J. Phyeiol. (1976), 261, pp. 647-671 647With 1 plate and 8 text-figure.Printed in Great Britain

INHIBITORY EFFECTS OF ACETYLCHOLINE ON NEURONESIN THE FELINE NUCLEUS RETICULARIS THALAMI

By Y. BEN-ARJ,* R. DINGLEDINE, I. KANAZAWA ANDJ. S. KELLY

From the MRC Neurochemical Pharmacology Unit,Department of Pharmacology, Medical School, Hills Road,

Cambridge CB2 2QD

(Received 25 March 1976)

SUMMARY

1. Short iontophoretic pulses of acetylcholine (ACh) inhibited almostevery spontaneously active cell encountered in the nucleus reticularisthalami of cats anaesthetized with a mixture of halothane, nitrous oxideand oxygen. On 200 cells the mean current needed to eject an effectiveinhibitory dose of ACh was 67 + 2 nA. When the ACh-evoked inhibitionwas mimicked by y-aminobutyric acid (GABA) or glycine on the samecell, the current required to release ACh was found to be approximatelytwice as great as that required to release an equally effective dose ofGABAor glycine.

2. The ACh inhibitions developed with a latency which was very muchshorter than that for ACh excitation in cells of the ventrobasal complex.The latency of the ACh-evoked inhibition was as rapid as the onset andoffset of the excitation of the same cells by glutamate and their inhibitionby GABA or glycine.

3. The firing pattern of ACh-inhibited neurones in the nucleus reticu-laris was characterized by periods of prolonged, high frequency bursts,and their mean firing frequency was 22 Hz. Raster dot displays and inter-spike interval histograms showed that whereas ACh suppressed thespikes that occurred between bursts much more readily than those thatoccurred during bursts, all spikes were equally sensitive to the depressantaction ofGABA and glycine. Large doses of ACh provoked or exaggeratedburst activity.

4. ACh-evoked inhibition was extremely sensitive to blockade by shortiontophoretic applications of atropine, which had no effect on the inhibi-tions evoked on the same cell by equipotent doses ofGABA or glycine. The

* Present address: CNRS Laboratoire de Physiologie Nerveuse, Gif-sur-Yvette91190, France.

Y. BEN-ARI AND OTHERSACh-evoked inhibitions were also antagonized by dihydro-fl-erythroidinereleased with slightly larger currents. When tested on the same cell,small iontophoretic applications of picrotoxin and bicuculline metho-iodide blocked the inhibition evoked by GABA but had no effect on thatevoked by ACh. Iontophoretic strychnine only rarely affected the in-hibition evoked byACh, while readily blocking the inhibition evoked on thesame cell by an equipotent dose of glycine. In two cats, intravenousstrychnine (1.2 mg/kg) had no effect on the ACh-evoked inhibition, whilegreatly reducing the sensitivity of the cell under study to glycine.

5. Only four out of forty-eight ACh-inhibited cells tested were inhibitedby iontophoretic applications of either guanosine or adenosine 3':5'-phosphate.

6. Cells of the nucleus reticularis have been shown to have an inhibitoryaction on the thalamic relay cells, which are excited by ACh. It is sugges-ted that the presence of both ACh excited and inhibited cells in differentnuclei of the thalamus could be of considerable functional significance ingating sensory transmission through the thalamus.

INTRODUCTION

Although a number of authors have reported that cells in differentregions of the thalamus respond differently to iontophoretic acetylcholine(ACh) (Andersen & Curtis, 1964a; McCance, Phillis & Westerman, 1968b;McLennan, Huffman & Marshall, 1968; Phillis, 1971) these studies mainlyemphasized the ease with which iontophoretic ACh excites the deep asopposed to the superficial cells of the thalamus. Recently, however, anumber of reports have appeared in which a greater proportion of thalamiccells were found to be inhibited by iontophoretic ACh. ACh-inhibited cellspredominated in the medial geniculate body (Teb6cis, 1970), lateralposterior nucleus, pulvinar (Godfraind, 1975), centromedian and lateralposterior regions of the thalamus (Duggan & Hall, 1975a, b).The results of Duggan & Hall (1975a, b) are of particular interest since

they suggest that the apparent inhibitory action of ACh on these cells ismediated solely by a presynaptic action of ACh and that ACh has itsusual excitatory action on the cell under study. We would, therefore, liketo describe in some detail our results (Ben-Ari, Kanazawa & Kelly,1976b; Ben-Ari, Dingledine, Kanazawa & Kelly, 1976a), which showedACh to have an inhibitory action on cells of another thalamic nucleus, thenucleus reticularis thalami.The nucleus reticularis lies more anterior and lateral than other regions

of the thalamus so far investigated with multibarrelled micropipettes.Interposed between the external medullary lamina and the internal

648

ACh INHIBITED CELLS IN THE THALAMUScapsule, it forms a thin shell of relatively large neurones that surroundthe anterior pole of the thalamus. The spontaneous activity of cells in thenucleus reticularis is periodically interrupted by sustained high frequencybursts of spikes (Negishi, Lu & Verzeano, 1962; Mukhametov, Rizzolatti& Tradardi, 1970; Schlag & Waszak, 1970; Lamarre, Filion & Cordeau,1971; Steriade, & Wyzinski, 1972; Waszak, 1974). The inhibitory respon-ses of these cells to ACh is of particular interest since a decrease in theiractivity has been shown to be associated with hyperactivity in thalamicrelay nuclei, and appears to be a concomitant of cortical arousal evoked bystimulation of the mesencephalic reticular formation (Waszak, 1974).Although our knowledge of cholinergic transmission in the feline thalamusis still rudimentary (Phillis, 1971; Krnjevic, 1974), the nucleus reticularisdisplays intense cholinesterase staining in the rat (Shute & Lewis, 1967)and monkey (Olivier, Parent & Poirier, 1970). In the rat the level ofcholine acetyltransferase, a more specific marker of cholinergic nerveterminals, is twice as high in the nucleus reticularis as in the ventralthalamic nucleus (Brownstein, Kobayashi, Palkovits & Saavedra, 1975).

METHODS

Fourteen adult cats of either sex were anaesthetized with a mixture of halothane,nitrous oxide (65%) and oxygen, placed in a stereotaxic frame, paralysed withgallamine triethiodide and artificially ventilated with a halothane (0-5%), nitrousoxide and oxygen mixture. Throughout the experiment, anaesthesia was maintainedas described above and paralysis maintained by means of an intravenous infusion(3 ml/h) of gallamine triethiodide (13-3 mg/ml) in dextrose (4-3%) and saline(0 18%). The blood pressure was continuously monitored and experiments ter-minated if the pressure fell below 80 mmHg. The rectal temperature was main-tained at 370 C by a thermostatically controlled heating pad.

After reflexion of the skin and soft tissues of the scalp a small burr hole wasdrilled in the skull under stereotaxic control and the dura mater removed. A freshlyfilled multibarrelled micro-electrode, centred between the stereotaxic co-ordinatesAP 9-5-11-5 and L 790-8-5 (atlas of Snider & Niemer, 1961), was passed verticallydownwards through the burr hole into the brain until it reached the horizontal co-ordinate H 7-0 where recording began. The micro-electrodes were aligned on thestereotaxic frame during the experiment by setting them up vertically in space withtheir tip centred on the point AP - 40-0, L 0-0, H -10-0 by sighting the tip througha pair of hollow ear bars set up on the stereotaxic frame behind the head of the catexactly 40 mm caudal to the intra-aural line. The depth of the cells was read fromthe micrometer gauge of the micromanipulator.Five or seven barrelled micropipettes with tip diameters of 4-8 #sm, containing

strands of glass fibre, were filled by the method of Tasaki, Tsukahara, Ito, Wayner &Yu (1968) or by centrifugation at 1500 g for 20 min. The central barrel was filledwith 4-0 M-NaCl (resistance 7-10 MCI) and used for single unit recording. The outerbarrels were used for iontophoresis and contained: 1 M AChCl or AChBr, pH 4-5;1 M Na-L-glutamate, pH 7-8, 1 m y-aminobutyric acid, pH 4-0; 1 M glycine, pH3-5; 5 mm bicuculline methoiodide in 165 mm saline, pH 3-5 (cf. Pong & Graham,

649

650 Y. BEN-ARI AND OTHERS1972); 5 mm picrotoxin in 165 mx saline, pH 5*5 (cf. Davidoff & Aprison, 1969);10 mm strychnine sulphate in 165 mx saline, pH 6-0; 5 or 50 mx atropine sulphateor methonitrate in 165 mx saline, pH 6-2; 5 mx dihydro-fl-erythroidine (gift fromDr C. A. Stone of Merck Sharp and Dohme Ltd) in 165 mx saline, pH 4-3; 1 M-NaCl;0-2 m solutions of guanosine and adenosine 3' :5'-phosphate (Na-salts), pH 7*0.

Micro-iontophoretic currents were generated by an operational amplifier basedtechnique in which the command voltages, applied to the input of the operationalamplifier chain, were compared and automatically corrected for the voltage dropacross a 10 MCI resistor placed in series with, and situated immediately above, thedrug-containing barrel of the micro-electrode (Kelly, Simmonds & Straughan,1975). The total return current was routinely displayed on one channel of an oscillo-scope and a strip chart recorder.A backing current of 25 nA was applied continuously to the drug containing

barrels of the micro-electrode. The effects of current on the cells under study weretested by automatic summing of the currents in the drug-containing barrels andpassing a current of opposite sign and equal magnitude through a barrel containing1 m-NaCl.Individual action potentials were converted by an adjustable voltage window

discriminator into standard pulses that were monitored by an audio amplifier andused to brighten the selected action potentials with respect to the background acti-vity by modulating the z axis input to the oscilloscope. The frequency of the outputpulses of the window discriminator was registered continuously by a resettablecounter connected to a rectilinear strip chart recorder. The standard pulses alsointerrupted a PDP-12 computer programmed to display on line raster dot displaysand caused the occurrence time of the individual spikes to be read into core from a1 kHz crystal clock. At 1 or 2 s intervals an interrupt pulse from a Digitimer, whichwas also used to reset the rate-meter connected to the strip chart recorder men-tioned above, caused the clock of the PDP-12 to be reset to zero and an analogue-to-digital converter to read and store the current being passed either by a singlechannel of the iontophoresis unit or the total current being passed by all the chan-nels. Off-line the occurrence time of the spikes and the magnitudes of the ionto-phoretic current could be recalled from incremental magnetic tape and displayedeither as rate-meter records or raster dot displays (Text-figs. 3 and 5). Two smallcursor bars, moved along these displays by means of potentiometers connected toanalogue-to-digital converters, were used to define the beginning and end of theepoch that contained the occurrence time of the spikes to be manipulated by thecomputer. As the cursors were moved across the display the duration of the epochbetween them and the total number of spikes in the epoch were shown on a digitaldisplay. Striking a teletype key caused an interspike interval histogram to be com-puted from the occurrence times of the spikes in the epoch and displayed on theoscilloscope (Text-figs. 3 and 5). The cursors were routinely used to define epochs ofequal duration before, during and after the iontophoretic application of a drug todetermine the percentage change in firing rate caused by the drug (Text-fig. 8).On each cell, the currents required to pass an equally effective dose of ACh and

GABA or glycine were estimated either by a bracketing procedure in which positivepulses of current approximately 20 s in duration were applied alternately to the AChand GABA or glycine barrels of the micro-electrode and the amplitude of the currentapplied to one or other barrel adjusted until the responses evoked by each sub-stance in turn were of similar magnitude (usually 30-80% inhibition), or a moreexhaustive method in which a range of currents were applied to both barrels in orderto construct current response curves (Text-fig. 8). As shown in Text-figs. 6, 7 and 8the action of antagonists was tested on single cells by mimicking the inhibition

ACh INHIBITED CELLS IN THE THALAMUS 61evoked by a 20 s application of ACh by an equipotent dose of GABA or glycine andrepeating this procedure at intervals of approximately 808s and applying the antago-nist continuously until the response to one or other of the agonists was blocked. Onlywhen this blockade was reversible was the test considered valid.At the end of each experiment the animal was anaesthetized more deeply with

intravenous pentobarbitone (50 mg/kg), the thorax opened and the animal per-fused with 300 ml saline followed by 300 ml 10% formal-saline solution injecteddirectly into the left ventricle of the heart. The animal was then decapitated andthe head kept in formal-saline overnight before removal of the brain. Frozen sec-tions of the whole brain were cut on a freezing microtome, stained with cresylviolet or carmine acetate and mounted on glass slides. Each slide was projectedon to a screen and photographs taken from all slides containing micro-electrodetracks.

RESULTS

Location of ACh-inhibited, cells in the nucleus reticularisACh was tentatively suggested to have an inhibitory action on the cells

of the nucleus reticularis when short iontophoretic pulses of ACh werefound to inhibit the spontaneous discharge of virtually every cell found tolie above the horizontal plane H 4-0 and within the region bounded by thestereotaxic co-ordinates AP 9-5-11-0 and L 7-0-8-5 (Snider & Niemier,1961). As shown in Text-fig. 1 the depth at which the largest number ofACh inhibited cells was encountered coincided with the stereotaxic co-ordinates of the nucleus reticularis, through which histological analysisshowed the majority of the micro-electrode tracks to have passed (P1. 1).This suggestion was reinforced in individual experiments in which histo-logical results showed that micro-electrode tracks in which only ACh-inhibited cells were encountered lay within the nucleus reticularis (P1. 1 Aand B). Similarly in micro-electrode tracks in which ACh-excited cellswere found to lie deeper than the ACh-inhibited cells, the histology(P1. 1 C and D) showed that the micro-electrode had passed through thenucleus reticularis and entered the ventrobasal complex of the thalamus,a region known to contain ACh-excited cells (Andersen & Curtis, 1964 a, b).In- Text-fig. 1 B the upper border of the region containing ACh-excitedcells was demarcated by plotting the depth distribution of the most super-ficial ACh-excited cells encountered in each micro-electrode track, usingdata from twenty-two separate micro-electrode tracks in ten cats. Clearlythe greatest number of ACh-inhibited cells was found at the depth wherethe width of the nucleus reticularis is greatest and the chance of meetingan ACh-excited cell increased at depths below H 4-0, where the nucleusreticularis thins out to cover the lateral curvature of the ventrobasalcomplex.The conclusion that the majority of the ACh-inhibited cells lay within

the nucleus reticularis was also strongly supported by their firing pattern.The average firing frequency of the ACh-inhibited cells was 22 Hz (S.E.

651

Y. BEN-ARI AND OTHERS

of mean = 1P4; n = 114) and their on-going activity was interrupted bycharacteristic long duration, high frequency bursts in which the maximumfrequency often exceeded 400 Hz. The firing pattern of the ACh-inhibitedcells was typical of that attributed to cells of the nucleus reticularis by

A B

cc~~ ~ ~ ~ ~ ~ ~ 7

.40

30

200

Text-fig. 1. The depth distribution of ACh inhibited and excitedcells encountered in the thalamus between the stereotaxic co-ordinatesAP 9-5--li5 and L 7-0-8-5. A, the line drawing was traced from thephotomicrograph designated stereotaxic plane AP 9-5 of Snider &; Niemer(1961): nucRis the nucleus reticularis; VBCis the ventrobasal complex, com-prising the ventral anterior, ventral posterior and ventral lateral nuclei;DM is the dorsal medial nucleus; LD is the dorsal lateral nucleus; F, thefornix ; CC, the corpus callosurn; Cd, the dorsal head ofthe caudate ; 8, thestriamedullaris thalami; and v, the third ventricle. B, the open bars of the histo-gram show the number of ACh-inhibited cells encountered at each depthand the filled bars, the number of times that the first ACh-excited cell en-countered was discovered at that particular depth, beneath a layer ofACh-inhibited cells. In ten cats, 200 ACh-inhibited cells were encounteredduring twenty-two micro-electrode tracks.

many other groups of workers (cf. Negishi et al. 1962; Mukhametov et at.1970; Schiag &r Waszak, 1970; Lamarre et at. 1971; Steriade &Z Wyzinski,1972; Waszak, 1974). On the other hand, the cells found to be excited byACh had a significantly lower average firing frequency of 6-0 Hz (s.E. of

652

ACh INHIBITED CELLS IN THE THALAMUS

mean = 0-8, n = 46) and the highest frequency attained during theirrather less prominent periods of burst activity rarely exceeded 100 Hz.Very much earlier Andersen & Curtis (1964a) drew attention to the slowfiring and the short duration burst activity of the ACh-excited cells in theventrobasal complex.

Nature of the ACh-evoked inhibitionThe ACh-evoked inhibition of spontaneously active cells in the nucleus

reticularis could not be mimicked by passing much larger amounts ofpositive currents through an adjacent NaCl-containing barrel of the micro-electrode (Text-fig. 2D and K). The inhibition was only rarely attenuatedwhen the positive current through the ACh barrel was balanced or com-pensated by passing simultaneously an equal amount of negative currentthrough an adjacent NaCl-containing barrel of the micro-electrode. Onthirteen occasions a significant degree of inhibition was evoked simply byreducing the level of retaining current (25 nA) applied to the ACh-containing barrel of the micro-electrode (Text-fig. 2C and J). The averagecurrent used to eject sufficient ACh to elicit 30-80% inhibition in 200 cells,the depth distribution of which is shown in Text-fig. 1 B. was 67 + 2 nA.No significant change occurred with depth in either the firing frequency orthe ACh sensitivity of the cells in Text-fig. 1 B. In contrast, ACh releasedfrom the same micro-electrodes with similar amounts of current (59 + 4 nA).excited forty-six cells encountered as they passed deep to the nucleusreticularis and entered the ventrobasal complex. On a number of occasionsACh was applied to rather more slowly firing nucleus reticularis cells, thefiring rate of which had been enhanced by a continuous application ofglutamic acid (approximately 40 nA). In all of these cases considerablyless current appeared to be required to release an effective dose of ACh.For instance, in one such experiment in which the activity of all sixteencells encountered in the same micro-electrode descent was enhanced bythe release of glutamate, the current required to release sufficient ACh tocause 50 % inhibition was only 38 + 5 nA, compared to the 55 + 7 nA thatwas required to cause a similar inhibition of thirteen spontaneously activecells encountered during an earlier descent by the same micro-electrodethrough the contra-lateral nucleus reticularis.

Since the amount of current required to release an effective dose of asubstance may well be more a characteristic of the micro-electrode thanthe potency of the substance (cf. Ben-Ari & Kelly, 1976), the current usedto eject ACh was compared in the same micro-electrode with that requiredto pass an equally effective dose of GABA and glycine. This may well beof value since the majority of cells in the central nervous system are in-hibited by one or other of these amino acids (cf. Curtis & Johnston, 1974;

653

654 Y. BEN ARI AND OTHERS(1) A

_111UkSM92.&Ijd flilt I IIk itiiiwiX ikibiiliACh 80

B

ACh 40

C

ACh 25

1200SV~~~~~~~~~~~~~00uNa+64

(2) E

1 luil 1llll 114ii Wlill ill I i ii ii;j 1liiiiiU I.l ~I duIIiil

F ACh'80c1 i iiIIUIIIii 111111Miii i li 1,1 111 111111 1111iIN III1111II;11L 1iiiii;

ACh 32CG

l ll lill 1 i| l i.j 4 l 1;k!l dl:; 1 a|! W| j || |[|li |1 i150 UV

Glutamate 16c

(3) H

Biikihilhd~ilhhiI~i~ii ;1 1 i i I.;1 111!Ill thU 11111iltiiflUIIIIUIACh 48c

mildbIUiI11ii il"IiIiii il i Inn ;t: i Ii Ili lIa Iw4ACh 32c

J

^ ~IIhI-^SdhJ hai 1b448 i, Ii 1Iki1 ili lIllI lII idi II|iI^II hI

ACh 24cK

m~k~ iumUI~h~iSMI hMSl lldhflU1rn|11iimibnu1115500 V

NatS80 lOs

Text-fig. 2. For legend see facing page.

ACh INHIBITED CELLS IN THE THALAMUSKrnjevi6, 1974; Kelly & Beart, 1975) and the published values for thetransport number for both amino acids and ACh appeared to be the same(cf. Kelly, 1975). On forty-nine occasions when the currents used torelease equally effective doses of ACh and GABA were compared, thecurrent applied to the ACh-containing barrel of the micro-electrode was2-6 + 0-2 times as great as that applied to the barrel containing GABA. Asimilar ratio (2.3 + 0-2) was obtained on thirty-nine more cells when thecurrents used to release equally effective doses of ACh and glycine werecompared.The onset of the ACh-evoked inhibition of spontaneously active cells

in the nucleus reticularis was rapid. Indeed, on many occasions on thesame cell the latency of the onset of the ACh-evoked inhibition was asshort as that for the onset of a glutamate-evoked excitation (Text-fig. 2 G)or of an inhibition evoked by equally effective doses of GABA (Text-figs.5 and 6) or glycine (Text-figs. 5, 7 and 8). The latency of onset for theACh-evoked inhibition on a number of cells encountered by a micro-electrode during its descent through the nucleus reticularis often appearedto be shorter than that of the ACh-evoked excitations encountered by thesame micro-electrode as it passed through the ventrobasal complex (cf.Text-figs. 2 and 4).

Increased burst activity during ACh-evoked inhibitionAlthough we suggested earlier that ACh-evoked inhibition could be

mimicked by iontophoretic pulses of GABA or glycine and an attempt was

Text-fig. 2. The spontaneous activity and ACh-evoked inhibition of threecells (1-3) of the nucleus reticularis recorded on moving film. A-K, showthe characteristic high firing frequency of cells of the nucleus reticularisand their periodic burst activity. In all three cells both the onset and theoffset of the inhibition occurred within a few seconds of the beginning andend of the ACh application. The inhibition was dose dependent, could notbe mimicked by positive current ejected from an adjacent NaCl-containingbarrel (D and L). In E-F and H-J the current used to eject ACh was com-pensated for by passing a negative current of equal magnitude throughan adjacent NaCl-containing barrel of the micro-electrode. G, shows aglutamate evoked excitation of a cell, the spontaneous activity of whichhad been depressed by a continuous application of 32 nA ACh during thewhole of the record. The onset of excitation occurred with approximatelythe same latency as the onset of ACh-evoked inhibition of the same cell.In this and subsequent Text-figs. the duration ofeach iontophoretic applica-tion is shown by a horizontal bar and the numerals signify the magnitudeof the ejecting current in nA. The letter c signifies that current com-pensation was employed. Since a 25 nA retaining current was appliedcontinuously to all drug-containing barrels, an ejection current of 25 nAsignifies the passage of no net current and smaller values the partialwithdrawal of the retaining current.

655

Y. BEN-ARI AND OTHERS

made to compare the potency of ACh with that of GABA and glycine, theinhibitions evoked by ACh were not strictly comparable with thoseevoked by the two amino acids. Whereas on most cells the magnitude ofthe inhibition evoked by GABA or glycine was linearly and steeply relatedto the amplitude of the ejecting current (Text-fig. 8), attempts to relatethe degree of inhibition evoked by ACh to the magnitude of the ejectioncurrent were often complicated by a dramatic increase in burst activityassociated with the ejection of larger doses of ACh. For example, Text-fig.

Al, control (615)

56 32 24 A3, control (548)

50A7, control (498)

I &.... -

B1, control (779)

12 24-

F IB3, control (910)

A2, ACh 56 nA (124)

A4, ACh 32 nA (259)

A6,.ACh 24 nA (410)

50

A"_ |FL..........L. I. -

0 20 50 100B2, ACh 48 nA (247)

A1C1 1n

84, ACh 32 nA (303)

|5OHZ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ii.r I

86, control (900) B7, ACh 24 nA (523)

llbl 1500 20 50 100Interspike interval

Text-fig. 3. For legend see facing page.

48 3m -

1617 18

60s

6f56

V I.... .I

ACh INHIBITED CELLS IN THE THALAMUS3A shows the rate-meter record and interspike interval histograms of anACh-inhibited cell, the spontaneous activity of which showed very littleburst activity. The modes of the control interspike interval histogram laybetween 15 and 18 ms. The inhibition evoked by relatively small applica-tions of ACh (24 or 32 nA) was shown by the interspike interval histogramto have occurred at the expense of spikes with short interspike intervals.During these ACh-evoked inhibitions the modes of the interspike intervalhistogram moved to the right and lay between 22 and 26 ms. However,when the ACh ejecting current was increased to 56 nA the spikes thatoccurred during the ACh-evoked inhibition were fired in bursts. Spikeswith short interspike intervals now dominated the interspike intervalhistogram so that the mode moved to the left and lay between 6 and 10 ms.The bursts, which appeared for the first time during the larger applica-tions of ACh, were extremely difficult to inhibit by further increases in theACh ejecting current. The records in Text-fig. 4B are from a less typicalcell in which high frequency bursts appeared to account for most of thespontaneous activity and spikes with short interspike intervals dominatedthe interspike interval histogram. Inhibitions evoked by relatively smalldoses ofACh were associated with a reduction in the number of spikes withthe longer interspike intervals. In addition larger doses of ACh caused anabsolute increase in the number of spikes with an interspike interval of less

Text-fig. 3. Changes in the firing pattern of two cells of the nucleus reticu-laris that accompanied a partial inhibition of their firing by ACh. Theformat of this and Text-fig. 5 consists of displays photographed from theoscilloscope of a PDP-12 computer. The firing frequency and the firingpatterns of each cell in the presence and absence of a drug application areshown by a rate-meter display that is divided into a series of numberedtime epochs, and interspike interval histograms, compiled for controlepochs between drug applications and from the spikes that occurredduring the drug evoked inhibitions. Above each interspike interval histo-gram is shown in parentheses the number of spikes in the epoch fromwhich it was compiled. A, the inhibitions evoked by ACh ejected withcurrents of 24 and 32 nA were accompanied by a reduction in the numberof spikes with short interspike intervals and the modes of the correspondinginterspike interval histograms (A6 and A4) were shifted to the right. How-ever, the increase in inhibition caused by ACh 56 nA was complicated bythe appearance of burst activity, which is shown by an increase in thenumber of spikes with a short interspike interval in the interspike intervalhistogram (A2), the mode of which now lies to the left of that of the con-trol interspike interval histograms (Al, A3 and A7). B, the displays arefrom a cell in which the bulk of the spontaneous activity occurred duringirregular bursts. During inhibitions evoked by ACh (24, 32 and 48 nA),spikes with a long interspike interval were suppressed and the number ofspikes with short interspike intervals increased to a level where they ex-ceeded those that occurred during the control epochs.

657

Y. BEN-ARI AND OTHERS

than 4 ms. Clearly ACh provoked an increase in the number of spikes thatoccurred during high frequency bursts.The increased burst activity that occurred during ACh-evoked inhibi-(1)__A

ACh 40c

ACh 32c

C

ACh 24c

(2) D

_1 I I --i !iI 1 6 ,

£ACh 60c

il in I .a.1 .t I

AIh . -

ACh 40c

(3) F,, m a1 M . :.. k UJi.aAl.a-Ji . . . *-

ACh 48G

IIii I~hl~ibiastagflhftlI_ -IIilk n hu

ACh 32H

ACh 24

I 1: j !

M II

ACh 56c ACh 48c

10sText-fig. 4. For legend see facing page.

(4) I

I

V61" .| AM a

658

ACh INHIBITED CELLS IN THE THALAMUStion was not merely the result of a reduction in cell firing rate, since asimilar change in firing pattern was rarely seen during similar degrees ofinhibition evoked by GABA or glycine. Similar records from two morecells in which the effect of ACh on the firing pattern of the cells was com-pared with that of an equipotent dose of GABA or glycine are shown inText-fig. 5. Whereas the ACh-evoked inhibition was associated with ashift of the mode of the interspike interval histogram to the left, in eachcase the reduction in the firing frequency evoked by the amino acid wasaccompanied by a flattening of the interspike interval histogram. Thisobservation is consistent with the idea that the spikes occurring duringbursts, i.e. those with short interspike intervals, were almost as sensitiveto the inhibitory action of the amino acid as those occurring during theinterburst intervals, i.e. those with long interspike intervals. Increases inburst activity during ACh-evoked inhibitions but not during inhibitionsevoked by GABA or glycine were also apparent in the interspike intervalhistogram computed from the firing of the five cells whose firing pattern isshown by means of continuous film and rate-meter records in Text-figs.6, 7 and 8. This was a consistent feature of virtually all cells (thirty) inwhich the changes in firing pattern accompanying inhibition by ACh andGABA or glycine were compared.

Pharmacology of the ACh-evoked inhibitionACh-evoked inhibitions proved extremely sensitive to iontophoretic

applications of atropine. Indeed, the larger current (67 + 2 nA) required torelease an effective dose of ACh in the present series of experiments thanused in our previous series of experiments (58 + 3 nA; Ben-Ari et al. 1976b)was attributed to the presence of atropine in one of the barrels ofour micro-electrodes. On many occasions during the initial experiments, the mag-nitude of the ACh-evoked inhibition declined steadily during the first 5or 6 min of recording, as if atropine were diffusing from the electrodes inspite of the use of 25 nA retaining current. The concentration of atropinein the micro-electrodes was then reduced from 50 to 5 mm, and thereafterwe did not notice this artifactual fade of ACh-evoked inhibition.

Text-fig. 4. The spontaneous activity and ACh-evoked excitation on cellsof the ventrobasal complex recorded on moving film. A-I, are from fourcells (1-4) encountered deeper than the nucleus reticularis and excited withACh released from the same micro-electrode used to record from the ACh-inhibited cells shown in Text-fig. 2. The spontaneous activity of the cellsin the ventrobasal complex was slow and the onset and offset of the AChexcitation was greatly delayed compared with that of the ACh-evokedinhibition in Text-fig. 2. D and E, are continuous and the long latencyfiring evoked by a large dose of ACh in D accounts for the enhanced firingseen at the beginning of record E.

659

Y. BEN-ARI AND OTHERS

Al, control (628)

N ,a.I J..'|I Irft

A3, control (502)

1~~~~~~ ..

I~t

A2, ACh 40 nA (318)

LI..,I,,",..I... -. "1I'M, GABA 28 nA (330)

0 20 50 100

B-B1, control (638)

Ii+t INLn

B3, control (666)

*JN...,, . I'..

82, GLY 20 nA (100)

I'r"l r... '.a

B4, ACh 36 nA (119)

.Ik... I -' I0 20 50 100Interspike interval

Text-fig. 5. Differences in the firing patterns of two cells in the nucleusreticularis that occurred during inhibitions evoked by ACh as opposed toGABA or glycine (GLY). A, the inhibition evoked by ACh is associatedwith an exaggeration of burst activity. The interspike interval histogramA2 shows an increase to have occurred in the number of spikes with shortinterspike intervals and a decrease in the number with long interspikeintervals so that its mode now lies to the left of those of the control inter-spike interval histograms A 1 and A3. On the other hand, a similar degree ofinhibition evoked by GABA 28 nA caused a decrease in the number ofspikes with both long and short interspike intervals and no definite changeoccurred in the mode of interspike interval histogram A4. B, the inhibitionevoked by ACh 36 nA was accompanied by intense burst activity and thecorresponding interspike interval histogram B4 was made up almost en-

tirely of spikes with interspike intervals that were shorter than any thatoccurred in the control interspike interval histograms B1 and B3. In con-

trast the inhibition evoked by an equipotent dose of glycine can be seen

in interspike interval histogram B2 to be accompanied by a suppression ofspikes with both short and long interspike intervals.

A

ACh GABA* a

hAl . U

60s 50

GLY ACh

60s

660

ACh INHIBITED CELLS IN THE THALAMUS

On twelve out of thirteen cells atropine completely and reversiblyblocked the ACh-evoked inhibition. In eight out of eight cells tested, theantagonism was specific in that atropine was without effect on a similarinhibition evoked alternately with the ACh-evoked inhibition by an equi-potent dose of GABA or glycine (Text-figs. 6 and 7; Table 1).On three of four cells tested the atropine-sensitive ACh-evoked inhibi-

tion was also reversibly blocked by an iontophoretic application ofdihydro-fi-erythroidine (DH/JE), suggesting that the ACh receptor mediating in-hibition in the nucleus reticularis may be of mixed muscarinic-nicotinic

_ I 1U 1I

Picrotoxin GABA 16C on 3 min.

A L L fiW1t fL i IIj I*

E off, GABA 16Sfl d_ 1. 'ii ;

14 minC recovery GABA 20

GABA 16

ACh GABA80 36 80 36 80 36 80 36 80 36 80 36

Picrotoxin

B4"41 ijfnl 11 I I W'IW.itI'l IFNI 1iw 'J. 11I r 'v,- .1

ACh 36 Picr'otoxin off0t Atropine on;|id, X It s 11 110If:11 I I _ __ I 1 111, 21 IW bI4 im r itr4,-.

F ACh 36lttii E;11{tiul i Wrll whii uIiMiiil i 1 sfl ' N

H ACh 48it__iit ii d li IIW R 20 Dim

ACh 48 -- S

10

1*aXACh GABA

80 36 80 36 80 36 80 36 80 36 80 36_8Atropine 32 60 s

Text-fig. 6. The specific antagonism of GABA and ACh-evoked inhibitionsby iontophoretic picrotoxin and atropine respectively. A-H, show theresponse of a spontaneously active cell of the nucleus reticularis to alter-nate iontophoretic applications of GABA and ACh. C-D approximately3 min after the start of an iontophoretic application of picrotoxin theresponse to GABA, but not that to ACh, was almost completely blocked.E, recovery occurred approximately 14 min later. F, approximately 1 minafter the end of a 2-3 min application of atropine with a current of 32 nAthe response to a larger dose of ACh than used during the control periodwas still partially blocked and, H, partial recovery had occurred some15 min later. I-J, rate-meter records from another cell of the nucleusreticularis inhibited by alternate applications of GABA and ACh, whichshowed the response to GABA but not that to ACh to be blocked reversiblyby a short application of picrotoxin. J, an equally short application ofatropine blocked the response to ACh but not that to GABA, but full re-covery did not occur for another 14 min (not shown).

661

Y. BEN-ARI AND OTHERS

type, as found in other regions of the thalamus (Andersen & Curtis, 1964b)and cerebral cortex (Jordan & Phillis, 1972).The possibility that ACh-evoked inhibition is artifactual in the sense

that it might be caused by spread of ACh to neighbouring cells, theexcitation of which leads to inhibition of the neurone under study byrelease of a transmitter other than ACh, has been discussed by severalgroups (Randic, Siminoff & Straughan, 1964; Phillis & York, 1967a, b,1968; Jordan & Phillis, 1972; Duggan & Hall, 1975a, b). Indeed, ACh-

TABLE 1. Pharmacology of ACh-evoked inhibition in the nucleus recticularis asrevealed by specific antagonists

Dihydro-fl-erythroi- Picro- Methyl Strych-

Atropine dine toxin bicucculine nine

Blocks ACh 12/13, 3/4b 2/7 0/5 1/12Blocks GABA 0/4 - 9/9, 5/6dBlocks glycine 0/6 - 13/13,Blocks ACh but not 8/8GABA/glycine

Blocks GABA but not - 4/6 4/4ACh

Blocks glycine but not - 11/12ACh

The denominator of each fraction shows the number of cells on which the anta-gonist, shown at the top of the column, was adequately tested, and the numeratorthe number of cells in which the inhibition evoked by the agonist, shown on theleft, was completely and reversibly blocked. Cells were only included in the analysisif the blockade evoked by the antagonist proved to be reversible.

The mean magnitudes and durations of the currents used to release the anta-gonists and the time taken from the response of the agonists to recovery were, re-spectively, in: (a) 52+7nA, 2-4+0-3min and 4-6±0-8min; (b) 100±2OnA,5-3+0-3min and 4-4+2-0min; (c) 45+4nA, 24+0-44min and 3-3±0-5min;(d) 4-6±7nA, 4-1+0-6min and 40+1lOmin; and (e) 51+4nA, 25±0-4min and4-8+ 07 min.

evoked inhibitions in the cerebral cortex were reported to be blocked byiontophoretic applications of strychnine and could not be differentiated bystrychnine from inhibitions caused by either glycine or 5-hydroxytrypt-amine (Phillis & York, 1967a, b; Jordan & Phillis, 1972). Stone (1972),however, was unable to block ACh-evoked inhibitions in the cerebralcortex with iontophoretic strychnine. It therefore seemed important inthe present study to show whether or not the inhibition evoked by AChcould be distinguished on the same neurone from that evoked by GABAand glycine by the use of the appropriate amino-acid antagonists (cf.review by Curtis & Johnston, 1974). As shown in Text-fig. 6 and Table 1

662

ACh INHIBITED CELLS IN THE THALAMUS

I r 0 IllStrychnine on,

{ W'l is I. i' V.!,W if kg I- 1, .I IF., ! F T

Strychnine Glycine 44 ACh 80off after 1-3 min v.

Vuii~~i~~ii'HL~~I I I luiiumwaklmmS~~~~r1 Vd Iv.d'Strychnine

off after 2-3 min to

Atropineoff after 1.5 min v

11iiil AhI, Iii II

.atkitwiS^al d. bil..A,..t Aif A I . .1 m1 i l Vaim MiAl Us a'-ILLUW HEfVIR111-LN 141 . 1 A

I1 -m9 N A'±t *lU im i fiixi1lfStA inhmi i

Atropineoff5-7min Itz&& I- &LtILA-h A L- L 1 1Ails '|250pm

F a 5svStrychnine off 7-7 minb~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-1

Glycine ACh 40 20 44 20 44 20 44 40 Glycine ACh20 44 20 Strychnine 40 20 40 20 20 36

H

ii S-MGlycine ACh

20 36 20 36 20 36 20 36 20 36 20 36

Atropine 40

I vAtropine off 10-7 min

MM~~~l1~50ACh Glycine36 20 40 20 44

60 s

Text-fig. 7. The specific antagonism of glycine and ACh-evoked inhibitionby iontophoretic applications of strychnine and atropine respectively.A-E, alternate glycine and ACh-evoked inhibition of a spontaneouslyactive cell of the nucleus reticularis recorded on moving film to show theinhibition evoked by glycine but not that by ACh, B, to be blocked at theend of a 1-3 min application of strychnine (60 nA). C, recovery was notquite complete 2-3 min later. D, 2-5 min after the end of a 1-5 min applica-tion of atropine (96 nA) the response to ACh but not that to glycine wasstill blocked and E, full recovery did not occur for 6 min. F-I, rate-meter records from another spontaneously active cell of the nucleus reticu-laris that was inhibited by alternate applications of glycine and ACh. F,the response to glycine but not that to ACh was blocked by a short ionto-phoretic application of strychnine (40 nA). G, approximately 7-7 min laterthe response to glycine had recovered. Iontophoretic atropine graduallyblocked the response to ACh without affecting the response to glycine and,I, recovery occurred 10-7 min. later.

.-*-- "M IP== _ . 663

22 P HY 26I

Y. BEN-ARI AND OTHERS

A

e^ ~~~~~~~~~~~~~~~5 HzA,..-__M -

80 Glycne 22 72 20 88 22 80 20 72 22 72 22 64 22Strychnine

BL

40

kCh Glycine24 80 24 80 24 64 32 4880 24 80

A Strychnine (1.-2 mglkg w~.)

C100

90

80

70

60

50

40

30

20

D0

0

- -_n-

40 64 32 48 24 48 32

60 s

VV

0

I I ' ' I I

10 15 20 25 30 35 40Glycine releasing current (nA)

20 30 40 50 60 70 80 90ACh releasing current (nA)

Text-fig. 8. The effect of intravenous strychnine on dose-response curves

for glycine and ACh-evoked inhibition in the nucleus reticularis. A, selectedparts of the rate-meter record to show the blockade of the glycine-evokedinhibition but not that evoked by ACh by iontophoretic strychnine (40 nA).B, the effect of an intravenous infusion of strychnine that began approxi-mately 10 min earlier and reached a total dose of 1-2 mg/kg at the pointmarked *. C and D, dose response curves plotted from more extensiverecords of the same cell to show the relationship between the magnitudeof the inhibition evoked by glycine (C) and ACh -D) and the current usedto eject them before (A and V) and during (0 and [1) an intravenousinfusion of strychnine. The percentage inhibition was calculated by divi-ding twice the number of spikes that occurred during the 20 s drug applica-tion by the sum of the number of spikes that occurred in the two 20 s

intervals which occurred immediately before and after the drug application.

beS

._

4=0C0

._

c2.C

C

OO11

- -664

ACh INHIBITED CELLS IN THE THALAMUSa small iontophoretic application of picrotoxin, of sufficient intensity andduration to block the inhibitory action of GABA on all nine cells tested,had no effect on the inhibition evoked by an equally effective dose of AChon four out of six of these cells. In much the same way bicuculline metho-iodide proved to be a specific blocker of the inhibition evoked by GABAbut not that evoked by an equally effective dose of ACh on all four cellsso tested (Table 1).On eleven out of twelve occasions iontophoretic strychnine reversibly

blocked the inhibition evoked by glycine without affecting that evoked byACh (Table 1). For example, in Text-fig. 7 relatively short applicationsof strychnine are shown on two cells to differentiate between equallyeffective inhibitions evoked by glycine and ACh. In addition, in two experi-ments we also examined the effect of repeated small doses of intravenousstrychnine on the ACh-evoked inhibition. On both occasions, as illustratedin Text-fig. 8, infusions of strychnine to a total of 1-2 mg/kg were withouteffect on the sensitivity of the cells to ACh, even though current-responsecurves showed the glycine sensitivity of the cells to have been reduced toapproximately one half.Recent iontophoretic studies in the rat cerebral cortex have shown that

the ACh-excited pyramidal tract cells were also excited by guanosine3': 5'-phosphate (cyclicGMP) and the cells inhibited by noradrenaline weredepressed by adenosine 3': 5'-phosphate (cyclicAMP) (Stone, Taylor &Bloom, 1975). Stimulation of muscarinic cholinergic receptors in othertissues has also been shown to be associated with an increased level ofcyclicGMP (Kuo, Lee, Peyes, Walton, Donnelly & Greengard, 1972). Wetested whether either of these nucleotides might mediate the atropinesensitive ACh-evoked inhibition of the cells of the nucleus reticularis.Unfortunately, even though our tests were made on spontaneously activecells with newly filled micro-electrodes and every effort was made tofollow the methods of Stone et al. (1975), only four out of the forty-eightACh-inhibited cells tested were inhibited in a dose-dependent manner byone or other of the two cyclic nucleotides. We are therefore unable toconclude that the ACh-evoked inhibition of the cells of the nucleusreticularis is mediated by a rise or fall in one of the cyclic nucleotideintracellular messengers.

DISCUSSION

In this paper iontophoretic applications of ACh have been shown toinhibit both the spontaneous and glutamate-evoked activity of cells ofthe nucleus reticularis. This action of ACh is blocked by iontophoreticatropine and dihydro-fi-erythroidine but is resistant to the amino acidantagonists picrotoxin, bicuculline methoiodide and strychnine. The ease

22-2

665

Y. BEN-ARI AND OTHERS

with which the ACh-inhibited cells were encountered in the nucleusreticularis allowed us to show afresh the specificity of the amino acidantagonists, using their inability to antagonize the inhibitions evoked byACh as an additional criterion of their specificity. Earlier accounts of theactions of the GABA antagonists bicuculline and picrotoxin have beenconcerned almost exclusively with their ability to distinguish betweenGABA and glycine (Curtis & Johnston, 1974). The specificity of theseantagonists is of interest since Breuker & Johnston (1975) have drawnattention to the common structural elements in GABA and ACh andsuggested that a common structural feature in the active sites for GABAand ACh could account for the GABA antagonistic activity of (+)-tubocurarine (Hill, Simmons & Straughan, 1972).Our results are different from those obtained by Duggan & Hall (1975b)

who found ACh to have a differential effect whereby it enhanced theglutamate-evoked excitation of the cells while inhibiting their spon-taneous firing. In the present experiments ACh proved to be an even morepotent inhibitor of glutamate-evoked activity and we do not favour thesuggestion that the action of ACh is mediated presynaptically. Earliersuggestions (Randic et al. 1964; Duggan & Hall, 1975a, b) that the in-hibitory action ofACh might be mediated by the spread ofACh to adjacentinhibitory or excitatory cells also seems to be unlikely in the nucleusreticularis in view of our results that show the ACh-evoked inhibition ofcells of the nucleus reticularis to be as rapid in onset as the inhibitionevoked by GABA or glycine. Indeed the latency of onset of the ACh-evoked inhibition in the nucleus reticularis is considerably less than thatof the ACh-evoked excitation detected by the same micro-electrode in theventrobasal complex.The presence of both ACh excited and inhibited cells in different nuclei

of the thalamus could be of great functional significance. This may be ofparticular importance in the case of the nucleus reticularis and ventro-basal complex. There is an abundance of anatomical (Scheibel & Scheibel,1966; Minderhoud, 1971; Jones, 1975) and physiological (Schlag & Waszak,1971; Massion & Rispal-Padel, 1972; Waszak, 1974) evidence for the viewthat the nucleus reticularis has an inhibitory action on the thalamic relaycells and in this way serves to modulate the flow of sensory informationto the cortex. Indeed Purpura, McMurtry & Maekawa (1966) have shownthat cortical arousal evoked by stimulation of the mesencephalic reticularformation is associated with a facilitation of synaptic transmission throughthe thalamic relay nuclei. Furthermore, intracellular recordings haveshown the facilitation to be the result of a decrease in the amplitude of theinhibitory potentials, i.e. disinhibition of the inhibition which normallyfecds back on to the relay cells from neurones situated elsewhere in the

666

ACh INHIBITED CELLS IN THE THALAMUS

thalamus. To date most of the pharmacological evidence that suggests thepathway from the mesencephalic reticular formation to the thalamus ischolinergic also suggests that activation of the pathway results in theexcitation of neurones of the ventrobasal complex (Suzuki & Taira, 1961;McCance, Phillis, Tebecis & Westerman, 1968a; McCance et al. 1968b;Satisnky, 1968; Phillis, 1971). Phillis (1971), however, has also suggestedthat there may be inhibitory cholinergic terminals on cells inhibited byACh (cf. Jordan & Phillis, 1972).

Although the ionic mechanisms responsible for the ACh-evoked inhibi-tion in the nucleus reticularis will only be revealed by the use of intra-cellular electrodes, it is of interest to note that in the heart (Burgen &Terroux, 1953; Trautwein, Kuffler & Edwards, 1956), salivary gland(Schneyer, Young & Schneyer, 1972) and certain ganglion cells in Aplysia(Tauc & Gerschenfeld, 1962), the inhibition evoked by ACh is associatedwith a hyperpolarizing potential mediated by an increase in potassiumconductance. On the other hand the atropine sensitive hyperpolarizingaction of ACh on neuroblastoma cells in tissue culture (Nelson, Peacock &Amano, 1971; Nelson & Peacock, 1972) and on C-cells of the frog sym-pathetic ganglion (Weight & Padjen, 1973) is associated with an increasein membrane resistance that may well be mediated by a decrease in sodiumconductance. No clear change in membrane potential or permeability hasbeen observed during the initial depressant action of ACh on corticalneurones (Krnjevic, Pumain & Renaud, 1971). The exaggerated burstactivity (Text-figs. 3 and 5) that occurs during the ACh-evoked inhibitionof cells of the nucleus reticularis but not during inhibition of the samecells to a similar degree by GABA and glycine could be regarded as tenta-tive evidence for the view that changes in ionic conductance evoked byACh must differ from the large increases in chloride conductance that havebeen shown to be associated with the actions of GABA (Dreifuss, Kelly &Krnjevic, 1969) and glycine (Ten Bruggencate & Engberg, 1971). Althoughthe rapid onset of the ACh-evoked inhibition on cells of the nucleusreticularis is quite unlike the slow onset of the ACh-evoked inhibition offrog ganglion cells described by Weight & Padjen (1973), the high firingrate of the neurones in the nucleus reticularis could be the result of anabnormally high Na+ permeability and in this situation even smallreductions in Na+ permeability could cause the immediate onset of in-hibition. Indeed Krnjevic (1974) has already suggested that the initialdepressant action of ACh on ACh-excited cells is a special feature ofcells with a high spontaneous firing rate.

Krnjevic et al. (1971) have also suggested that ACh promotes bursts bydelaying the repolarization of the membrane during the falling phase ofthe action potential. A decrease in the slope of the falling phase of the

22-3

667

668 Y. BEN-ARI AND OTHERS

action potentials could lead to an increase in repetitive firing triggeredeither by spontaneous spikes or by subliminal excitatory synaptic poten-tials. In this sense the inhibitory actions of ACh and the tendency ofACh-inhibited neurones to fire in bursts could be the result of two un-related actions of ACh on the neurones of the nucleus reticularis.

We are grateful to Dr L. L. Iversen and Professor D. W. Straughan for reading ourmanuscript and to George Marshall for technical assistance. Support is acknowledgedby Y. Ben-Ari from the CNRS and the EBBS, by R. Dingledine from the Pharma-ceutical Manufacturers Association Foundation and the Foundations Fund forResearch in Psychiatry and by I. Kanazawa from the Wellcome Trust.

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The Journal of Physiology, Vol. 261, No. 3

XcrAIa> .-.v ir

Y. BEN-ARI AND OTHERS (Facing p. 671)

Plate I

ACh INHIBITED CELLS IN THE THALAMUS 671

EXPLANATION OF PLATE

Photomicrographs from four different experiments in which subsequent histologicalanalysis showed the micro-electrode tracks to have penetrated the nucleus reticu-laris. A and B, the micro-electrode tracks lay solely within the nucleus reticularisand were from experiments in which only cells inhibited by ACh were encountered.C, is from an experiment in which both a superficial and deep population of ACh-inhibited cells were found to be separated by a layer of ACh excited cells. The layerofACh excited cells was presumed to correspond to the short trajectory of the micro-electrode through the lateral curvature of the ventrobasal complex. D, two micro-electrodes that penetrated the dorsal medial aspect of the nucleus reticularis andthen the underlying ventrobasal complex of the thalamus are from an experiment inwhich a layer of fast firing ACh-inhibited cells were found above more slowly firingcells excited by ACh.