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Prolonged daily inhalation of halothane modifies the dose-response pattern to acute administration of halothane
0028-3908/85 $3.00 + 0.00 Copyright 0 1985 Pergamon Press Ltd
Neuropharmacology Vol. 24, No. II, pp. 1033-1038, 1985 Printed in Great Britain. All rights reserved
PROLONGED DAILY INHALATION OF HALOTHANE MODIFIES THE DOSE-RESPONSE PATTERN TO ACUTE ADMINISTRATION OF HALOTHANE AN ELECTROPHYSIOLOGICAL
STUDY
G. N. FULLER, B. M. RIGOR, R. C. WIGGINS and N. DAFNY* Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77025, U.S.A. (Accepted 1 March 1985)
Summary-Sensory-evoked field potentials were obtained from freely moving rats implanted sterotaxically with permanent electrodes in the parafasciculus thalami (PF), mesencephalic central gray (CG), ventromedial hypothalamus (VMH) and somatosensory cortex (SCX). Animals were exposed to chronic, subanesthetic inhalation of halothane (0.5%, 3 hr/day, 5 days/week) for 56 days. The averaged acoustic evoked responses (AAER) were recorded on day 0, as well as at 28 and 56 days after a 48-hr halothane-free period (“control”) and after acute doses of halothane (0.25, 0.5 and 1.5%). In general, the averaged sensory-evoked responses from each structure were affected at day 0 of the experiment in dose-response manner, and suppression of the responses was the main effect of halothane. Chronic exposure to subanesthetic inhalation of halothane produced marked alteration of the “control” recording from 3 CNS structures; mainly from the mesencephalic central gray, the parafasciculus thalami and the somatosensory cortex and the direction (increase or decrease) of the averaged acoustic evoked responses in all the four CNS sites studied. The total responsiveness was modified as well, i.e. the recordings obtained from the mesencephalic central gray and somatosensory cortex exhibited hypersensitivity while the recordings obtained from the parafasciculus thalami and ventromedial hypothalamus exhibited tolerance. It is concluded that prolonged and intermittent inhalation of halothane can alter the electrophysiological properties of the four structures investigated. Key words: halothane, evoked potentials, thalamus, central gray, hypothalamus cortex.
Repetitive inhalation of halothane (2-bromo-2chloro- 1,l ,l -trifluorethane) has been reported to be hazardous to operating room personnel (Cohen, Brown and Bruce, 1974; Linde and Bruce, 1969; Whitcher, Cohen and Trudell, 1971), as well as to laboratory animals (Chang, Lee, Dudley and Katz, 1975,b). Halothane exerts numerous deleterious effects on the tissues of the central nervous system, including alterations of neuronal morphology (Chang, Dudley, Lee and Katz, 1976) and chemistry (Divakaran, Rigor and Wiggins, 1980a; Nahrwold, Lust and Passonneau, 1977; Ngai, Cheney and Finck, 1978). Impaired myelination of the brain (Wiggins, Fuller, Astrello and Rigor, 1979; Patsalos, Rigor and Wiggins, 1980), altered synaptogenesis (Quimby, Aschkenase, Bowman, Katz and Chang, 1974; Uemura and Bowman, 1980) and behavioral sequelae (Bowman and Smith, 1977; Quimby et al., 1974; Quimby, Katz and Bowman, 1975) have also been described following chronic exposure to halothane. Significant electrophysiological changes in the normal baseline electroencephalogram have recently been described (Reilly, Fuller, Wiggins, Rigor and Dafny, 1980) and sensory-evoked responses (Fuller, Rigor, Wiggins *Author to whom correspondence should be sent.
and reprint requests
and Dafny, 1980) taken from laboratory animals chronically exposed to subanesthetic amounts of halothane. The present experiments were designed to characterize further the relationship between exposure to halothane and altered neurophysiological parameters by examining the effect of chronic administration of halothane on the dose-response pattern to acute administration of halothane. METHODS
Surgical procedures Stainless-steel teflon-coated semi-microelectrodes (80 pm diameter) were permanently implanted stereotaxically in the brains of 12 male SpragueDawley rats, weighing 20&250 g, while they were under sodium pentobarbital anesthesia (50 mg/kg, i.p.). The electrodes were located in the somatosensory cortex (SCX), within the parafasciculus thalami (PF) in the ventromedial hypothalamus (VMH) and in the central gray (CG). The coordinates used throughout were: SCX A = 6280 pm, L = 4.0 mm, PF, H = 4.2 mm; A = 3290 pm, L= l.Omm, L = 0.5 mm, VMH, A = 4380 pm, H = 0.0 mm; H=-3.6mm; CG A=1170 pm, L=O.4mm, H = 0.0 mm. The atlas of Konig and Klippel (1970) was used for determining the coordinates and for
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G. N. FULLER et al.
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histological verification of the electrode placement. A reference (ground) electrode was implanted in the frontal sinus of each animal. Each electrode was fixed to the skull permanently with dental cement and was attached to an Amphenol metal pin lying inside a nylon plug. Recording procedures
After allowing 3-5 days recovery from surgery, each animal was placed in a closed, ventilated plexiglass box located within a shielded, sound-proofed electrophysiological chamber. Electrodes were connected via a commutator to an emitter follower while the animals moved freely about the plexiglass box. The amplified electrical activity was monitored with a Tektronik storage oscilloscope and fed into a four-channel NIC 1070 (signal averaging) minicomputer with a bin width (sampling time) set at 1 msec. A permanent record of the averaged responses from the NIC 1070 was produced by a Houston Instrument Omnigraphic 2000 X-Y plotter. The acoustic stimulation in the form of clicks was produced by a Grass ultralinear audiomonitor through a 15 cm remote loudspeaker located approx. 14cm from the center of the plexiglass box. The animals were allowed 1 hr to adapt to the test chamber and to the acoustic stimulation before the beginning of each experiment. Control recordings after acoustic stimulation were taken from the animal prior to exposure to halothane. Each set of recordings was composed of the average of a series of 32 repetitive stimuli which were presented 2.5 set apart (Dafny, 1975; Dafny and Rigor, 1978); four such sets were obtained. This procedure (four sets of recordings) lasted for approx. 40 min. Halothane was administered using the Fluotec Mark II vaporizer at a flow rate of 4 l/min of compressed air. Three concentrations of halothane were used in increasing order of concentration: 0.25x, 0.5% and 1.5% respectively. After completing the recording after the last dose of halothane (1.5x), the animals were removed from the cage to their permanent facilities. An identical procedure was re-
peated 28 and 56 days later. During the 56 days each rat was exposed to 0.5% halothane for 3 hr/days/week (Monday-Friday) in an exposure chamber. Recordings (day 26 and 56) were taken each time on Monday, i.e. 48 hr after the last daily treatment with halothane, to ensure clearance of halothane from the tissues (Divakaran, Joiner, Rigor and Wiggins, 1980b) prior to the recording sessions. Histology
At the end of the recording session, each rat was deeply anesthetized with sodium pentobarbital and perfused with 10% formalin solution containing 3% potassium ferrocyanide. A d.c. current was then passed to each electrode in order to make a small lesion (and a blue spot) at the tip of each electrode (Dafny, 1975, 1978). The brains were removed for histological verification and only the recordings from those electrodes located in the target areas were reported. Evaluation
The averaged acoustic evoked responses (AAER) were evaluated in terms of peak amplitudes (measured peak to peak; Dafny, 1975, 1978) of the various components. The mean and standard deviation for each component were compared for the four sets of pre-halothane (control) and the three experimental recording obtained at different doses (0.25, 0.5 and 1.5%) of halothane. An increase or decrease in amplitude of a particular component shown by an individual animal after exposure to halothane was considered significant if it was different from the control (pre-halothane recording) value by 2SE of the mean (P < 0.05). Each animal served as its own control. Significant changes within structures for all animals for the averaged acoustic evoked response and their latencies were determined using two-way analysis of variance, followed by Student-Newman-Keuls multiple range testing whenever a significant F-ratio was found.
Table 1. Direction of response exerted by three doses of the halothane on the P, component of the averaged acoustic evoked responses at day 0, 28 and 56 of the experiment (similar observations were obtained from P, and N, components) Day 0 Dose of halothane PF N=8 CC N=8 VMH N=5 sex N=8
0.25% (%) Iloo ;*, ;*w t 50 i 50
Day 28
0.5% (%)
1.5% (%)
0.25% (%)
0.5% (%)
20 80 100 100 100
100
61 33 50 50 100 67 33
50 50 80 20 67 33 50 50
100 100 100
Day 56 1.5% (%) 100 100 100 100
0.25% (%)
0.5% (%)
60 40 60 40 100
75 25 86 14 50 50 61 33
100
1.5% (%) 100 100 100 100
The direction of changes in response amplitudes by increase (t) or decrease (1) of component P3 after three doses of inhalation of halothane is given. The number of evoked responses affected by treatment with halothane (Fig. 2) was arbitrarily set at 100% for the purpose of comparing the preparations of evoked or decreases. PF = parafasciculus thalami; CG = central gray; responses increases showing VMH = ventromedial hypothalamus; SCX = sensorimotor cortex.
Chronic halothane modifies CNS activity
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HALOTHANE
CONTROL P3
0.25%
0.5%
DAY 0
DAY 28
DAY 56
Fig. 1. Representative averaged acoustic evoked responses (AAER) recorded from the mesencephalic central gray on days 0,28 and 56 of the experiment (controls) and after acute administration of halothane in three doses (0.25, 0.5 and 1.5%). Halothane (0.5%) was administered chronically (5 days/week) on days l-25 and 29-53.
Averaged evoked responses All the structures investigated exhibited similar response patterns in the averaged acoustic evoked responses (AAER), with differences found in latenties and amplitudes. The general pattern shown by all four structures consisted of an initial biphasic spike (positive-negative, P,, N,) succeeded by a large triphasic wave (positive-negative-positive, P,-N,-P,). Components P,-N,-P, of the triphasic responses demonstrated stability and consistency within and between the animals, and were evaluated statistically. Controls
Control recordings were obtained prior to acute treatment with halothane on days 0, 28 and 56, after repeated intervals of exposure to halothane between 1 and 25 and 29 and 53 days. Stable and consistent control averaged wave forms were obtained from all structures. Figure 1 shows representative recordings obtained from the central gray and the effects of the various acute doses of halothane. The Figure also illustrates the experimental procedure. The four control recordings (resting) demonstrate the stability and the consistency of the recording procedures. The “control” amplitudes recorded from the central gray
on day 56 were potentiated by 37%. The amplitude of the evoked response recorded in both parafasciculus thalamus (PF) and somatosensory cortex (SCX) were also potentiated at the 28th and 56th day of the experiment by 26% (PF), 31% (SCX), 37% (PF) and 43% (SCX) respectively. In contrast, “control” recordings obtained from the ventromedial hypothalamus (VHM) at days 28 and 56 of the experiment were not altered by the intervening exposure to halothane. Effects of acute halothane on the average acoustic evoked response Responsiveness. At day 0 of the experiment, halothane modified the evoked response of all four structures in a dose-related manner, i.e. with increased doses the percentage of animals showing a change in the evoked response also increased (Fig. 2). At days 28 and 56 of the experiment two phenomena were observed: supersensitivity and tolerance to acute treatment with halothane. The responses obtained from the central gray and somatosensory cortex became supersensitive, i.e. the evoked responses of animals which were not affected by halothane at day 0 of the experiment became sensitive and were affected by the initial two doses of halothane (0.25 and 0.5%) at days 28 and/or 56 (Fig. 2). The re-
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G. N. FULLER et al.
DOSE EFFECTS OF HALOTHANE
100%
r
100%
r
al
m i k
50% 0% h_L 02856
02856
0 2856
DAY
Fig. 2. Histograms show the percentage of animals in which component N, of the evoked response (see Fig. I) was
altered significantly (see Methods) by the schedule of chronic and acute exposure to halothane. Similar changes were observed in the other components of the evoked response.
shown by the P, component Table 1); however, mixed responses (both potentiation and suppression) were observed in the parafasciculus (0.5% halothane) and the cortex (0.25% halothane). On day 28 of the experiment mixed responses to the initial two doses of halothane were obtained from the recordings in central gray parafasciculus and cortex. In the hypothalamus all evoked responses affected by the evoked responses recorded from the 0.25% halothane were potentiated, whereas mixed results were obtained at 0.5% (Table 1). The dose of 1.5% halothane suppresses the evoked responses on all the experimental days and in all the four structures. On day 56 of the experiment similar phenomena to that obtained on day 28 were observed. The total responsiveness and the direction of the effects of the drug were compared between the four structures to find out whether the three doses of halothane at day 0, 28 and 56 exerted similar or different effects using the x2 tests. It was found that each structure was affected differently at the P = 0.05 level. Latencies. Latencies of the control evoked responses for components P,, N, and P, from the recordings obtained from the four sites are summarized in Table 2A. Chronic inhalation of halothane did not modify the controls obtained at day 28 and 56 of the experiment. Changes in latency were observed only after inhalation of 1.5% halothane and only in component P, of the evoked responses (Table 2B).
sponses obtained from the parafasciculus
DISCUSSION
Direction (increase or decrease in the amplitude of the evoked responses). The direction of response
(increase and/or decrease) of the evoked response recorded from the four sites are summarized in Table 1. At day 0 of the experiment most of the evoked responses affected by halothane were suppressed (as
In this study the dose-effects of halothane were recorded in naive animals (day 0) and in animals treated with halothane (0.5x), intermittently, over a prolonged period (up to 56 days). The use of a dose-response protocol, from a subanesthetic dose, that exerted effects on few responses, to an anesthetic dose that affected all the responses, provided a tool to observe whether the structures of the CNS responded identically or differently to a drug under
Table 2. Mean latencies in milliseconds and the + standard deviation of components P2, N2 and Pj of evoked responses recorded from parafasciculus thalami (PF), central gray (CG), ventromedial hypothalamus (VMH), sensorimotor cortex (SCX) prior to acute inhalation of halothane (control) (A) and following acute inhalation of halothane (B)
Control
6% Day 0
CC VMH
P, 38+_2 43+4 12
50 * 55 f 77+ 17
50+
Day 28 P, 79+4 + 7 107+_26
P* 39 f 39 * 54* 47*
4 5 10 14
Acute effects of halothane
(B) Day 0 Halothane PF CG VMH _..
sex
*P < 0.05.
N, 53 * 48 k 76? 62+
0.25% 82+5 84 + 8 115+32 98 f 24
5 6 14 18
Day 58
P, 76 + 4 82 + I 132+28 114k32
P2
N,
P,
36k4 38 +6 51+ 13 53rt 16
51*3 47 + 6 72f 18 68 + 37
81 k4 88 + 6 141 5 36 131+43
on P, component
Day 28
0.5%
1.5%
85 f 7 89 k 8 115+27 103; 26
98 _+8’ 112*9* 182+31* 210 + 33*
0.25% 78 f 4 80 + 8 114+26 97 + 29
Day 58
0.5%
1.5%
0.25%
72_+4 76 + 6 108+28 92 f 27
108 + 6’ 110 i 6* 196$-31’ 243 + 45*
74 f 6 76 + 4 128+28 109 f 28
0.5% 76 f 75 f 135? 101 k
1.5% 8 7 25 26
110+6* 112+6’ 207 _+32* 260 k 38’
Chronic halothane modifies CNS activity investigation and whether the drug exerted biphasic (increase and decrease) effects related to the dose of halothane. The results demonstrated that daily exposure to subanesthetic inhalation of halothane induced marked alteration of the control averaged acoustic evoked responses recorded from three central nervous structures; namely from the central gray parafasciculus thalamus and the sensorimotor cortex. The trend was toward potentiation of the amplitude. Moreover, prolonged inhalation of a subanesthetic dose of halothane also modified the direction (increase or decrease) of the evoked responses after acute treatment with the drug at days 28 and 56 of the experiment in all the four CNS sites studied, indicating toxic effects. The total responsiveness was modified as well, i.e. the recordings obtained from the central gray and cortex exhibited hypersensitivity while the recordings obtained from the parafasciulus and hypothalamus exhibited tolerance. Acute inhalation of halothane elicited differences in the number of responses affected by the drug between structures and components (only following the first two doses of halothane 0.25 and 0.5%). This could be explained by the fact that the neuronal organization of different structures of the CNS, as well as their role in producing anesthesia and/or analgesia, are different. Drugs and anesthetics that produce amnesia and anesthesia may inhibit the activity of inhibitory neurons, resulting in a net excitatory response at smaller doses (Borbely and Hall, 1970; Clark and Rosner, 1973; Dafny and Rigor, 1978; Yeoman, Moreno, Rigor and Dafny, 1980). There is evidence that the alteration of excitatory potentials by an anesthetic occurs at more than one site (Dafny, 1978; Rabe, Moreno, Rigor and Dafny, 1980), leading to a decrease in the excitatory transmitter (Weakly, 1969); or an increase in the release of inhibitory transmitter (Nicholl and Madison 1982); or a release of cyclic AMP, which was reported to suppress neuronal activity (DeFrance, Stanley, Marchand, Divakaran, Rigor and Wiggins, 1981). Supersensitivity and tolerance to acute treatment with halothane, resulting from prolonged subanesthetic inhalation of halothane, were obtained. It is possible to assume that chronic inhalation of halothane exerts various effects such as on (1) the release of neurostorage and/or synthesis, transmitters, (2) modification of the number of receptors and/or (3) modification of the sensitivity of the receptors to neurotransmitters (Berridge, 1975; Chapin, Waterhouse and Woodard, 1981; DeFrance et al., 1981; DeLorenzo, Freedman, Yohe and Maurer, 1979). The bipashic responses, i.e. suppression and/or potentiation of the evoked responses to small doses of halothane, probably resulted from suppression oi the inhibitory components, i.e. disinhibition. These observations are consistent with other reports that
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anesthetics may inhibit the activity of inhibitory interneurons, resulting in a net excitatory response at smaller doses of anesthetics (Borbely and Hall, 1970; Clark and Rosner, 1973; Dafny 1975, 1978; Eccles, Schmidt and Willis, 1963; Shimoji, Fujioka and Ebata, 1984; Yeoman et al., 1980). Larger doses of halothane blocked both the inhibitory and the excitatory components; therefore, the sensory-evoked responses were suppressed. Since potentiation was obtained in the four structures at days 28 and 58 of the experiment, it is possible that chronic inhalation exerted some slow effects, which may be permanent on the inhibitory interneurons. Confirmation of this conclusion, however, must await further investigation using different procedures. Slowing of the evoked responses in all the four CNS sites was observed only in the late components (component P3) of the amplitudes during all the experimental procedure. Slowing of all the components of the evoked responses (P2, N, and P,) resulting from large doses of anesthetic was reported (Chapin et al., 1981; Dafny 1975, 1978; Ritchie, Cohen and Dripps, 1975) and was attributed to the effects of the drug on axons such as the effects of local anesthesia which alters axonal electrical transmission (Dafny, 1980). Since only the late component (P3) was affected, it is concluded that, in the present study, neither acute nor prolonged inhalation of halothane in the doses used affected axonal properties, but rather affected synaptic or receptor properties. These modifications occurred within the site investigated and not in remote structures, since effects in remote structures would modify all the components of the evoked responses and not one component (PJ) selectively. Considerable caution is necessary when comparing experimental laboratory studies of chronic exposure to halothane with the clinical situation. The most important caveat to be kept in mind is that the concentration of halothane employed for daily exposure (0.5%) is considerably greater than ambient levels (l-20 ppm) reported for operating theaters (Linde and Bruce, 1969; Whichter et al., 1971). Moreover, the exposure regimen imposed (3 hr/day, 5 days/week, over 56 days) and the administration of only a single anesthetic agent (halothane) represents a different situation from that encountered by the operating room team. However, these results do reinforce the sensible contention that chronic exposure to waste anesthetics should be rigorously minimized. Acknowledgement-We
thank
D.
Parker
for
secretarial
assistance.
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Neuropharmacology Vol. 24, No. II, pp. 1033-1038, 1985 Printed in Great Britain. All rights reserved
PROLONGED DAILY INHALATION OF HALOTHANE MODIFIES THE DOSE-RESPONSE PATTERN TO ACUTE ADMINISTRATION OF HALOTHANE AN ELECTROPHYSIOLOGICAL
STUDY
G. N. FULLER, B. M. RIGOR, R. C. WIGGINS and N. DAFNY* Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77025, U.S.A. (Accepted 1 March 1985)
Summary-Sensory-evoked field potentials were obtained from freely moving rats implanted sterotaxically with permanent electrodes in the parafasciculus thalami (PF), mesencephalic central gray (CG), ventromedial hypothalamus (VMH) and somatosensory cortex (SCX). Animals were exposed to chronic, subanesthetic inhalation of halothane (0.5%, 3 hr/day, 5 days/week) for 56 days. The averaged acoustic evoked responses (AAER) were recorded on day 0, as well as at 28 and 56 days after a 48-hr halothane-free period (“control”) and after acute doses of halothane (0.25, 0.5 and 1.5%). In general, the averaged sensory-evoked responses from each structure were affected at day 0 of the experiment in dose-response manner, and suppression of the responses was the main effect of halothane. Chronic exposure to subanesthetic inhalation of halothane produced marked alteration of the “control” recording from 3 CNS structures; mainly from the mesencephalic central gray, the parafasciculus thalami and the somatosensory cortex and the direction (increase or decrease) of the averaged acoustic evoked responses in all the four CNS sites studied. The total responsiveness was modified as well, i.e. the recordings obtained from the mesencephalic central gray and somatosensory cortex exhibited hypersensitivity while the recordings obtained from the parafasciculus thalami and ventromedial hypothalamus exhibited tolerance. It is concluded that prolonged and intermittent inhalation of halothane can alter the electrophysiological properties of the four structures investigated. Key words: halothane, evoked potentials, thalamus, central gray, hypothalamus cortex.
Repetitive inhalation of halothane (2-bromo-2chloro- 1,l ,l -trifluorethane) has been reported to be hazardous to operating room personnel (Cohen, Brown and Bruce, 1974; Linde and Bruce, 1969; Whitcher, Cohen and Trudell, 1971), as well as to laboratory animals (Chang, Lee, Dudley and Katz, 1975,b). Halothane exerts numerous deleterious effects on the tissues of the central nervous system, including alterations of neuronal morphology (Chang, Dudley, Lee and Katz, 1976) and chemistry (Divakaran, Rigor and Wiggins, 1980a; Nahrwold, Lust and Passonneau, 1977; Ngai, Cheney and Finck, 1978). Impaired myelination of the brain (Wiggins, Fuller, Astrello and Rigor, 1979; Patsalos, Rigor and Wiggins, 1980), altered synaptogenesis (Quimby, Aschkenase, Bowman, Katz and Chang, 1974; Uemura and Bowman, 1980) and behavioral sequelae (Bowman and Smith, 1977; Quimby et al., 1974; Quimby, Katz and Bowman, 1975) have also been described following chronic exposure to halothane. Significant electrophysiological changes in the normal baseline electroencephalogram have recently been described (Reilly, Fuller, Wiggins, Rigor and Dafny, 1980) and sensory-evoked responses (Fuller, Rigor, Wiggins *Author to whom correspondence should be sent.
and reprint requests
and Dafny, 1980) taken from laboratory animals chronically exposed to subanesthetic amounts of halothane. The present experiments were designed to characterize further the relationship between exposure to halothane and altered neurophysiological parameters by examining the effect of chronic administration of halothane on the dose-response pattern to acute administration of halothane. METHODS
Surgical procedures Stainless-steel teflon-coated semi-microelectrodes (80 pm diameter) were permanently implanted stereotaxically in the brains of 12 male SpragueDawley rats, weighing 20&250 g, while they were under sodium pentobarbital anesthesia (50 mg/kg, i.p.). The electrodes were located in the somatosensory cortex (SCX), within the parafasciculus thalami (PF) in the ventromedial hypothalamus (VMH) and in the central gray (CG). The coordinates used throughout were: SCX A = 6280 pm, L = 4.0 mm, PF, H = 4.2 mm; A = 3290 pm, L= l.Omm, L = 0.5 mm, VMH, A = 4380 pm, H = 0.0 mm; H=-3.6mm; CG A=1170 pm, L=O.4mm, H = 0.0 mm. The atlas of Konig and Klippel (1970) was used for determining the coordinates and for
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G. N. FULLER et al.
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histological verification of the electrode placement. A reference (ground) electrode was implanted in the frontal sinus of each animal. Each electrode was fixed to the skull permanently with dental cement and was attached to an Amphenol metal pin lying inside a nylon plug. Recording procedures
After allowing 3-5 days recovery from surgery, each animal was placed in a closed, ventilated plexiglass box located within a shielded, sound-proofed electrophysiological chamber. Electrodes were connected via a commutator to an emitter follower while the animals moved freely about the plexiglass box. The amplified electrical activity was monitored with a Tektronik storage oscilloscope and fed into a four-channel NIC 1070 (signal averaging) minicomputer with a bin width (sampling time) set at 1 msec. A permanent record of the averaged responses from the NIC 1070 was produced by a Houston Instrument Omnigraphic 2000 X-Y plotter. The acoustic stimulation in the form of clicks was produced by a Grass ultralinear audiomonitor through a 15 cm remote loudspeaker located approx. 14cm from the center of the plexiglass box. The animals were allowed 1 hr to adapt to the test chamber and to the acoustic stimulation before the beginning of each experiment. Control recordings after acoustic stimulation were taken from the animal prior to exposure to halothane. Each set of recordings was composed of the average of a series of 32 repetitive stimuli which were presented 2.5 set apart (Dafny, 1975; Dafny and Rigor, 1978); four such sets were obtained. This procedure (four sets of recordings) lasted for approx. 40 min. Halothane was administered using the Fluotec Mark II vaporizer at a flow rate of 4 l/min of compressed air. Three concentrations of halothane were used in increasing order of concentration: 0.25x, 0.5% and 1.5% respectively. After completing the recording after the last dose of halothane (1.5x), the animals were removed from the cage to their permanent facilities. An identical procedure was re-
peated 28 and 56 days later. During the 56 days each rat was exposed to 0.5% halothane for 3 hr/days/week (Monday-Friday) in an exposure chamber. Recordings (day 26 and 56) were taken each time on Monday, i.e. 48 hr after the last daily treatment with halothane, to ensure clearance of halothane from the tissues (Divakaran, Joiner, Rigor and Wiggins, 1980b) prior to the recording sessions. Histology
At the end of the recording session, each rat was deeply anesthetized with sodium pentobarbital and perfused with 10% formalin solution containing 3% potassium ferrocyanide. A d.c. current was then passed to each electrode in order to make a small lesion (and a blue spot) at the tip of each electrode (Dafny, 1975, 1978). The brains were removed for histological verification and only the recordings from those electrodes located in the target areas were reported. Evaluation
The averaged acoustic evoked responses (AAER) were evaluated in terms of peak amplitudes (measured peak to peak; Dafny, 1975, 1978) of the various components. The mean and standard deviation for each component were compared for the four sets of pre-halothane (control) and the three experimental recording obtained at different doses (0.25, 0.5 and 1.5%) of halothane. An increase or decrease in amplitude of a particular component shown by an individual animal after exposure to halothane was considered significant if it was different from the control (pre-halothane recording) value by 2SE of the mean (P < 0.05). Each animal served as its own control. Significant changes within structures for all animals for the averaged acoustic evoked response and their latencies were determined using two-way analysis of variance, followed by Student-Newman-Keuls multiple range testing whenever a significant F-ratio was found.
Table 1. Direction of response exerted by three doses of the halothane on the P, component of the averaged acoustic evoked responses at day 0, 28 and 56 of the experiment (similar observations were obtained from P, and N, components) Day 0 Dose of halothane PF N=8 CC N=8 VMH N=5 sex N=8
0.25% (%) Iloo ;*, ;*w t 50 i 50
Day 28
0.5% (%)
1.5% (%)
0.25% (%)
0.5% (%)
20 80 100 100 100
100
61 33 50 50 100 67 33
50 50 80 20 67 33 50 50
100 100 100
Day 56 1.5% (%) 100 100 100 100
0.25% (%)
0.5% (%)
60 40 60 40 100
75 25 86 14 50 50 61 33
100
1.5% (%) 100 100 100 100
The direction of changes in response amplitudes by increase (t) or decrease (1) of component P3 after three doses of inhalation of halothane is given. The number of evoked responses affected by treatment with halothane (Fig. 2) was arbitrarily set at 100% for the purpose of comparing the preparations of evoked or decreases. PF = parafasciculus thalami; CG = central gray; responses increases showing VMH = ventromedial hypothalamus; SCX = sensorimotor cortex.
Chronic halothane modifies CNS activity
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HALOTHANE
CONTROL P3
0.25%
0.5%
DAY 0
DAY 28
DAY 56
Fig. 1. Representative averaged acoustic evoked responses (AAER) recorded from the mesencephalic central gray on days 0,28 and 56 of the experiment (controls) and after acute administration of halothane in three doses (0.25, 0.5 and 1.5%). Halothane (0.5%) was administered chronically (5 days/week) on days l-25 and 29-53.
Averaged evoked responses All the structures investigated exhibited similar response patterns in the averaged acoustic evoked responses (AAER), with differences found in latenties and amplitudes. The general pattern shown by all four structures consisted of an initial biphasic spike (positive-negative, P,, N,) succeeded by a large triphasic wave (positive-negative-positive, P,-N,-P,). Components P,-N,-P, of the triphasic responses demonstrated stability and consistency within and between the animals, and were evaluated statistically. Controls
Control recordings were obtained prior to acute treatment with halothane on days 0, 28 and 56, after repeated intervals of exposure to halothane between 1 and 25 and 29 and 53 days. Stable and consistent control averaged wave forms were obtained from all structures. Figure 1 shows representative recordings obtained from the central gray and the effects of the various acute doses of halothane. The Figure also illustrates the experimental procedure. The four control recordings (resting) demonstrate the stability and the consistency of the recording procedures. The “control” amplitudes recorded from the central gray
on day 56 were potentiated by 37%. The amplitude of the evoked response recorded in both parafasciculus thalamus (PF) and somatosensory cortex (SCX) were also potentiated at the 28th and 56th day of the experiment by 26% (PF), 31% (SCX), 37% (PF) and 43% (SCX) respectively. In contrast, “control” recordings obtained from the ventromedial hypothalamus (VHM) at days 28 and 56 of the experiment were not altered by the intervening exposure to halothane. Effects of acute halothane on the average acoustic evoked response Responsiveness. At day 0 of the experiment, halothane modified the evoked response of all four structures in a dose-related manner, i.e. with increased doses the percentage of animals showing a change in the evoked response also increased (Fig. 2). At days 28 and 56 of the experiment two phenomena were observed: supersensitivity and tolerance to acute treatment with halothane. The responses obtained from the central gray and somatosensory cortex became supersensitive, i.e. the evoked responses of animals which were not affected by halothane at day 0 of the experiment became sensitive and were affected by the initial two doses of halothane (0.25 and 0.5%) at days 28 and/or 56 (Fig. 2). The re-
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G. N. FULLER et al.
DOSE EFFECTS OF HALOTHANE
100%
r
100%
r
al
m i k
50% 0% h_L 02856
02856
0 2856
DAY
Fig. 2. Histograms show the percentage of animals in which component N, of the evoked response (see Fig. I) was
altered significantly (see Methods) by the schedule of chronic and acute exposure to halothane. Similar changes were observed in the other components of the evoked response.
shown by the P, component Table 1); however, mixed responses (both potentiation and suppression) were observed in the parafasciculus (0.5% halothane) and the cortex (0.25% halothane). On day 28 of the experiment mixed responses to the initial two doses of halothane were obtained from the recordings in central gray parafasciculus and cortex. In the hypothalamus all evoked responses affected by the evoked responses recorded from the 0.25% halothane were potentiated, whereas mixed results were obtained at 0.5% (Table 1). The dose of 1.5% halothane suppresses the evoked responses on all the experimental days and in all the four structures. On day 56 of the experiment similar phenomena to that obtained on day 28 were observed. The total responsiveness and the direction of the effects of the drug were compared between the four structures to find out whether the three doses of halothane at day 0, 28 and 56 exerted similar or different effects using the x2 tests. It was found that each structure was affected differently at the P = 0.05 level. Latencies. Latencies of the control evoked responses for components P,, N, and P, from the recordings obtained from the four sites are summarized in Table 2A. Chronic inhalation of halothane did not modify the controls obtained at day 28 and 56 of the experiment. Changes in latency were observed only after inhalation of 1.5% halothane and only in component P, of the evoked responses (Table 2B).
sponses obtained from the parafasciculus
DISCUSSION
Direction (increase or decrease in the amplitude of the evoked responses). The direction of response
(increase and/or decrease) of the evoked response recorded from the four sites are summarized in Table 1. At day 0 of the experiment most of the evoked responses affected by halothane were suppressed (as
In this study the dose-effects of halothane were recorded in naive animals (day 0) and in animals treated with halothane (0.5x), intermittently, over a prolonged period (up to 56 days). The use of a dose-response protocol, from a subanesthetic dose, that exerted effects on few responses, to an anesthetic dose that affected all the responses, provided a tool to observe whether the structures of the CNS responded identically or differently to a drug under
Table 2. Mean latencies in milliseconds and the + standard deviation of components P2, N2 and Pj of evoked responses recorded from parafasciculus thalami (PF), central gray (CG), ventromedial hypothalamus (VMH), sensorimotor cortex (SCX) prior to acute inhalation of halothane (control) (A) and following acute inhalation of halothane (B)
Control
6% Day 0
CC VMH
P, 38+_2 43+4 12
50 * 55 f 77+ 17
50+
Day 28 P, 79+4 + 7 107+_26
P* 39 f 39 * 54* 47*
4 5 10 14
Acute effects of halothane
(B) Day 0 Halothane PF CG VMH _..
sex
*P < 0.05.
N, 53 * 48 k 76? 62+
0.25% 82+5 84 + 8 115+32 98 f 24
5 6 14 18
Day 58
P, 76 + 4 82 + I 132+28 114k32
P2
N,
P,
36k4 38 +6 51+ 13 53rt 16
51*3 47 + 6 72f 18 68 + 37
81 k4 88 + 6 141 5 36 131+43
on P, component
Day 28
0.5%
1.5%
85 f 7 89 k 8 115+27 103; 26
98 _+8’ 112*9* 182+31* 210 + 33*
0.25% 78 f 4 80 + 8 114+26 97 + 29
Day 58
0.5%
1.5%
0.25%
72_+4 76 + 6 108+28 92 f 27
108 + 6’ 110 i 6* 196$-31’ 243 + 45*
74 f 6 76 + 4 128+28 109 f 28
0.5% 76 f 75 f 135? 101 k
1.5% 8 7 25 26
110+6* 112+6’ 207 _+32* 260 k 38’
Chronic halothane modifies CNS activity investigation and whether the drug exerted biphasic (increase and decrease) effects related to the dose of halothane. The results demonstrated that daily exposure to subanesthetic inhalation of halothane induced marked alteration of the control averaged acoustic evoked responses recorded from three central nervous structures; namely from the central gray parafasciculus thalamus and the sensorimotor cortex. The trend was toward potentiation of the amplitude. Moreover, prolonged inhalation of a subanesthetic dose of halothane also modified the direction (increase or decrease) of the evoked responses after acute treatment with the drug at days 28 and 56 of the experiment in all the four CNS sites studied, indicating toxic effects. The total responsiveness was modified as well, i.e. the recordings obtained from the central gray and cortex exhibited hypersensitivity while the recordings obtained from the parafasciulus and hypothalamus exhibited tolerance. Acute inhalation of halothane elicited differences in the number of responses affected by the drug between structures and components (only following the first two doses of halothane 0.25 and 0.5%). This could be explained by the fact that the neuronal organization of different structures of the CNS, as well as their role in producing anesthesia and/or analgesia, are different. Drugs and anesthetics that produce amnesia and anesthesia may inhibit the activity of inhibitory neurons, resulting in a net excitatory response at smaller doses (Borbely and Hall, 1970; Clark and Rosner, 1973; Dafny and Rigor, 1978; Yeoman, Moreno, Rigor and Dafny, 1980). There is evidence that the alteration of excitatory potentials by an anesthetic occurs at more than one site (Dafny, 1978; Rabe, Moreno, Rigor and Dafny, 1980), leading to a decrease in the excitatory transmitter (Weakly, 1969); or an increase in the release of inhibitory transmitter (Nicholl and Madison 1982); or a release of cyclic AMP, which was reported to suppress neuronal activity (DeFrance, Stanley, Marchand, Divakaran, Rigor and Wiggins, 1981). Supersensitivity and tolerance to acute treatment with halothane, resulting from prolonged subanesthetic inhalation of halothane, were obtained. It is possible to assume that chronic inhalation of halothane exerts various effects such as on (1) the release of neurostorage and/or synthesis, transmitters, (2) modification of the number of receptors and/or (3) modification of the sensitivity of the receptors to neurotransmitters (Berridge, 1975; Chapin, Waterhouse and Woodard, 1981; DeFrance et al., 1981; DeLorenzo, Freedman, Yohe and Maurer, 1979). The bipashic responses, i.e. suppression and/or potentiation of the evoked responses to small doses of halothane, probably resulted from suppression oi the inhibitory components, i.e. disinhibition. These observations are consistent with other reports that
1037
anesthetics may inhibit the activity of inhibitory interneurons, resulting in a net excitatory response at smaller doses of anesthetics (Borbely and Hall, 1970; Clark and Rosner, 1973; Dafny 1975, 1978; Eccles, Schmidt and Willis, 1963; Shimoji, Fujioka and Ebata, 1984; Yeoman et al., 1980). Larger doses of halothane blocked both the inhibitory and the excitatory components; therefore, the sensory-evoked responses were suppressed. Since potentiation was obtained in the four structures at days 28 and 58 of the experiment, it is possible that chronic inhalation exerted some slow effects, which may be permanent on the inhibitory interneurons. Confirmation of this conclusion, however, must await further investigation using different procedures. Slowing of the evoked responses in all the four CNS sites was observed only in the late components (component P3) of the amplitudes during all the experimental procedure. Slowing of all the components of the evoked responses (P2, N, and P,) resulting from large doses of anesthetic was reported (Chapin et al., 1981; Dafny 1975, 1978; Ritchie, Cohen and Dripps, 1975) and was attributed to the effects of the drug on axons such as the effects of local anesthesia which alters axonal electrical transmission (Dafny, 1980). Since only the late component (P3) was affected, it is concluded that, in the present study, neither acute nor prolonged inhalation of halothane in the doses used affected axonal properties, but rather affected synaptic or receptor properties. These modifications occurred within the site investigated and not in remote structures, since effects in remote structures would modify all the components of the evoked responses and not one component (PJ) selectively. Considerable caution is necessary when comparing experimental laboratory studies of chronic exposure to halothane with the clinical situation. The most important caveat to be kept in mind is that the concentration of halothane employed for daily exposure (0.5%) is considerably greater than ambient levels (l-20 ppm) reported for operating theaters (Linde and Bruce, 1969; Whichter et al., 1971). Moreover, the exposure regimen imposed (3 hr/day, 5 days/week, over 56 days) and the administration of only a single anesthetic agent (halothane) represents a different situation from that encountered by the operating room team. However, these results do reinforce the sensible contention that chronic exposure to waste anesthetics should be rigorously minimized. Acknowledgement-We
thank
D.
Parker
for
secretarial
assistance.
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