices exhibited spontaneous activity of cyclic discharges at frequencies from 20 up to 150 spikes per second. Although Okamoto and Quastel [12] also observed spontaneous activity of Purkinje neurons in the guinea-pig cerebellar slices, the patterns of their spontaneous discharges were not described Present study shows that approximately half of the rat cerebeUar
CEREBELLAR PURKINJE CELLS
FIG 2 Spontaneous activity of two cerebellar Purlonje cells_ (A) A steady firing unit with a discharge rate of approximately 45/sec (B) Acuvity of a cycling umt during the finng period The d~scharge rate was approximately 120/sec
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FIG 3 Alterations of cerebeUar Purklnje cell activity foUowlng ethanol administration (A) A steady unit with a basehne finng rate ~: approximately 30 per second. Ethanol, 0 05%, produced a 50% reduction in finng rate w~thln 2 minutes The inhibition lasted throughout ethanol exposure The u m t activity returned to baseline finng approximately 6 minutes after resumptnon of the control meduum IB) a, steady unit with a b a s e h n e discharge rate of approximately 50/sec Ethanol, 0 1%, resulted in an imtnal excitation followed by a depression Note the brief bursts of activity prior to the o n s e t of inhibition The f i n n g rate returned to baseline approximately 6 minutes after ethanol was removed (C) A cycling unit with a f'tnng rate of greater than 100/sec and a firing period of approximately 1 minute Ethanol, 0 05%, caused a reduction m both firing rate and period This u m t returned to control finng pattern several minutes after r e s u m p t i o n of the control solulnon The upward arrow indicates beginning of ethanol perfusnon and downward arrow lndncates return to control m e d m m H o n z o n t a l b a r ~ 1 mm, and vert~c,d b a r = 100 spikes/second
357
CEREBELLAR PURKINJE CELLS TABLE 1 EFFECTS OF ETHANOL ON SPONTANEOUS ACTIVITY OF CEREBELLAR PURKINJE CELLS Steady Units (25)* Ethanol Dose (% v/vl
% Excited
% Inhibited
0 0 0 0
40(2) 13(1) 0 0
60(3) 87(7) 100(6) 100(6)
05 1 2 3
Cycling Units (11) % Change* 20 ± -42 ± -74 ± -90±
30 14 10 4
% Inhibited
% Change
% Change m DurationS:
100(5) 100(3) 100(1) 100(2l
-39 ± 14 -50 ± 7 0 -100
-35 ± 12 -58 ± 12 - 40 -100
*The numbers m parentheses indicate the numbers of Purkmje cell units SThe mean and S.D of the percentage change m finng rate of all units for the particular ethanol concentration Minus indicates a decrease in finng rate :~The mean and S D of the percentage change m the firing duration of all units for the particular ethanol concentration. Minus indicates a decrease In firing duration
Purkmje cells exhibited cycling dtscharges, but the remainmg ones had steady firing with a m e a n rate of 39/sec which was almost identtcal to that o f 41/sec o b s e r v e d in w v o study from our laboratory [2]. H o w e v e r , the charactensttcs of cyclic finng o f rat cerebellar Purkinje cells w e r e stmilar to t h o s e of guinea-pig Purlonje cells [10, l l , 20]. The steady firing Purkmje cells were generally inhlbtted by ethanol in dose-related manner. The inhibttton o f the steady units was apparent e v e n with low doses of ethanol. The finding is in contrast to in vivo studies which usually s h o w e d that the p r e d o m m a n t responses o f cerebellar Purkmje cells to low doses of ethanol were excitatory [2,16]. All of the cycling units w e r e mhlbtted by both low and i'ugh doses of ethanol, suggesting that cyclic finng Purkmje cells w e r e more susceptible to ethanol than w e r e steady firing Purkmje cells. Study by Llinas and Sugimorl [10,11] in guinea-pig bram slices has s h o w n that cyclic bursting discharges o f Purkanje cells w e r e generated by Ca2+-dependent splkmg and K ÷ conductance. In hippocampal slices, ethanol a u g m e n t e d Ca2+-mediated m e c h a n i s m s including hyperp o l a n z a t i o n and increased K ÷ c o n d u c t a n c e [1] It is possible that ethanol exerts its inhibitory effects on the cyclic discharges of Purkmje cells by those Ca2+-mediated mechanisms.
Compatible results from this study and the study using mtcro-lontophoresis and mtcropressure injection [ 15] suggest that the o b s e r v e d mhlbition by ethanol on Purkinje cells were most likely a dtrect effect of ethanol H o w e v e r , the inlubitory effects of ethanol in brain slice study appear to be m o r e p r o n o u n c e d w h e n c o m p a r e d to those m m vivo studies [2, 14, 16]. The e n h a n c e d mlubitlon may be in part due to mterruptton of excitatory climbing and m o s s y fiber mputs. Since ethanol inhibits noradrenergic locus coeruleus and serotonergic raphe neurons [3, 4, 13], interruption of these ascending mhtbitory p a t h w a y s [8,19] also r e m o v e s excitation due to dislnhibltion when ethanol is administered via systemtc route. In s u m m a r y , the present data indtcate that the predominant effects o f ethanol on cerebellar Purkmje cells w e r e inhibitory. The inhibition was d o s e - d e p e n d e n t at concentrations which are within the ranges of ethanol levels necessary for behavioral responses The advantage o f brain slice methods relative to in vivo methods o f electrophyslologlcal analysis, suggest that brain slice techniques are useful in determining the relative importance of systemic and m e t a b o h c variables revolved in the effects of ethanol on the CNS
REFERENCES 1 Carlen, P L., V Gurevich and D Durand. Ethanolin low doses augments calcmm-medlated mechamsms measured intracellularly in hlppocampal neurons. Science 215: 306--308, 1982 2 Chu, N-S Effects of ethanol on rat cerebellar Purkinje cells lnt J Neuroscl 21: 265-278, 1983 3 Chu, N-S. and C. L Keenan. Effects of ethanol on spontaneous activity of dorsal raphe neurons m vitro Soc Neurosci Abstr 7: 1232, 1983. 4 Chu, N-S Responses of mldbrmn raphe neurons to ethanol Brain Res, in press 5 Dingledine, R , J Dodd and J S. Kelly. The m vttro brain shce as a useful neurophysiological preparation for mtracellular recording Ann Neurosct Methods 2: 323-362, 1980 6. Eldelberg, E_, M L Bond and A. Kelter Effects of alcohol on cerebellar and vestibular neurons. Arch In* Pharmacodyn 192: 213-219, 1971 7 Grupp, L A_ and E_ Perlancki Ethanol-reduced changes in the spontaneous act~wty of single units in the hippocampus of the awake rat A dose-response study Neuropharmacology 18: 63-70, 1979
8 Hoffer, B. J_, G R. SIggms, A P. Ohver and F_ E Bloom. Activation of the pathway from locus coeruleus to rat cerebellar Purkmje neurons' Pharmacological evidence of noradrenerglc central inlubRlon J Pharmacol Exp Ther 184: 553-569, 1973 9 Klemm, W. R , C G Mallarl, L_ R Dreyfus, J C Fiske, E Forney and J. A Mikeska. Ethanol-induced regional and doseresponse differences m multiple-unit activity m rabbits Psyc hopharmacology (Berlin) 49: 235-244, 1976 10 Lhnas, R and M. Suglmon Electrophyslological properttes of tn wtro Purkmje cell somata in mammahan cerebellar shces J Physlol 305: 171-195, 1980. 11. Lhnas, R and M Suglmori_ Electrophysiologlcal properties of m vttro Purlonje cell dendrites m mammalian cerebellar slices_ J Phys~ol 305: 197-213, 1980 12 Okamoto, K. and J. H. Quastel Spontaneous action potentials in isolated guinea-pig cerebellar slices Effects of amino actds and conditions affecting sodmm and water up-take Proc R Soc Btol 184: 83-90, 1973.
358
13 Pohorecky, L A and J Brick Actlwty m neurons m the locus coeruleus of the rat Inhibition by ethanol Brain Re~ 131: 174179, 1977 14 Rogers, J , G R Slggms, J A Schulman and F E Bloom Physiological correlates of ethanol intoxication, tolerance, and dependence in rat cerebellar Purkmje cells. Bram Re~ 196: 183-198, 1980 15 Slggms, G R and E. French Central neurons are depressed by ~ontophoretlc and m~cropressure apphcation of ethanol and tetrahydropapaverolome Drug Al~ ohol D e p e n d 4. 239-243, 1979 16, Sinclair, J. G and G F Lo The effects of ethanol on cerebellar Purkinje cell discharge pattern and inhibition evoked by local surface stimulation Brain Re~ 204: 465-471. 1981
GEORGE
AND CHL'
17 Sinclair, J G , G F Lo and A F TLen The effects of ethanol on cerebellar Punkmje cells m naive and alcohol-dependent rat~ ( an I Ph)',~ud Pharma~ ol 58: 429-432, 1980 18 Snlder, R S , S Teramoto and J T Ban Activity of Purkmjc and basket cells in chromcally isolated t.erebellal foha I ~:, Neurol 19: 667-683, 1967 19 Strahlendorf. J. C H K S t r a h l e n d o r f a n d C Barne', Modulation of cerebellar neuronal activity by raphe st~mulatlon Brain Re~ 169: 565-569. 1979 20 Yamamoto, C Electrical actLvjty observed tn ~lJo m thin ~ectlons from guinea pig cerebellum Ipn I Ph', st,~l 2 4 : [ 7 7 188 1974
0741-8329/84$3 00 + 00
Effects of Ethanol on Purkinje Cells Recorded From Cerebellar Slices FRANK GEORGE AND NAI-SHIN CHU"
D e p a r t m e n t o f Neurology, University o f Cahfornia lrvine M e d w a l Center, Orange, CA 92668 R e c e i v e d 11 M a y 1984 GEORGE, F AND N -S CHU Effects of ethanol on Pur,~tnje tells recordedfrorn cerebellar shces ALCOHOL 1(5) 353-358, 1984 --The effects of ethanol on rat cerebellar Purklnle cells were studied using brain shce preparatmns_ The spontaneous actlwty of Purklnje cells exhibited either steady or cyclic finng patterns The steady units were generally lnh~b~tedby ethanol, but some units were excited by low doses The cychng units showed a dose-related decrease In finng rate and the duratmn of cychc finng_ The data indicate that ethanol in concentrations which produce behaworal effects is primarily inhibitory to cerebellar Purklnle cells. Ethanol
Cerebellar Purklnje cells
Brain slice
ATAXIA is the prominent symptom in ethanol intoxication, suggesting that cerebellum may be one of the major sites of ethanol action Single unit study also indicates that cerebellar cortex is one of the most sensitive brain regions to ethanol [9]. However, the effects of ethanol on cerebellar Purklnje cells in m wvo studies have been variable Ethanol is inhibitory to cerebellar Purkinje cells m some studies [6,9], but excitatory in others [14, 16, 17]. Apparently, the observed effects were complex ones The altered actwlty of cerebellar Purkinje cells might be in part due to dosage, the systemic effects of ethanol and its metabohtes, influences from other parts of the brain, and the use of anesthetics [2, 14, 15, 16, 17]. It is Important that a different approach is used to elucidate the direct actions of ethanol on cerebellar Purkinje cells. In recent years, brain slice methods have become increasingly useful for electrophyslological and pharmacological studies [5] The technique eliminates the blood-brain barrier and the systemic and synaptic interactmns. In many cases, cells and their regional architecture can be identified visually. Properly prepared, it is possible to retain most, if not all, of the local neuronal circuitry. It has advantage over mlcroiontophoresls to allow more precise dose-response study. Thus, this method is ideal for examining drug effects on neuronal actlwty Recently, this technique was utilized to demonstrate the actmns of ethanol in hippocampus slices [1] There are also several electro-physiological studies with cerebellar slice preparations [10, 11, 12, 20] In this study, cerebellar slices were used to examine the dose-response relationship between ethanol and cerebellar Purkmje cell activity. METHOD Male Slmonsen rats between 30 and 50 days old and
weighing 150-180 grams were used. Animals were decapitated between l0 a.m and 12 p.m. Whole brain, including brainstem, was quickly excised and placed in ice-cold artificial CSF which was composed of NaCl 124.0, KCI 3 3, KH2PO4 l 2, MgSO4 2 4, CaCI2 2.5, NaHCO~ 25 7, and D-glucose 10.0 in raM. The brain was cooled for approximately 2 to 4 minutes to reduce the possibility of anoxia before it was removed from solution and grossly dissected to isolate the cerebellum Several 300/zm slices were cut saggitally from the vermis using a mechanical tissue chopper and immediately immersed in the ice-cold artificial CSF. The slices were gently transferred to a nylon mesh platform in a recording chamber which was located in the center of a larger heating chamber filled with normal saline (Fig. 1). The slices were rested on the nylon mesh such that the flow and the level of the solution could be vaned with little effect on the tissue. With the opening of the larger chamber covered, the temperature of the bathing solution was gradually increased from 34°C to and malntamed at 37°C Heated and humidified air consisting of 95% 02 and 5% CO~ was continuously circulated through the recording chamber. Fluid level was maintained at or just above the top of the slices. This method typically provided viable tissues for at least 5 hours. Solutions were introduced into the recording chamber through a valve system. Complete exchange of perfusion solution within the recording chamber was achieved in approximately 2 minutes with a gravity-assisted flow rate at 5 ml/min which was manually controlled with an IV infusion dnp-clamp apparatus The slices were maintained in the recording chamber for 1 hour prior to the recording. A tungsten mlcroelectrode with 1 /zm tip and an impedance of 9-12 MO was lowered through the central opening of the cover into the Purkinje cell layer under direct visual
~Thls research was supported by a grant from Nauonal Instnute on Alcohol Abuse and Alcoholism (K02-AA00049-04)to Dr Chu. -'Requests for reprints should be addressed to N -Shin Chu, Department of Neurology, University of California Irvme Medical Center, 101 City Dnve South, Orange, CA 92668
353
354
GEORGE AND CHU
Oscilloscope Polygraph
Mmroscope I I
Stimulator
I
I
Temperature
Controller
U Outflow
FIG 1 Schemat=c for m
vitro
recording system See text for detads
guidance using a low-power microscope. Extracellular action potentials were amplified through a dual stage amplification system and displayed on an oscilloscope Wellisolated action potentials were converted to pulses of constant voltage via a window dlscrirmnator. The constant pulses were then counted by a ratemeter and summed over 1 sec intervals and displayed on a polygraph. Purkinje cells were verified by climbing fiber responses following white matter stimulatmn with a Grass SD9 stimulator using a bipolar tungsten electrode Baseline spontaneous activity of the Purkmje cell unit was observed for 30 minutes using control CSF medium. One of several ethanol solutions ranging in concentrations from 0 05% v/v (40 mg%), 0 1% (80 mg%), 0.2% (160 mg%) to 0.3% (240 mg%) was then introduced into the recording chamber. After approximately 10 minutes, the solution was again switched to control medium. Continuous recording of the unit activity during this entire period allowed for comparison of neuronal activity between control, ethanol and recovery phases RESULTS
Of a total of 63 Purkinje cell units, 30 displayed a steady firing pattern with a mean discharge rate of 3 9+- 16 per second (mean-+S.D.) (Fig. 2A) and the remmnlng 33 exhibited cychc discharges. The cycling units typically fired for a period which ranged from several seconds to several minutes (Fig. 3C) However, the majority of the units had a firing period of approximately 1 minute The firing period was followed by a brief silence period of approximately 10-20 seconds. This firing pattern was consistent within a given cell, but there was considerable variation between cells. Within these cycling units, there appeared to be two groups of fast fining and slow firing cells. The fast units (N=21) had a mean f-tnng rate of 143-+32 per second during their discharge periods (Fig 2B), whereas the slow units (N= 12) had a mean fining rate of 40 + - 19 per second, a rate similar to the steady units. The recordings were usually maintained for 1 to 4 hours without alterations in the size and shape of the spikes The effects of several ethanol doses on spontaneous Pur-
kinje cell activity were successfully observed in 36 units (Table 1). Ethanol generally produced consistent, doserelated responses The onset of Purkinje cell responses to ethanol became evident 2 to 3 minutes following introduction of the ethanol solution and could be accounted for by the time required for media exchange and tissue diffusion [1]. In steady units, the 0.05% ethanol solution produced either a mild excitation or a mild inhibition (Table l and Fig. 3A and 3B). Higher ethanol concentrations resulted in a dose-related decrease in spontaneous firing All Purklnje cell units were inhibited by high ethanol doses of 0 2% and 0 3% There was also a dose-related progressive increase in the degree of inhibition of finng rates (Table I). In addition to decreased finng rates, some units showed periodic bursting discharges (Fig. 3B). All cycling units showed dose-related decrease in both firing rate and the duration of cyclic finng (Table l and Fig 3C). Even at low ethanol dose of 0.05% the discharge rates of all 5 units studied were decreased to a mean of 39% The cyclic firing was completely inhibited by the high ethanol dose of 0 3%_ An exception was the response of a unit to 0 2% ethanol dose (Table l) The discharge rate was not decreased, but the duration of cychc finng was decreased by 40% Finally, while the finng periods of these units were decreased, their silent periods were almost not changed at all by ethanol. DISCUSSION
Autorhythmicity of cerebellar Purkinje cells has been observed in both acutely and chromcally Isolated cerebellar tissues [10, l l , 12, 18, 20] The studies by Yamamoto [20] and by Llinas and Suglmon [10,11] have shown that the majority of Purkinje cells in guinea-pig cerebellar slices exhibited spontaneous activity of cyclic discharges at frequencies from 20 up to 150 spikes per second. Although Okamoto and Quastel [12] also observed spontaneous activity of Purkinje neurons in the guinea-pig cerebellar slices, the patterns of their spontaneous discharges were not described Present study shows that approximately half of the rat cerebeUar
CEREBELLAR PURKINJE CELLS
FIG 2 Spontaneous activity of two cerebellar Purlonje cells_ (A) A steady firing unit with a discharge rate of approximately 45/sec (B) Acuvity of a cycling umt during the finng period The d~scharge rate was approximately 120/sec
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FIG 3 Alterations of cerebeUar Purklnje cell activity foUowlng ethanol administration (A) A steady unit with a basehne finng rate ~: approximately 30 per second. Ethanol, 0 05%, produced a 50% reduction in finng rate w~thln 2 minutes The inhibition lasted throughout ethanol exposure The u m t activity returned to baseline finng approximately 6 minutes after resumptnon of the control meduum IB) a, steady unit with a b a s e h n e discharge rate of approximately 50/sec Ethanol, 0 1%, resulted in an imtnal excitation followed by a depression Note the brief bursts of activity prior to the o n s e t of inhibition The f i n n g rate returned to baseline approximately 6 minutes after ethanol was removed (C) A cycling unit with a f'tnng rate of greater than 100/sec and a firing period of approximately 1 minute Ethanol, 0 05%, caused a reduction m both firing rate and period This u m t returned to control finng pattern several minutes after r e s u m p t i o n of the control solulnon The upward arrow indicates beginning of ethanol perfusnon and downward arrow lndncates return to control m e d m m H o n z o n t a l b a r ~ 1 mm, and vert~c,d b a r = 100 spikes/second
357
CEREBELLAR PURKINJE CELLS TABLE 1 EFFECTS OF ETHANOL ON SPONTANEOUS ACTIVITY OF CEREBELLAR PURKINJE CELLS Steady Units (25)* Ethanol Dose (% v/vl
% Excited
% Inhibited
0 0 0 0
40(2) 13(1) 0 0
60(3) 87(7) 100(6) 100(6)
05 1 2 3
Cycling Units (11) % Change* 20 ± -42 ± -74 ± -90±
30 14 10 4
% Inhibited
% Change
% Change m DurationS:
100(5) 100(3) 100(1) 100(2l
-39 ± 14 -50 ± 7 0 -100
-35 ± 12 -58 ± 12 - 40 -100
*The numbers m parentheses indicate the numbers of Purkmje cell units SThe mean and S.D of the percentage change m finng rate of all units for the particular ethanol concentration Minus indicates a decrease in finng rate :~The mean and S D of the percentage change m the firing duration of all units for the particular ethanol concentration. Minus indicates a decrease In firing duration
Purkmje cells exhibited cycling dtscharges, but the remainmg ones had steady firing with a m e a n rate of 39/sec which was almost identtcal to that o f 41/sec o b s e r v e d in w v o study from our laboratory [2]. H o w e v e r , the charactensttcs of cyclic finng o f rat cerebellar Purkinje cells w e r e stmilar to t h o s e of guinea-pig Purlonje cells [10, l l , 20]. The steady firing Purkmje cells were generally inhlbtted by ethanol in dose-related manner. The inhibttton o f the steady units was apparent e v e n with low doses of ethanol. The finding is in contrast to in vivo studies which usually s h o w e d that the p r e d o m m a n t responses o f cerebellar Purkmje cells to low doses of ethanol were excitatory [2,16]. All of the cycling units w e r e mhlbtted by both low and i'ugh doses of ethanol, suggesting that cyclic finng Purkmje cells w e r e more susceptible to ethanol than w e r e steady firing Purkmje cells. Study by Llinas and Sugimorl [10,11] in guinea-pig bram slices has s h o w n that cyclic bursting discharges o f Purkanje cells w e r e generated by Ca2+-dependent splkmg and K ÷ conductance. In hippocampal slices, ethanol a u g m e n t e d Ca2+-mediated m e c h a n i s m s including hyperp o l a n z a t i o n and increased K ÷ c o n d u c t a n c e [1] It is possible that ethanol exerts its inhibitory effects on the cyclic discharges of Purkmje cells by those Ca2+-mediated mechanisms.
Compatible results from this study and the study using mtcro-lontophoresis and mtcropressure injection [ 15] suggest that the o b s e r v e d mhlbition by ethanol on Purkinje cells were most likely a dtrect effect of ethanol H o w e v e r , the inlubitory effects of ethanol in brain slice study appear to be m o r e p r o n o u n c e d w h e n c o m p a r e d to those m m vivo studies [2, 14, 16]. The e n h a n c e d mlubitlon may be in part due to mterruptton of excitatory climbing and m o s s y fiber mputs. Since ethanol inhibits noradrenergic locus coeruleus and serotonergic raphe neurons [3, 4, 13], interruption of these ascending mhtbitory p a t h w a y s [8,19] also r e m o v e s excitation due to dislnhibltion when ethanol is administered via systemtc route. In s u m m a r y , the present data indtcate that the predominant effects o f ethanol on cerebellar Purkmje cells w e r e inhibitory. The inhibition was d o s e - d e p e n d e n t at concentrations which are within the ranges of ethanol levels necessary for behavioral responses The advantage o f brain slice methods relative to in vivo methods o f electrophyslologlcal analysis, suggest that brain slice techniques are useful in determining the relative importance of systemic and m e t a b o h c variables revolved in the effects of ethanol on the CNS
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