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Effects of pentobarbital on the visual evoked response in the avian optic tectum
Physiology and Behavior, Vol. 8, pp. 1193--I196, Brain Research Publications Inc., 1972. Printed in Great Britain.
BRIEF COMMUNICATION Effects of Pentobarbital on the Visual Evoked Response in the Avian Optic Tectum HERMAN
H. S A M S O N A N D L A W R E N C E L A V I N E
Department of Psychology, Rutgers University, New Brunswick, New Jersey, 08903, U.S.A. (Received 26 N o v e m b e r 1971) H. H. AND L. LAVINE. Effects of pentobarbital on the visual evoked response in the avian optic tectum. PHYSIOL. BErtAV. 8 (6) 1193-1196, 1972.--Pentobarbital sodium altered the effect of constant retinal illumination upon the photic evoked potential in the optic tectum of the pigeon. As the dose was increased, suppression by background illumination of the amplitude of the response decreased. With a dose level of 15 mg/kg, constant retinal illumination (background) potentiated rather than suppressed the response amplitudes. SAMSON,
Pentobarbital
Evoked potential
Visual evoked potential
ON~ oF many barbiturates which has been shown to affect the photic evoked potential is pentobarbital. Brazier [1 ] reported that the potentials were increased in size and were less complex following an anesthetic level dose. These results have been confirmed and elaborated by other investigators [6, 7, 8, 9]. In determining the effects of constant retinal illumination upon the photic evoked response, Heron and Samson [4] found a gross difference between the anesthetized and unanesthetized bird. In the unanesthetized animal, the response to the photic stimulus was reduced in amplitude (suppressed) by constant retinal illumination. However, in the same preparation, anesthetized with sodium pentobarbital (Nembutal, 25 mg/kg), the response was increased in amplitude (potentiated) by the presence of the same constant retinal illumination. Because of this reversal in effect of constant retinal illumination by pentobarbital, it was decided that a more complete study of the effects at various doses of pentobarbital was needed. This study reports the results for four dose levels on the change in the photic evoked response by constant background illumination. METHOD
Animals Five White Carneaux pigeons were maintained in individual cages with food and water freely available. The room was temperature controlled at 72°F ( ~ 5°), and illuminated by 24-hr fluorescent light.
Surgical Procedure Under sodium pentobarbital (Nembutal) anesthesia (25 mg/kg), the animal was placed in a Revzin headholder held 1193
by a K o p f stereotaxic instrument. Stainless steel electrodes insulated with an epoxy resin (Epoxylite Corp.) were implanted into the optic tectum at the level of the S G F (stratum griseum et fibrosum superficiale [5]). An indifferent electrode was placed into the frontal bone. Stainless steel screws and dental acrylic were used to anchor the electrodes. In addition, a small bolt and plastic rings around each eye were attached to the skull cap for placement of the head during recording of evoked potentials. Following surgery, all animals were allowed a one-month recovery period prior to the experiment.
Stimulus Apparatus In order to obtain repeated measures over a number of days, an apparatus was constructed so that the restrained bird could be presented monocularly with a flash which was superimposed upon a light or dark background. The evoking stimulus was delivered from a Grass PS-2 photo stimulator onto a translucent, white diffusion screen which had a 49 % transmittance factor. On the other side of this screen was a camera bellows (4 × 3 in.) with an aperture milled such that the plastic ring around the animal's eye would just fit. The distance of the flash head from one side of the screen was 14 in, and the length of the bellows on the other side was 6 in. The screen and bellows were arranged so that only the flash which was within the 3 x 4 in. opening of the bellows was viewed by the bird. With a flash setting of 4 on the photostimulator, the approximate intensity of the flash was determined as 1,950 ml (Gamma Scientific photometer, using a flash rate of 60 per sec). The constant background illumination was provided by a GE-47 car reading bulb ran at 0.5 amp with a DC 6 V source. It was placed 2 ft from the translucent screen and had an intensity of 8 ft-c as measured on the bird's side of the screen.
II 94
Recording Apparatus Evoked responses were amplified by a factor of 1,000 with a Grass P-15 preamplifier, monitored by a Tektronix 502 oscilloscope and led into a Digital Equipment Corporation Lab 8 computer. They were also stored on magnetic tape. The computer performed on-line averages which were used in the final data analysis.
SAMSON AND LAVINE removed and 50-~ frozen sections were taken. These were stained with thionin, and electrode placements were determined.
130-
Procedln'e The animal was restrained by wrapping in a cloth and the head positioned into the stimulus apparatus through the use of the bolt and eyepiece. All stimuli were presented monocularly, with the recording done from the contralateral tectum only. The bird was given a period of adaptation (15 rain) to both the physical restraint and to the level of light that was on the screen (same as the background condition). The rest of the room was dark. Following adaptation, ten sets of flashes were presented, consisting of fifty flashes per set delivered at a rate of one per second. Following each set, a 4-rain adaptation period was given. On half of the sets the background illumination was absent. The order of background on or off was random for each electrode location, but was kept the same at each location over sessions. This was done to evaluate order effect within treatment, as well as in a particular session. Four sessions per week were given with the following regime. On day one, the standard session was given (the presession). On the next day, the drug was administered 15 rain before the session began (the drug session). Then five hr after the drug was administered a third session was given (the 5-hr post-session). On the next day a 24-hr post-session was then given. Ten weeks of testing were conducted. All injections were in the intraperitoneal cavity. In the first two weeks each bird received a dose of isotonic saline (0.25 ml) during the drug session. In the third and fourth weeks the dose was 4 mg/kg pentobarbital, with the dose increased to 8 mg/kg in the fifth and sixth weeks, and to 15 mg/kg in the seventh and eighth weeks. For the last two weeks, a dose of 25 mg/kg was used. The use of an incremental dosage schedule minimized any tolerance effect. Within any week only one electrode location per bird was tested.
Data Analysis From the computer-averaged responses the largest negative and positive deflections were determined. An analysis was then performed by comparing the response amplitudes with and without background. By forming a ratio of the amplitude with background against the amplitude without background, an assessment of the suppression by background could be made, independent of change in absolute amplitude. This ratio is called the suppression ratio and is symbolized by B/NB. If there was no effect of background, this ratio was 1.00. As the response amplitudes were suppressed by background, the ratios approached zero. Ratios were computed for the positive peak, negative peak, and peak to peak amplitudes. These ratio scores were then used to compute a two-way analysis of variance [3] across drug dose and session variables.
l-listology At the conclusion of the study the animals were sacrificed and perfused with 1 0 ~ formal-saline. The brains were
i~ i
o----o
salve
: : 0----0 e--*-e o--.--o
4mg/kq
G mg/Itg |5mg/kg 25mg/kg
12.0 =
/
lie=
/
I00-
',\
,/ !' :/
go-
\0
,
0
,
\ \
X
0
//
80-
l-nc
",ix,
m Z
70-
60"
i
I
PRE
DRUG
| 5~r POST
I 2 4 nr POST
SESSION
FIG. 1. Drug dose-effect curves for B/NB ratios (mean data for all birds).
RESULTS
Figure 1 shows the mean results for all birds. Figure 2 shows the results of a single bird, illustrating that the group data are representative of the results for single animals (cf. Fig. 1). The control saline result shows that the injection process did not produce a change. Three points are evident in both Figures 1 and 2. One, the suppression by background is decreased as the dose of pentobarbital is increased up to 8 mg/kg pentobarbital. Two, in doses of 15 mg/kg or greater, the background does not suppress the photic evoked response, but rather potentiates it. Three, while the maximally effective potentiating dose used in this series was 15 mg/kg for the drug session, the largest dose (25 mg/kg) had the largest residual effect at the 5-hour post-session.
PENTOBARBITAL AND VISUAL EVOKED RESPONSE
o -o.---~0 e.---,.l o-.-.o
120" • it I° t 'l~'1 II0-
P
1195 the effect of the 15 mg/kg dose was seen to be slightly greater than the 25 mg/kg dose, but at the 5-hr post, the larger dose was the one with the largest effect. This would indicate that the 15 mg/kg dose produced a maximum effect, and the attenuation of the suppressive effect of the background by the larger dose (25 mg/kg) became evident at the 5-hr postsession, by which time the effective level of the other dosages had declined, There were no significant differences between all pre- and post-sessions or among any of the saline sessions. An analysis performed on the positive peak and the negative peak was found to be exactly the same as that for the total peak-to-peak scores. This indicates that the response components are not affected independent of one another. The histology showed all electrodes were within the S G F layer of the optic rectum.
SALINE 4 mglkg e mg/kg 15 mglkg 25 mg/l~9
tt
4\
P
~
I00 ~
b
90--
I ~J
c2_
l\
,
DISCUSSION
BO--
OE El Z -% m
I
l
\ ', .,, - ,
:;/ 70-
O0--
pRIr
DRUG
5h¢ ~
24hr Pg~T
SESSION
FIG. 2. Drug dose-effect curves for B/NB ratio (Bird 3, left tectum).
An analysis of variance was significant at greater than the 0.01 level for both drug and session variables. When specific means were tested against their appropriate control presessions, all drug session treatments were significantly changed. The significance levels were 0.05 for the 4 mg/kg dose and 0.01 for all other doses. The two 5-hour post-sessions that were significantly different from the saline control weie the values 15 mg/kg (0.05 level) and 25 mg/kg (0.01 level). At the drug session,
From the data it is clear that an anesthetic dosage of pentobarbital reverses the effect of constant retinal illumination upon the photic evoked response. Normally the response is suppressed, but under narcosis with pentobarbital the responses are actually potentiated. The effects of doses lower than those producing narcosis show a dose-dependent relation which indicates that the stlppressive effect of background illumination is antagonized in a graded fashion by increasing doses of pentobarbital. The maximum effect lies in the region of 15 mg/kg. A significant decrease in the amount of suppression can be found at the 4 mg/kg level, which shows little if any overt behavioral effect. The results confirmed the danger of using barbiturate anesthesia for the acute study of the photic evoked potential, which had been pointed out by earlier investigators [1, 2]. However, the dose-dependent effect found in this study might provide a method for the systematic determination of components of the evoked potential itself, as well as the effects of light adaptation upon the visual system. The change in the effect of the constant retinal illumination as a function of drug state may also offer a partial explanation for the conflicting results concerning the role of adaptation state on the photic evoked potential. However, further work employing a range of flash intensities, background illuminations, and other drugs is needed before this explanation could be considered. One point is clear: The interaction between the drug state and the stimulation state can be related not only to the evoking stimuli but also to the state of adaptation of the receptive system as determined by the background illumination conditions.
REFERENCES 1. Brazier, M. B. A. Studies of evoked responses by flash in man and cat. In: Reticular formation of the brain, edited by H. H. Jasper: Boston: Little Brown, 1958. 2. Bremer, F. Neurogenic influences on evoked potentials. In: Sensory communication, edited by W. A. Rosenblith, New York: Wiley, 1961. 3. Edwards, A. L. Experimental design in psychological research, New York: Holt, Rinehart and Winston, 1963, 4. Heron, W. and H. H. Samson. Some effects of perceptual isolation on the CNS. Paper presented at Eastern Psychological Association, Atlantic City, 1965. 5. Karten, H. J. and W. Hodos A Stereotaxic Atlas o f The Brain o f the Pigeon (Columba Livia), Baltimore: Johns Hopkins University Press, 1967.
6. Olds, M. E. and G. Baldrighi. Effects of meprobamate, chlordiazepoxide, diazapam, and sodium pentobarbitai on visually evoked responses in the tecto-tegmental area of the rat. Int. J. Neuropharmac. 7: 231-239, 1968. 7. Samson, H. H. Photic stimulation and the evoked response in the avian visual system. Unpublished Ph.D. dissertation, University of Waterloo, 1968. 8. Suzuki, T. A., Y. Masuda and J. H. Jacobsen. Distinct spike discharges in the evoked cortical response of the light adapted cat. Vision Res, 7: 415-426, 1967. 9. Yonemura, D., K. Kawasaki, and Y. Tsuchida. Three-four differential vulnerability of the ERG components to pentobarbital. Proceedings of the 4th ISCERG symposium. Jap. J. Opt. Vol. 10 suppl., Tokyo, pp. 155-166, 1966.
BRIEF COMMUNICATION Effects of Pentobarbital on the Visual Evoked Response in the Avian Optic Tectum HERMAN
H. S A M S O N A N D L A W R E N C E L A V I N E
Department of Psychology, Rutgers University, New Brunswick, New Jersey, 08903, U.S.A. (Received 26 N o v e m b e r 1971) H. H. AND L. LAVINE. Effects of pentobarbital on the visual evoked response in the avian optic tectum. PHYSIOL. BErtAV. 8 (6) 1193-1196, 1972.--Pentobarbital sodium altered the effect of constant retinal illumination upon the photic evoked potential in the optic tectum of the pigeon. As the dose was increased, suppression by background illumination of the amplitude of the response decreased. With a dose level of 15 mg/kg, constant retinal illumination (background) potentiated rather than suppressed the response amplitudes. SAMSON,
Pentobarbital
Evoked potential
Visual evoked potential
ON~ oF many barbiturates which has been shown to affect the photic evoked potential is pentobarbital. Brazier [1 ] reported that the potentials were increased in size and were less complex following an anesthetic level dose. These results have been confirmed and elaborated by other investigators [6, 7, 8, 9]. In determining the effects of constant retinal illumination upon the photic evoked response, Heron and Samson [4] found a gross difference between the anesthetized and unanesthetized bird. In the unanesthetized animal, the response to the photic stimulus was reduced in amplitude (suppressed) by constant retinal illumination. However, in the same preparation, anesthetized with sodium pentobarbital (Nembutal, 25 mg/kg), the response was increased in amplitude (potentiated) by the presence of the same constant retinal illumination. Because of this reversal in effect of constant retinal illumination by pentobarbital, it was decided that a more complete study of the effects at various doses of pentobarbital was needed. This study reports the results for four dose levels on the change in the photic evoked response by constant background illumination. METHOD
Animals Five White Carneaux pigeons were maintained in individual cages with food and water freely available. The room was temperature controlled at 72°F ( ~ 5°), and illuminated by 24-hr fluorescent light.
Surgical Procedure Under sodium pentobarbital (Nembutal) anesthesia (25 mg/kg), the animal was placed in a Revzin headholder held 1193
by a K o p f stereotaxic instrument. Stainless steel electrodes insulated with an epoxy resin (Epoxylite Corp.) were implanted into the optic tectum at the level of the S G F (stratum griseum et fibrosum superficiale [5]). An indifferent electrode was placed into the frontal bone. Stainless steel screws and dental acrylic were used to anchor the electrodes. In addition, a small bolt and plastic rings around each eye were attached to the skull cap for placement of the head during recording of evoked potentials. Following surgery, all animals were allowed a one-month recovery period prior to the experiment.
Stimulus Apparatus In order to obtain repeated measures over a number of days, an apparatus was constructed so that the restrained bird could be presented monocularly with a flash which was superimposed upon a light or dark background. The evoking stimulus was delivered from a Grass PS-2 photo stimulator onto a translucent, white diffusion screen which had a 49 % transmittance factor. On the other side of this screen was a camera bellows (4 × 3 in.) with an aperture milled such that the plastic ring around the animal's eye would just fit. The distance of the flash head from one side of the screen was 14 in, and the length of the bellows on the other side was 6 in. The screen and bellows were arranged so that only the flash which was within the 3 x 4 in. opening of the bellows was viewed by the bird. With a flash setting of 4 on the photostimulator, the approximate intensity of the flash was determined as 1,950 ml (Gamma Scientific photometer, using a flash rate of 60 per sec). The constant background illumination was provided by a GE-47 car reading bulb ran at 0.5 amp with a DC 6 V source. It was placed 2 ft from the translucent screen and had an intensity of 8 ft-c as measured on the bird's side of the screen.
II 94
Recording Apparatus Evoked responses were amplified by a factor of 1,000 with a Grass P-15 preamplifier, monitored by a Tektronix 502 oscilloscope and led into a Digital Equipment Corporation Lab 8 computer. They were also stored on magnetic tape. The computer performed on-line averages which were used in the final data analysis.
SAMSON AND LAVINE removed and 50-~ frozen sections were taken. These were stained with thionin, and electrode placements were determined.
130-
Procedln'e The animal was restrained by wrapping in a cloth and the head positioned into the stimulus apparatus through the use of the bolt and eyepiece. All stimuli were presented monocularly, with the recording done from the contralateral tectum only. The bird was given a period of adaptation (15 rain) to both the physical restraint and to the level of light that was on the screen (same as the background condition). The rest of the room was dark. Following adaptation, ten sets of flashes were presented, consisting of fifty flashes per set delivered at a rate of one per second. Following each set, a 4-rain adaptation period was given. On half of the sets the background illumination was absent. The order of background on or off was random for each electrode location, but was kept the same at each location over sessions. This was done to evaluate order effect within treatment, as well as in a particular session. Four sessions per week were given with the following regime. On day one, the standard session was given (the presession). On the next day, the drug was administered 15 rain before the session began (the drug session). Then five hr after the drug was administered a third session was given (the 5-hr post-session). On the next day a 24-hr post-session was then given. Ten weeks of testing were conducted. All injections were in the intraperitoneal cavity. In the first two weeks each bird received a dose of isotonic saline (0.25 ml) during the drug session. In the third and fourth weeks the dose was 4 mg/kg pentobarbital, with the dose increased to 8 mg/kg in the fifth and sixth weeks, and to 15 mg/kg in the seventh and eighth weeks. For the last two weeks, a dose of 25 mg/kg was used. The use of an incremental dosage schedule minimized any tolerance effect. Within any week only one electrode location per bird was tested.
Data Analysis From the computer-averaged responses the largest negative and positive deflections were determined. An analysis was then performed by comparing the response amplitudes with and without background. By forming a ratio of the amplitude with background against the amplitude without background, an assessment of the suppression by background could be made, independent of change in absolute amplitude. This ratio is called the suppression ratio and is symbolized by B/NB. If there was no effect of background, this ratio was 1.00. As the response amplitudes were suppressed by background, the ratios approached zero. Ratios were computed for the positive peak, negative peak, and peak to peak amplitudes. These ratio scores were then used to compute a two-way analysis of variance [3] across drug dose and session variables.
l-listology At the conclusion of the study the animals were sacrificed and perfused with 1 0 ~ formal-saline. The brains were
i~ i
o----o
salve
: : 0----0 e--*-e o--.--o
4mg/kq
G mg/Itg |5mg/kg 25mg/kg
12.0 =
/
lie=
/
I00-
',\
,/ !' :/
go-
\0
,
0
,
\ \
X
0
//
80-
l-nc
",ix,
m Z
70-
60"
i
I
PRE
DRUG
| 5~r POST
I 2 4 nr POST
SESSION
FIG. 1. Drug dose-effect curves for B/NB ratios (mean data for all birds).
RESULTS
Figure 1 shows the mean results for all birds. Figure 2 shows the results of a single bird, illustrating that the group data are representative of the results for single animals (cf. Fig. 1). The control saline result shows that the injection process did not produce a change. Three points are evident in both Figures 1 and 2. One, the suppression by background is decreased as the dose of pentobarbital is increased up to 8 mg/kg pentobarbital. Two, in doses of 15 mg/kg or greater, the background does not suppress the photic evoked response, but rather potentiates it. Three, while the maximally effective potentiating dose used in this series was 15 mg/kg for the drug session, the largest dose (25 mg/kg) had the largest residual effect at the 5-hour post-session.
PENTOBARBITAL AND VISUAL EVOKED RESPONSE
o -o.---~0 e.---,.l o-.-.o
120" • it I° t 'l~'1 II0-
P
1195 the effect of the 15 mg/kg dose was seen to be slightly greater than the 25 mg/kg dose, but at the 5-hr post, the larger dose was the one with the largest effect. This would indicate that the 15 mg/kg dose produced a maximum effect, and the attenuation of the suppressive effect of the background by the larger dose (25 mg/kg) became evident at the 5-hr postsession, by which time the effective level of the other dosages had declined, There were no significant differences between all pre- and post-sessions or among any of the saline sessions. An analysis performed on the positive peak and the negative peak was found to be exactly the same as that for the total peak-to-peak scores. This indicates that the response components are not affected independent of one another. The histology showed all electrodes were within the S G F layer of the optic rectum.
SALINE 4 mglkg e mg/kg 15 mglkg 25 mg/l~9
tt
4\
P
~
I00 ~
b
90--
I ~J
c2_
l\
,
DISCUSSION
BO--
OE El Z -% m
I
l
\ ', .,, - ,
:;/ 70-
O0--
pRIr
DRUG
5h¢ ~
24hr Pg~T
SESSION
FIG. 2. Drug dose-effect curves for B/NB ratio (Bird 3, left tectum).
An analysis of variance was significant at greater than the 0.01 level for both drug and session variables. When specific means were tested against their appropriate control presessions, all drug session treatments were significantly changed. The significance levels were 0.05 for the 4 mg/kg dose and 0.01 for all other doses. The two 5-hour post-sessions that were significantly different from the saline control weie the values 15 mg/kg (0.05 level) and 25 mg/kg (0.01 level). At the drug session,
From the data it is clear that an anesthetic dosage of pentobarbital reverses the effect of constant retinal illumination upon the photic evoked response. Normally the response is suppressed, but under narcosis with pentobarbital the responses are actually potentiated. The effects of doses lower than those producing narcosis show a dose-dependent relation which indicates that the stlppressive effect of background illumination is antagonized in a graded fashion by increasing doses of pentobarbital. The maximum effect lies in the region of 15 mg/kg. A significant decrease in the amount of suppression can be found at the 4 mg/kg level, which shows little if any overt behavioral effect. The results confirmed the danger of using barbiturate anesthesia for the acute study of the photic evoked potential, which had been pointed out by earlier investigators [1, 2]. However, the dose-dependent effect found in this study might provide a method for the systematic determination of components of the evoked potential itself, as well as the effects of light adaptation upon the visual system. The change in the effect of the constant retinal illumination as a function of drug state may also offer a partial explanation for the conflicting results concerning the role of adaptation state on the photic evoked potential. However, further work employing a range of flash intensities, background illuminations, and other drugs is needed before this explanation could be considered. One point is clear: The interaction between the drug state and the stimulation state can be related not only to the evoking stimuli but also to the state of adaptation of the receptive system as determined by the background illumination conditions.
REFERENCES 1. Brazier, M. B. A. Studies of evoked responses by flash in man and cat. In: Reticular formation of the brain, edited by H. H. Jasper: Boston: Little Brown, 1958. 2. Bremer, F. Neurogenic influences on evoked potentials. In: Sensory communication, edited by W. A. Rosenblith, New York: Wiley, 1961. 3. Edwards, A. L. Experimental design in psychological research, New York: Holt, Rinehart and Winston, 1963, 4. Heron, W. and H. H. Samson. Some effects of perceptual isolation on the CNS. Paper presented at Eastern Psychological Association, Atlantic City, 1965. 5. Karten, H. J. and W. Hodos A Stereotaxic Atlas o f The Brain o f the Pigeon (Columba Livia), Baltimore: Johns Hopkins University Press, 1967.
6. Olds, M. E. and G. Baldrighi. Effects of meprobamate, chlordiazepoxide, diazapam, and sodium pentobarbitai on visually evoked responses in the tecto-tegmental area of the rat. Int. J. Neuropharmac. 7: 231-239, 1968. 7. Samson, H. H. Photic stimulation and the evoked response in the avian visual system. Unpublished Ph.D. dissertation, University of Waterloo, 1968. 8. Suzuki, T. A., Y. Masuda and J. H. Jacobsen. Distinct spike discharges in the evoked cortical response of the light adapted cat. Vision Res, 7: 415-426, 1967. 9. Yonemura, D., K. Kawasaki, and Y. Tsuchida. Three-four differential vulnerability of the ERG components to pentobarbital. Proceedings of the 4th ISCERG symposium. Jap. J. Opt. Vol. 10 suppl., Tokyo, pp. 155-166, 1966.