Evoked interaction patterns in chronically implanted cats following chlorpromazine

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EVOKED

1965, 4, 47-64 PergamonPress. Printed in Gt. Britain. [I table, 9 figs.,48 refs.]

INTERACTION

IMPLANTED

PATTERNS

CATS FOLLOWING

IN CHRONICALLY

CHLORPROMAZ1NE*

J. H. CIUN, E. K. KILLAMand K. F. K1LLAM Department of Pharmacology, Stanford University School of Medicine, Palo Alto, California

Summary--In five unrestrained cats with electrodes chronically implanted in brain structures, relatively stable reticular-auditory interaction patterns were recorded in the auditory pathway over a period of 2-3 months. In the following 4-month period single doses of cblorpromazine, pentobarbital and reserpine were tested in each cat. In some animals, amphetamine and meprobamate were also tested. Chlorpromazine (1-I0 mg/kg) enhanced the reticular inhibitory modulation of the responses to click in the auditory pathway. In contrast, following pentobarbital, reticular formation (RF) stimulation was generally less effective in reducing the auditory potentials at suprathreshold, submaximal intensities; occasionally the reticular influence was converted to facilitation. The effects of meprobamate (50-75 mg/kg) were similar, but not as pronounced as those seen after pentobarbital. The effects of reserpine (100 lag/kg) on the evoked interaction pattern were complex. In cats in which reticular stimulation reduced the auditory responses in auditory cortex and in the brachium of inferior colliculus, as well as the longer latency responses in medial geniculate, RF stimulation was less effective after the drug. In contrast, the RF-induced inhibition of dorsal cochlear nucleus and the shorter latency medial geniculate responses were enhanced by reserpine. The effects of amphetamine on the reticular interaction in auditory cortex were similar to those of reserpine.

INTRODUCTION

MANY STUDIES suggest that the reticular formation, or pathways within it, plays an important role in the modulation of sensory input (see reviews of LIVINGSTON, 1959; HERNXNOEz-PE6N, 1961; KILLAM, 1962). Most of the early studies showed potentials evoked by a peripheral stimulus to be reduced by reticular stimulation even at the first synapse in the classical sensory pathway. In contrast, when the relay nuclei were stimulated directly, the centrally elicited, sensory responses were facilitated by reticular stimulation. The evidence that chlorpromazine enhanced the reticular modulating system by lowering the threshold of reticular stimulation required to reduce auditory potentials (KILLAM, 1957; KILLAMand KILLAM, 1958) suggested that these mechanisms should be further investigated as a site of the drug's action. To eliminate variables due to carbon dioxideoxygen levels, loss of proprioceptive inflow, changing circulatory patterns with neuromuscular blockade and other artifacts of the acute, immobilized preparation, the freely-moving chronic animal was selected as the preparation of choice. Factors altering reticularsensory interaction at relays of the auditory pathway under these conditions were first investigated (CHIN et al., 1962, 1965). Changes in levels of arousal during cycles of sleep * This investigation was supported by USPHS Research Grant MHO-3374. 47 B

48

J.H. CHJN,E. K. KILLAMand K. F. KILLAM

and wakefulness were shown to be associated with marked alterations in evoked auditory activity and in its modulation by reticular stimulation. The following investigation was therefore designed to study the effects of chlorpromazine on the reticular mechanisms altering auditory inflow in the unrestrained cat under the restricting condition that sensory potentials be evoked only at one defined level of the sleep-wakefulness continuum, the "drowsy state." A further purpose of this study was to compare the actions of pentobarbital, reserpine, amphetamine and meprobamate to those of chlorpromazine under the same conditions. Comparative data were obtained from acute experiments on immobilized animals using those drugs not studied earlier under such conditions (KILt,AMand KILLAM,1958). METHODS Electrodes were implanted in both cortical and deep structures in five cats as described in a previous paper (CHIN et al., 1965). Evoked potentials were recorded from dorsal cochlear nucleus in four cats throughout all studies. Medial genic~late nucleus responses were recorded in three cats thioughout, ectosylvian responses in t~¢o cats and responses from the brachium of the inferior colliculus in two. Electroencephalographic monitoring was carried out simultaneously from the following areas in all cats: ectosylvian and lateral cortex, reticular formation, dorsal coehlear nucleus, lateral geniculate nucleus, and the medial geniculate nucleus or the brachium of the inferior colliculus. The one-way mirrored observation chamber and stimulating and recording apparatus were those previously described. Auditory potentials were evoked by click; reticular stimuli were applied from a Grass stimulator with isolation unit or from Tektronix wave-form and pulse generators. A "drowsy state" condition was defined for each cat. Criteria included a quiet, relaxed posture, quiet, regular respiration and spontaneous electrical activity of the brain of reasonably low amplitude and moderate frequency: that is, an L E G intermediate between the desynchrony seen in the alert state and the synchrony of the sleep stage. Experimental sessions: whefi cats were judged to be in the "drowsy" condition, clicks were presented every 40 sec. Following six to twelve control auditory evoked responses, the mesencephalic reticular formation (RF) at the intra-aural plane was stimulated 70 msec before presentation of the next click. RF stimuli consisted of a 50 msec train of monophasic pulses, 0-1 msec duration, 200 or 400 c/s. Regardless of whether there was a reticular interaction or not, a minimum of six more responses to click alone were recorded each time. The RF was then stimulated again at a higher intensity but with the same temporal relation to a click presentation. Routinely, a series of stimuli to R.F. ranged in intensity from below threshold to six times that value. Intervals between series were sufficiently variable to avoid habituation or conditioning effects. Following 2-3 months of control observations, a dose-response analysis of chlorpromazine effect was constructed for each animal by administration of single doses (between 1 and 10 mg/kg) on days interspersed at random among days of control studies. At least five days elapsed between drug administrations except for a few three-day to four-day intervals following ineffective doses. Saline solution was injected on some days and a schedule of repeated testing was followed similar to that used when drug was administered. For comparison, single doses of pentobarbital (10 mg/kg), meprobamate (50-75 mg/kg) and amphetamine (0.5 mg/kg) were administered on a similar schedule. Reserpine (100 /~g/kg) was tested last in each animal one week prior to sacrifice. Drug administration in each animal is summarized in Table 1.

49

Ex.oked interaction patterns in chronically inaplantcd cats following chlorpronaazine TABLI: [.

Aninaal IV V1 VII VIII XII

T O T A L CONTROL AND D?.LIG TEqF SE~ION~ IN CHi~ONIC ANIM,MN

Coch., BIC Coch., MG, AC Coch., MG Coch., B1C A.C., M G

Total IV VI VII VIII XIi

Control days before any drugs

Electrodes

Pentob. Pentob. Pentob. Pentob. Pentob.

Control days between drugs

CPZ given (nag/kg)

Total sessions

35 100 t05 49 81

97 120 132 97 104

2.5,5,5", I0 2,2,2:~:,5,10 2,2,2",5,10 I, 1,1 ~,2,2,5,10 2,2,2',5,10

140 229 245 156 194

370

550

CPZ 27 doses

964

Other Drugs Tested (dose/ks) l0 mg.; Meprob. 50 mg.; Reserp. 100 lag l0 nag.; 10 rag.; Anaphet. 0-5 nag.; Reserp. 100 lag l0 nag., 10 nag. ; Reserp. 100 lag 10 nag., 10 nag. ; Reserp. 100 gg l0 nag.; Meprob. 75 nag.; Anaphet. 0.5 ms.; Reserp. 100 lag

* Terminal acute experiment Coch. : cochlear nucleus BIC brachiuna of inferior colliculus CPZ chlorpronaazine Pentob. pentobarbital Meprob. naeprobanaate

MG AC Reserp. Anaphet.

medial geniculate nucleus auditory cortex reserpine amphetamine

At the t e r m i n a t i o n o f the series o f chronic experiments an acute experiment was perf o r m e d on each cat. U n d e r ether anesthesia the brachial vein was c a n n u l a t e d for drug injections and the femoral artery for b l o o d pressure recordings. Tile a n i m a l was i m m o b i lized with gallamine triethiodide and placed on artificial respiration. G a l l a m i n e triethiodide was reinjected on a regular schedule to maintain c o m p l e t e immobilization. T e m p e r a ture was m a i n t a i n e d at 3 6 . 5 - 3 7 . 5 C. The w o u n d margins and all pressure points were infiltrated with procaine. A t least 2 hr were allowed for recovery from the ether before recordings were begun. The e x p e r i m e n t a l p r o c e d u r e was exactly similar to that carried out in the early daily experiments on the unrestrained animals. Doses of 1-5 mg/kg o f c h l o r p r o m a z i n e were given intravenously or intraperitoneally. F o r c o m p a r i s o n with these data, twelve acute experiments were performed in other animals. These were similar to terminal experiments in the chronic animals except that during surgery, holes were drilled in the c a l v a r i u m a n d the electrodes oriented into the various structures with c o n c o m i t a n t recording on a M n e m o t r o n C o m p u t e r of Average Transients (C.A.T.) to aid in placement. In each drug experiment, chronic or acute, at least two, but usually three, c o n t r o l series of responses to click, to reticular stimulation and to the pattern o f their interaction were taken before drug was given. Only those areas showing consistent patterns during the c o n t r o l period were used in evaluating drug effects. A f t e r drug a d m i n i s t r a t i o n the series o f click and reticular stimuli were repeated at suitable intervals to establish the time course of drug effect. Reserpine (100 t~g/kg) was given to five animals, a m p h e t a m i n e (I mg/kg) to six animals, and m e p r o b a m a t e (75 mg/kg) to one animal. All drugs were given intravenously. Reserpine" and a m p h e t a m i n e were dissolved in distilled water and m e p r o b a m a t e in polyglycol E400. The d a t a in acute experiments were evaluated fi'om potentials recorded on lilm and s i m u l t a n e o u s l y ~'averaged" using the c o m p u t e r o f average transients (C.A.T.). Since this Serpasil (R) Phosphate Lyophilized dissolved at 2 nag/cc in distilled water and diluted to 10')/~g/cc for injection.

50

J. H. (_Tm~, E. K. KILLAMand K. F. KIL.LAM

computer actually computes sums rather than true averages, ten responses taken before reticular stimulation were summated using a 250 msec analysis time. The single response immediately after RF stimulation was then displayed at a tenfold magnification by increasing the vertical range sensitivity of the instrument to facilitate visual comparison with the summed control response. A 40-sec writeout time on the Mosely ink recorder was used for display. Analyses of evoked potentials in the chronic animals were carried out in two ways. In some experiments the C.A.T. was used as described above. In a majority the evoked potentials were measured from oscilloscopic traces recorded on fihn. Points at 1.5 or 3 msec intervals along each trace were measured by hand using a Benson-Lehner decimal converter. True average response amplitudes were then calculated at each time interval and the resultant potential was plotted. This method allows assessment of variability which is not available if the C.A.T. is used. RESULTS

In the "drowsy" state defined earlier, the effect of reticular stimulation (RF-s) on response to a subsequent click was assessed at four levels of intensity: below threshold, threshold (response to click irregularly or slightly reduced), supra-threshold (response to click consistently reduced by the RF-s) and submaximal (RF-s caused EEG and/or behavioral arousal plus reduction of response to click). The effects of varying the reticular stimulus at the three lower intensities upon sensory potentials in the dorsal cochlear nucleus and the brachium of the inferior colliculus (BIC) are illustrated in Fig. 1. Coohi¢o

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FiG. 1. Greater effectiveness of increasing intensity of the reticular stimulus upon the potential evoked by click in the dorsal cochlear nucleus and in the brachium of the inferior colliculus (BIC) in the drowsy state. Digitized averages (solid lines) before stimulation and single response (dashed lines) immediately after R F stimulation.

Evoked interaction patterns in chronically implanted cats following chlorpromazine

51

The solid line represents the average response curve of six potentials. The dashed line is the single auditory potential altered by prior stimulation of the mesencephalic reticular formation. Although the degree of RF effect varied in different recording areas of the same cat as well as from cat to cat, a suprathreshold intensity which decreased the response to click without EEG or behavioral arousal could be found in each animal (see Fig. l, 2 v), particularly if the intensity of the click was adjusted to a level close to threshold. The five chronic animals from which data are reported in this study were selected for the investigation of drug action from a larger series of animals because responses to click alone were stable for at least two months and reproducible interaction patterns of RF-s and click were obtained for the same period. Each animal served as his own control to obviate the inter-animal variation and slight differences in electrode placement. An example of the stability of response over time is given in Fig. 2. Contt-ol CPZ

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FIG. 2. Daily variability of the response to click and its interaction with reticular stimulation in the dorsal cochlear nucleus on 4 different days (left). Effects of chlorpromazine (5 mg/kg) on the evoked interaction pattern in the dorsal cochlear nucleus on control day 4 (right). Amplitudes were measured at 1 '5 or 3 msec intervals over each entire potential using a Benson-Lehner decimal converter. The solid line represents the average response curve of six potentials. The dashed line is the single potential altered by stimulation of mesencephalic reticular formation at the threshold voltage o f I V. The dotted line is a single potential 40 sec after stimulation.

Chlorpromazine Twenty-seven single doses ranging from 1 to 10 mg/kg given among the five animals over a period of four months are summarized in Table 1. Doses of 1 and 2 mg/kg had no major behavioral effect. Although four of five animals were somewhat more quiet after 2 mg/kg, no motor deficit could be observed. At 5 mg/kg, these four appeared slightly ataxic and hyperexcitable. They chewed on recording cables, climbed the walls of the

52

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KttJ..,,xt

and K. t:. Ku.t.Ax~

recording c h a m b e r and vocalized. At 10 m g i k g there was definite ataxia and three of the four showed hyperexeitability just described. One cat vomited on one occasion (2 mg/kg), but the same and higher doses did not p r o d u c e the eft'eel again. In all five cats single doses o f c h l o r p r o m a z i n e at 1 and 2 mg/kg, as well as at higher doses, enhanced the effectiveness o f R F in reducing response to click in all areas studied. F i g u r e 2 illustrates such an effect at 5 mg/kg. C o r r e c t i n g for alteration in baseline, a m a r k e d increase in effectiveness o f threshold RF-s (dashed line) can be noted at 90 rain and three hr after drug. The effectiveness was equivalent to that o f twice the control RF-s intensity. Figure 3 illustrates a similar effect of a lower dose o f c h l o r p r o m a z i n e (2 mg/kg) t h r o u g h out the a u d i t o r y pathway. At the time o f peak effect, a previously ineffective stimulus to the R F reduced a u d i t o r y potentials at the dorsal cochlear nucleus, the medial geniculate

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CONTROL Ac " ~x~,

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CPZ 2 mg

Peok Effect

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Pt~i. 3. Effect of chlorpromazinc (2 mg/kg) on the evoked interaction pattern in the auditory pathway. Oscilloscopic recordings in "A': six control responses to click before stimulation: "B': the auditory potential 20 msec after the end of reticular stimulation; 'C': six control responses after those in 'B'. The recordings in the auditory cortex (AC) and the medial geniculate nucleus (MG) are from the same cat and those in the dorsal cochlear nucleus (COCH) from another animal. Note oscilloscope sweep speed in COCH twice that in AC and MG.

E~oked interaction patterns in chronically implanted cats following chlorpromazine

53

nucieus, and the a u d i t o r y cortex although the evoked potentials themselves were unchanged by the drug. Six successive potentials in response to click are shown at ' A ' . The next click was preceded by 70 msec by R F - s ('B') and then six successive responses to click alone are shov,'n at 'C'. Recovery after c h l o r p r o m a z i n e usually occurred within 6 hr. Doses o f 10 mg/kg of the drug were no more efl'ectivc in enhancing the reticular action on the a u d i t o r y stimulus. A c u t e terminal experilnents in the same animals p e r f o r m e d during gallamine triethiodide paraly,,,is gave results similar to the foregoing. D a t a from typical experiments are shown in Figs. 4 and 5. These are from the same animals as those shown in Fig. 3. The t o p line (Fig. 4) shows essentially no change in the potentials following the threshold

A

B

C

CONTROL (chronic) MG

CONTROL (acute) ~%

r-

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---I 7K;qC

Flu. 4. Comparison of the evoked interaction patterns m the auditory pathway in unrestrained cats and in the same animals paralyzed with gallamine triethiodide. Oscilloscopic recordings in 'A': six control responses to click before stimulation; 'B": the auditory potential 20 mscc after the end of reticular stimulation: 'C': six control responses after those in 'B'. The recordings in the auditory cortex (AC) and the medial geniculate nucleus (MG) are from the same cat and those in the dorsal cochlear nucleus (COCH) from another animal. Some 60 cycle interference occurred in the recording from MG at the time of the acute preparation. reticular stimulus {1 V) in the unrestrained animal before surgery. Similar results were o b t a i n e d in the paralyzed animal 2,~, hr after the end o f ether administration (Fig. 4, 2nd line). A t the time of tile peak effect o f 2 m g / k g intraperitoneally (Fig. 5) there was a definite decrease o f tile a u d i t o r y potentials l\}llowing the reticular stimulus which had either little or no elTect during the control period. The b l o o d presstlre at this time was 75 m m Fig c o m p a r e d to 80 m m Hg in the control period. Recovery was seen in 3 hr. Pentobarbital A t least one 10 m g / k g dose o f p e n t o b a r b i t a l was administered to each o f the chronic a n i m a l s ; to measure reproducibility o f the drug effects, the dose was repeated oil a different d a y in three o f the animals. In all cats, 10 m g / k g of p e n t o b a r b i t a l p r o d u c e d some ataxia

54

.I. H, ('Hr~, E. K. KIt+LAMand K. F. K.ILLAM

A

CONTROL

B

C

(acute)

CPZ 2 mg

Peak

Effect

Recovery '

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50JJ~

Fxo. 5. Effects of chlorpromazine upon evoked interaction patterns throughout the auditory pathway in the paralyzed unanesthetized preparation. Control recordings are the same as those shown in Fig. 4.

within 10 min following intravenous or intraperitoneal injection. Four of five animals exhibited excessive licking during the first hr after drug administration so that recording during this period was very difficult. The data were recorded only when the animals sat quietly so that movement artifacts did not confound the results obtained. Pentobarbital decreased the effectiveness of RF-s in reducing evoked auditory potentials. A typical experiment is illustrated in Fig. 6. During the control period, stimulation of the reticular formation at a suprathreshold intensity produced a definite decrease of the auditory potentials in the auditory cortex, the medial geniculate nucleus and the dorsal cochlear nucleus (Fig. 6 'B'). At the time of the peak effect of 10 mg/kg of pentobarbital this intensity of reticular stimulation was almost ineffective in reducing the auditory potentials. Recovery was seen within 6 hr. In two animals, pentobarbital induced an increase of the auditory potential following a stimulation of RF previously found to reduce the potential. In one of the animals the increase of potential was seen in both the brachium of the inferior colliculus and the later components of the response from the dorsal cochlear nucleus. In the other animals, the cochlear potential was recorded from an electrode spanning the dorsal and ventral portions of the nucleus. After administration of 10 mg/kg of pentobarbital, a definite enhancement of the potential in the cochlear nucleus occurred following a RF-s which previously reduced the potential. The same reticular stimulus was less effective than control in reducing the potential recorded simultaneously in the auditory cortex.

Evoked interacuon patterns in chronically implanted cats following chlorpromazine

CONTROL

A

COCH" ~ ' ~ ' ~

B

55

C

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PENT I0 mg

Pea k Effect

Re c overy

FIG. 6. Pentobarbita[ elfect on the evoked interaction pattern throughout the auditory pathway. Oscilloscopic recordings are shown as in Fig. 3.

Meprobamate Two of the chronic animals received single doses of meprobamate. 50 mg/kg caused no particular behavioral changes although the cats seemed quieter than usual. Seventy-five mg/kg produced definite ataxia and some licking similar to that induced by 10 mg/kg of pentobarbital, in both animals 30 rain following meprobamate there was less reduction of the late component of the response in the dorsal cochlear nucleus by RF-s than in the control. These effects were similar, but not as pronounced as those seen after pentobarbital. In one of the animals the early negative potential was markedly increased by reticular stimulation 30 rain after 75 mg/kg of meprobamate. Similar results from an acute experiment on a naive cat, immobilized with gallamine triethiodide and placed on artificial respiration are illustrated in Fig. 7. At "A' and "C', ten consecutive potentials recorded in the dorsal cochlear nucleus are shown summed. ~B, and "B2' represent single responses to click preceded by RF-s at 70 msec and 45 sec respectively. By appropriate gain change these are made equivalent to the average response of the ten potentials. Control

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nucleus in an immobilized animal on artificial respiration. The traces are write-outs from the Mnemotron Computer of Average Transients (C.A.T.) as recorded on a Moseley ink recorder. 'A' and 'C' are sunanaed response curves for ten consecutive potentials evoked in response to click. 'BI' is the write-out of the single potential 20 msec following a reticular stimulus of 2 V. 'B2' represents a single potential 45 sec following the one in 'B~'. Both 'BI' and 'Be' were amplified tenfold for comparison with 'A' and 'C'. responses before drug showed a definite decrease o f the potential ('B~') i m m e d i a t e l y following 2 V. stimulation o f the reticular f o r m a t i o n , delayed enhancement 45 sec later ('B2') and return to condition ' A ' in the following ten responses ('C'). T h i r t y rain after slow injection o f 75 m g / k g o f m c p r o b a m a t e , the a u d i t o r y potential was definitely enhanced by R F stimulation ('B~'). A t 45 sec the potential remained enhanced ('B~'). Partial recovery from the drug effect was seen within 90 rain. Since the drug was administered intravenously over a period o f five rain, there was no d r o p in b l o o d pressure; in fact the b l o o d pressure rose From 130 m m Hg to 170 m m Hg.

Reserpine A single dose of 100/~glkg o f reserpine was g~ven intraperitoneally to each o f the five chronic animals one week before termination o f the experiments. Within 2 - 4 hr all animals had diarrhea, m a r k e d ptosis, and miosis. 3-he animals were much quieter t h a n usual and did not return to their n o r m a l behavior until 3 days later. Since k n o w n long t e r m effects o f reserpine precluded study of additional doses in chronic animals, s u p p l e m e n t a r y d a t a were o b t a i n e d from five acute p r e p a r a t i o n s on naive animals. The effects o f reserpine on the evoked interaction pattern were complex. The results varied with recording area and the type and degree o f interaction o b t a i n e d between click

Evoked interaction patterns in chronically implanted cats follm~ing ch[orpromazine

57

and RF-s. In two of five chronic and two of five acute experiments in which RF-s produced a decrease in the response to click in the auditory cortex, the effect of the RF-s was reduced by the drug; that is, there was less decrease of the attditory potential following RF-s. This decreased effectiveness of reticular stimulation following reserpine was also seen in two other animals with recording sites in the medial geniculate nucleus. The iatencies of the responses in the medial geniculate were longer than those in which reserpine had opposite elt'ects. Typical experiments are shown in Figs, 8 and 9. Figure 8 shows potentials A CONTROL

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Flc;. 8. Comparison of reserpine effects upon the evoked interaction pattern in the auditory cortex (AC) and the medial geniculate nucleus (MG) with those in the dorsal cochlear nucleus (COCH) in animals with chronically implanted electrodes. Oscillo:~copicrecordings in "A': control responses to click before stimulation; 'B': the auditory potentials 20 msec after reticular stimulation, "C': six control potentials after those in 'B'. Controls I and 2 are taken on two different days to illustrate the day to day variability of the response. The superimposcd traces show the variability during the same recording ses'.qon. A dose of 100 pg,/kg of reserpine was given intraperitoneally. Recordings in AC, MG and COCH are from the same animals illustrated in Figs• 3, 4, 5 and 6. Note opposite effects of reserpine in AC and MG when compared to Ihat in COCH.

trom the same chronic animals as those sho\~n in Fig. 3. The two control ,ccordings were taken on consecutive days: control 2 was just prior to reserpine administration. A reticular stimulus of 2 V. produced a definite decrease both in the potential in the auditory cortex and the second phase of the medial gcniculate r~:sponse. Four and one-half hr following reserpine, this reticular stimuhts ~vas definitely less clt'cctive in reducing both auditory potentials. Recovery of the interaction pattern was seen 24 hr after reserpine although the

58

.I.H. (.ttJN, k. K. KILLAM and K. F. Kit, i,,x,~

animal still showed gross behavioral signs or" the drug. Similar effects of reserpine are even more clearly defined using the C.A.T. averaging technique in an acute preparation (Fig. 9). The figure is similar in pattern to Fig. 7. 'A' and 'C' represent summed potentials A

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animal on artificial respiration. The traces are write-outs from the C.A.T. illustrated as in Fig. 7. Those in the upper portion of the figure are from the auditory cortex (AC); the reticular stimulus preceding 'B 1' - 6 V. The tracings in the middle portion are from the medial geniculate nucleus (MG); the reticular stimulus preceding 'B 1' = 8 V. Recordings in the lower portion are from the dorsal cochlear nucleus; the reticular stimulus preceding ' B 1 ' = 4 V.

and 'B 1' and 'B2' an amplified single auditory potential immediately after RF stimulation. The voltage of reticular stimulation necessary differed for different levels of the auditory path. In the controls, a maximum depression of response to click in auditory cortex by RF-s (6 V.) is shown in Fig. 9, 'B 1' with recovery within 45 sec ('B2'). Four hr after 100 t,g/kg of reserpine intravenously the same reticular stimulus only very slightly reduced the auditory potential. In the same experiment, (Fig. 9, middle portion) in the control period a RF-s of 8 V. completely eliminated the auditory potential in rnedia.1 geniculate ('BI'), but partial recovery of the potential occurred 45 sec later ('B2'). Four hr after reserpine this RF-s was definitely less effective in decreasing the potential in the medial geniculate than during the control period although the effect was less striking than on the cortex. In two chronic and one acute experiment of five in which auditory potentials were recorded in the brachium of the inferior colliculus, the effect of RF-s in reducing the amplitude of the potentials was also decreased by reserpine.

Evoked interaction patterns in chronically implanted cats followingchlorpromazinc

59

In contrast an increase in auditory potential immediately following stimulation of the dorsolateral portion of the RF was recorded in the dorsal cochlear nucleus (three acute cats). Again, reserpine reduced the RF interaction. An opposite effect of the same dose of reserpine was recorded in some of the experiments. In two chronic and one acute experiment the effectiveness of RF-s in reducing auditory potentials at medial geniculate was enhanced by reserpine. Similar results were obtained on potentials recorded from the dorsal cochlear nucleus (four chronic, two acute cats) when ventromedial areas of the RF were stimulated. The similarity of this effect to that of chlorpromazine is shown in the right-half of Fig. 8. At 'B' in the controls a threshold RF-s is shown barely to influence the evoked auditory potential in the dorsal cochlear nucleus while 4 hr after reserpine administration the same stimulus interacts markedly with click to reduce the evoked potential. Similar results were also observed in the dorsal cochlear nucleus in an acute preparation (Fig. 9, lower portion) following reserpine. The threshold reticular stimulus was ineffective in reducing the auditory potential during the control period, but the same RF stimulus became effective at the time of peak reserpine action, in three acute experiments in which auditory responses recorded from cortex were enhanced by interacting reticular stimuli, reserpine again had a positive action, that is, stimulation of RF produced a greater increase in response than before the drug. Amphetamine Preliminary studies in two chronic and four acute experiments indicated that amphetamine at 0,5-1 mg/kg at first reduced (30 min) the effectiveness of RF-s in reducing auditory responses at cortex. In the two chronic experiments amphetamine later enhanced this reticular interaction. In two animals in which interaction enhanced the auditory potential, no early reduction of effect was seen. DISCUSSION Many authors have postulated the existence of an inhibitory effect of the reticular formation on the first synaptic relay within each of the classical afferent systems (HAGBARTH and KERR, 1954; GRANtT, 1955; KERR and HAGBARTH, 1955; GALAMBOS,1956; JOUVET and DESMEDT, 1956; HERN/~NDEZ-PE6N et al., 1957). KJLLAM and KILLAr~t(1958) suggested that this suppressive influence is a mechanism for the filtering of incoming information in favor of that to which attention is directed. HERN~NDEZ-PEdN(1961) has further postulated that an anatomical basis for such a mechanism may be feedback loops, with an ascending branch from the second-order sensory neurons to the RF and a descending limb in the opposite direction. An important factor in the assessment of drug action in the conscious, freely moving animal has been the recognition of the variability of central representation of single and interacting stimuli with alterations in level of consciousness. In the unrestrained animal both the amplitude of potentials which were evoked in response to click and the degree and type of reticular interaction at any level of stimulus intensity varied with behavioral state of the animal as well as with background bioelectric activity monitored from cortex and deep structures of the brain (CHIN et al., 1962, 1965). Responses were most stable and reproducible when the animal was quiet and relaxed and the EEG was intermediate between the desynchrony characteristic of alerting and the synchrony of high

v,O

.i t1.( tit\., t]. K. K i l l ~nland K F Klli ~l

:lml~lilude spindles. Such a stair' was ,hereFore delined :1,, closely as pos-;ible and all ('on{tel and post-drug series oF ~ 11! .llJ V.CIO presenled undo, s~ch c ;ndiiion,. ! lieder these cc-nditioi>. '~c coillirn]ed (Sill" :':lrlicr limtings -ind ~;hnilar lindings o f o l h c r wc;rker!, mentioned, thai is, lhat reticular stinlulcltion generall b caused ',i reduction in late colnponents oP a closely-followhlg a u d i t o r y response. There ~ c l e some discrepancies. notably the a u d i t o r y cortex and medial geniculate and cochlear nucleus in five acute

animals in each of which potentials in response to click were enhanced by the prior RP stimulation. Slight differences in electrode placement oF the recording and stimulation sites identified by histological examination may have been responsible: in any event, in such work, each animal nmst serve as his own control. Each animal reported herein was therefore subjected to control studies several times a week for more than 2 months. As ;i further control, the same control studies were repeated terminally in each animal under acute experilnenlal conditions. Paralysis with gallamine iriethiodide did not alter the effect of RF stimulation indicating that contraclion of middle ear muscles was not a Factor as had earlier been suggested (Ht;Gl-l,IN et al., 1960: Mo/~Sllt~!(;IA~ et al., 1961: AI~('ARAZ et al., ! 962). Our data indicate that the reticular liltering mechanism as demonstrated in the auditory pathway is enhanced by chlorpromazine in low (lose; thus we confirm and extend earlier findings (K1LLAM, 1957: KH_i.AM and Kill,tiM. 1958) by showing that the drug has this action in unrestrained, unanesthetized animals whose general c o m p o r t m e n t , while somewhat less active, indicates no motor deficil. The drug effect was reproducible on different days in each animal. Doses more than 2-5 mg/kg, however, did not produce greater effect in any particular animal. Secondary influences of the drug on alertness were eliminated by careful use of a single controlled state. By comparison, peniobarbital in low doses partially blocked thc inhibitory effects of stimulation of RF in ti~c freely-moving animal. This data is in agreement with earlier findings in immobilized cats (KJtiAM, 1957: KILLAM and KILI_A5L 1958). Additionall), in two of the animals a threshold inhibitory effect of RIC:-s on ihe auditory potential was converted to a facilitory action by the drug. A similar enhancement of sensory potentials :_tftcr pentobarbital has been attributed to a release of ionic inhibitory inl]uences upon sensory transmission during wakefulness (HAGBARTH and KERR, 1954; HERNb,NDi-Z-PEdN et al., 1956). tIAGBARTtiand KERR (1954) reported lack of effect of central stimulation upon spinal afferent responses and an increase in control size of afferent ventral column responses following 15 mg, kg of pentobarbital Following 30 m g k g of pentobarbiial, HtcR.~XNI)ez-PEdN, el a/. (1956) observed similar enhancement of evoked potentials at the trigeminal sensory, gracilis and lateral geniculate nuclei. In addition, ionic centrifugal facilitat.ory influences upon sensory transmission were also eliminated by barbilurates (HERNXNDEZ-PEdN el al., 1960: HAGBARTHand Fex, 1959). The opposite effects of chiorpromazine and pentobarbital upon the reticular modulation of afferent responses in the unrestrained animal support earlier hypotheses that different mechanislns are involved in the behavioral depression by these two drugs (KILt,AM, 1957: K~LLAM and KILLAM. 1958). It was suggested that chlorpromazine enhanced the filtering or modulating effect of the reticular Formation on incoming sensory signals with little effect on the reticular mechanisms acting on state of awareness of the organism, In contrast, pentobarbital depressed the mechanisms of input control as well as the general

Exoked interaction patterns in chronically implanted cats f¢~llowingchlolplomazinc

61

reactivity of the reticular formation as earlier described by FR~!NClt e t al. (1953). Arousal responses from this area (ARI)t!INI and Ai~l)tJtNr, 1954: KIN(I, 1954, 1056: DOMINO, 1955: and others) were also blocked to a point at which atlenti~m :rod consciousness were disturbed. The effects of a moderate dose of meprobamate upon reticular modulation of sensory input \~ere similar to those of pentobarbital. These results are in agreement :~.ith those of PFHmSR el al. (1957) and GAN¢;1OrV (1959) who reported barbiturate-like properties of meprobamate. Unlike chlorpromazine, reserpine appeared to have different effects depending upon the level of interaction in the auditory pathway. Inhibitory effects of RF stimulation at dorsal cochlear nucleus and in distal regions of the medial geniculate nucleus were facilitated following reserpine, just as following chlorpromazine. However, reserpine blocks the reticular inhibition of potentials in the auditory cortex and more rostral medial geniculate nucleus. On the other hand, under conditions in which potentials in the auditory cortex were increased by reticular stimulation during the control period, reserpine produced a further enhancement. Stimulating effects of reserpine on the arousal mechanisms of the reticular formation have been shown for the rabbit (RINALD1 and HIMww~k 1955a,b). floweret, in the cat, reserpine produced no change in the threshold or duration of EEG arousal following peripheral or reticular stimulation (K1LLAM and K u l ,~M. 1957: KILLAM cl al., 1957: HAMEt and KAELBAR, 1961). Effects similar to those of reserpine on cortical potentials were also observed after amphetamine. The similarity of effects of these two drugs on the reticular modulation of cortical potentials may be related to the level of circulating amines. Biochemical correlates substantiating such a hypotl'esis are beyond the scope of this study. Evidence for and against an adrenergic mechanism at the reticular level has been summarized by Du,L (1960) and KILLAS,I (1962). Amphetamine which mimics some of the actions of norepinephrine (NE) has been shown to act on arousal mechanisms of the reticular i'ormation (Hu!BEL el ct/.. 1954: BRADI.tiY and El K~:s, 1957: R~N,\I D1 and HIMWICtf, 1955c: Ku_l AM el al.. 1959). In both unrestrained and immobilized animals there was a tendency for a decrease in the amplitude of the potentials ew)ked by click alone following 0.5 1 m g k g of amphetamine. It may be that this tendency, although not found in all experiments, represented an effect secondary to a stimulating action of anlphetamine on the reticular formation. The data from our experiments suggest opposite effects of amphetamine on reticular influences on auditory potentials depending on whether the latter were facilitating or inhibitory. The exceptional cases in our series were those in which RF stimulation induced facilitory effects on the response to an auditory stimulus. This effect, however, is reported to be the most consistent result of RF interaction with central stimuli inducing cortical (BREMER and STOUPEL, 1959a; DUMONI and DELL, 1960) and subcortical (BREMER and SfOUpl2t., 1959b) responses. BREMt!Rand STOUPH, (1959a) showed I mg/kg of amphetamine to block such reticular elt'ects, an action which they attributed to occlusion resulting from extreme cortical activation. Such activation was suggested as due to a combined stirnulus-induced and pharmacologically-induced reticular arousal. A somewhat similar hypothesis could explain the opposing effects of amphetamine on RF modulation of auditory responses in different cats. It is possible that the combined activating effect of amphetamine and of electrical stimulation on the reticular system triggers the negative feedback mechanisms from the

~2

.I.H. (-HI';, E. K. KILl.AM and K. F. KILLAM

c e r e b r a l c o r t e x (HuGEI,IN a n d Bt)!',VAI.LEI, 1957a, b, c) o r f r o m t h e m e d u l l a (BAHNI et al., 1958, 1959: MAGNI et al., 1959; CORDEAU a n d MANCIA, 1959; BONVAt.LET a n d BLO('tL 1960, 1961). T h u s t h e R F m a y i n c l u d e m e c h a n i s m s b o t h f o r t h e m o d u l a t i o n o f s e n s o r y input and for control of such modulation.

A c k n o w l e d g e m e n t s - - T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e t h e a s s i s t a n c e o f M r . ADAIR HEATH in c a r r y i n g o u t s o m e o f t h e e x p e r i m e n t s , M r . NOEL FLEM1NG f o r t h e i l l u s t r a t i o n s , a n d M i s s e s GEORGIA SCHERRER, PETRASUL~fEMEYERa n d VALERIE NORMAN f o r t h e h i s t o l o g y .

R~sum~--Chez cinq chats libres, porteurs d'61ectrodes implant6s ~. demeure dans diff6rentes structures c6r~brales, des interactions r6ticulo-auditives relativement stables ont 6t6 enregistr6qs dans la vole auditive centrale pendant 2-3 mois. Au cours des 4 mois suivants, chaque chat a requ, en dose unique, de la chlorpromazine, du pentobarbital et de la r6serpine. L'amph6tamine et le m6probamate ont 6t6 6galement test~s chez certains animaux. La chlorpromazine (1-10 mg.kg) intensifie la modulation r6ticulaire inhibitrice sur la r6ponse au signal sonore dans la vole auditive, lnvers6ment, apr6s administration de pentobarbital, la stimulation de la Formation R~ticulaire (FR) 5. des intensit~s supraliminales, submaximales, entraine g6n6ralement une r6duction moins importante des potentiels auditifs; dans certains cas, I'influence r6ticulaire se transforme en facilitation. Les effets du m6probamate (50-75 mg.kg), bien que moins intenses, sont similaires 5_ ceux induits par le pentobarbital. La r6serpine (100/~g.kg) d~termine une r6ponse complexe sur se type d'interaction 6voquee. Chez les chats, o/da stimulation r6ticulaire r6duit la r6ponse sp~cifique dans le cortex auditif et dans le brachium du colliculus inf6rieur ainsi que la r6ponse 5. latence prolong~e au corps genouill6 m6dian, cette stimulation r~ticulaire est moins efficace apr6s r6serpine. Par contre, I'inhibition induite par la FR sur le noyau chochl6aire dorsal ainsi que la r6ponse g6nicul6e latence plus courte sont augment6es par la r6serpine. L'amph6tamine pr~sente des effets similaires fi la r6serpine sur l'interaction reticulaire au nivau du cortex auditif.

Zusammenfassung--ln f f n f freibeweglichen Katzen, denen man Elektroden permanent in das Gehirngewebe eingepflanzt hatte, wurden fiber eine Zeitspanne von zwei bis drei Monaten relativ stabile retikul~.r-geh6r Wechselwirkung in der H6rbahn verzeichnet. WS_hrend der folgenden vier Monate wurden Einzeldosen von Chlorpromazin, Pentobarbital und Reserpin in jeder Katze getestet. In einigen Tieren wurden ausserdem die Wirkungen von Amphetamin und Meprobamat untersucht. Chlorpromazin (1-10 mg/kg) verstS_rkte die retikul~re Hemmungs-modulation der Rcaktionen zu einem Ticken in der H6rbahn. lm Gegensatz dazu war nach Verabfolgung yon Pentobarbital eine Reizung yon Retikul~r formation (RF) bei fiberschwelligen, submaximalen lntensit~iten im allgemeinen weniger wirksam hinsichtlich der Reduzierung des H6rpotentials; gelegentlich verkehrte sich der retikuRire Einfluss zugunsten einer Erleichterung. Die Folgeerscheinungen yon Meprobamat (50-75 mg/kg) waren ~ihnlich, doch nicht so ausgepr~gt, wie die nach Pentobarbital. Die Wirkungen yon Reserpin (100 Lug/kg) auf das hervorgerufene Wechselwirkungen war komplex. In Katzen, in denen retikul~ire Reizung die Geh6rreaktionen im kortikalen Geh6rzentrum und im Brachium colliculi inferioris reduzierte, und auch die lSngeren Latenzreaktionen im medialen KniehScker verringerte, ergab sich nach Verabreichung der Droge eine geringere Wirksamkeit yon RF-Reizung. lm Gegensatz dazu wurde die RF-induzierte Hemmung des dorsalen Cochlearkerns und die Reaktionen (in Form yon ktirzerer Latenz) des medialen Knieh6ckers nach Reserpin verstfirkt. Die Wirkungen yon Amphetamin auf die retikul~iren Wechselwirkungen im kortikalen Geh6rzentrum waren ~.hnlich wie die von Reserpin.

Evoked interaction patterns in chronically implanted cats following chlorpromazine

63

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J. H. Cl-ll~,, E. K. K|Lt,~I and K. F. Kn.I .xxl

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