- Email: [email protected]
Retrograde amnesia produced by electrical stimulation of the amygdala: Attenuation with adrenergic antagonists
Bruin Research, 211(1981) 59-65 0 Elsevier/North-Holland Biomedical Press
RETROGRADE AMNESIA PRODUCED OF THE AMYGDALA: ATTENUATION ANTAGONISTS
DEBRA B. STERNBERG*
59
BY ELECTRICAL STIMULATION WITH ADRENERGIC
and PAUL E. GOLD**
Department of Psychology Gilmer Hall University of Virginia Charlottesville, Vu. 22901 (U.S.A.) (Accepted September l&h, 1980) Key words: retrograde amnesia - memory storage - amygdala ance training - catecholamines - memory
adrenergic antagonists -
avoid-
SUMMARY
Subseizure electrical stimulation of the amygdala produced retrograde amnesia for a visual discrimination shock-motivated task. Animals pretreated with the aadrenergic antagonist phenoxybenzamine, or the p-adrenergic antagonist propranolol, did not develop amnesia. The findings indicate that adrenergic antagonists attenuate amnesia produced by amygdala stimulation for visual discrimination training. These results are consistent with previous evidence indicating that adrenergic antagonists attenuate the amnesias produced by a variety of agents, and thus, suggest that adrenergic mechanisms may be involved in the production of retrograde amnesia.
INTRODUCTION
Many treatments, including electroconvulsive shock, convulsant drugs and direct electrical stimulation of specific brain regions, produce retrograde amnesiarO>ss. The findings of several studies indicate that subseizure electrical stimulation of discrete brain sites may be particularly effective in impairing later retentionsa~se~s*.In particular, post-training, subseizure amygdala stimulation can produce amnesia for many learned responses - i.e. for inhibitory (passive) avoidancesJrJ4*25, active avoidancess, visual discriminationr5 and taste aversion 1924training. Others have found that pharmacological manipulation of cholinergics4, adrenergicsJ@rJs, opiate* and protein synthesis2 systems in the amygdala also disrupt memory formation. * Present address: Department of Psychobiology, University of California, Irvine, Calif. 92717, U.S.A. ** To whom correspondence and reprint requests should be addressed.
60 A common interpretation of such findings is that the amygdala is one of the neuroanatomical substrates of learning and memory 2s,2s,32. According to this view, localized chemical or electrical stimulation impairs memory processing occurring at the electrode site or at a brain area anatomically coupled to the stimulation site. An alternative explanation
of the above data is that the amygdala
manipulations
may elicit widespread
brain changes that alter memory storage processing in many brain areas. For example, manipulations ofthe amygdala may elicit central or peripheral nervous system responses that contribute view, electrical
to the mechanisms that produce amnesia. Of relevance to this point of (or chemical) stimulation of the amygdala produces many alterations
in the autonomic nervous system. For example, amygdala stimulation appears to cause central and peripheral release of norepinephrine 7,s1 which may interfere with the processes involved in memory formation. If one pharmacologically attenuates the effect of excessive norepinephrine release, then one may attenuate the production of retrograde amnesia. Recently we demonstrated that phenoxybenzamine (PBZ), an a-adrenergic antagonist, attenuates amnesia produced by various treatments, including amygdala stimulationls. Since a single drug can block the memory-impairing actions of these various treatments, the results suggest that many, if not all, amnestic treatments may act through a common neurobiological mechanism. Thus, many treatments, including amygdala stimulation, may impair memory not through their putative ‘major’ action but by altering the activity of neuroendocrine systems shortly after training. The present experiment focusses on the use of adrenergic antagonists to attenuate retrograde amnesia produced by subseizure electrical stimulation of the amygdala. The findings indicate that peripheral injections of either an a-adrenergic antagonist, phenoxybenzamine, or a /3-adrenergic antagonist, propranolol, can attenuate amygdala stimulation-produced retrograde amnesia for visual discrimination escape training. METHODS
Male Sprague-Dawley rats (70-90 days old) weighing 250-300 g were used. Animals were individually housed upon arrival from the supplier (Flow Laboratories). The animals were maintained on a 12 h light-dark cycle (08.00 h on-20.00 h off) with ad libitum access to food and water. Surgery was performed under Nembutal anesthesia (45-50 mg/kg). Bipolar amygdala electrodes made of twisted stainless steel wire (250 pm) were implanted bilaterally in 62 rats. The wire was coated with enamel except at the tips. Stereotaxic coordinates were 0.8 mm posterior to bregma, +4.5 mm lateral to the midline, and 8.9 mm below the surface of the cortex 30. In addition, 4 stainless steel screws were placed in the skull over frontal (2.0 mm anterior to bregma, *2.0 mm lateral to the midline) and posterior (7.0 mm posterior to bregma, 12.0 mm lateral to the midline) cortex. The left frontal and right posterior screws and the bipolar electrodes were connected to leads from an Amphenol microminiature connector strip. The entire assembly was affixed to the skull with dental acrylic cement. Animals received i.p. injections of either saline, phenoxybenzamine (2 mg/kg,
61 0.2 mg/lOO g) or propranolol (0.5 mg/kg, 0.1 ml/100 g) 30 min prior to training. All drugs were dissolved in isotonic saline. In pilot experiments, we examined the doseresponse curves for the effects of these pre-trial injections on retention. For each drug in this experiment, we chose a dose known to block adrenergic function*,5*27Js that does not itself affect retention performancess. The groups of animals used were as follows: saline + stimulation (n = 14); saline + no stimulation (n = 11); phenoxybenzamine + stimulation (n = 9); phenoxybenzamine + no stimulation (n = 6); propranolol + stimulation (n = 8); propranolol + no stimulation (n = 8); and unoperated controls - saline + no stimulation (n = 6). Training began l-2 weeks following the completion of surgery. The animals were trained to escape footshock in a visual discrimination Y-maze. Each arm was a troughshaped alleyway 63 cm long x 13.5 cm high, and was covered with a dark translucent plexiglass roof. The width was 4.5 cm at floor level and 7.5 cm at the top. The inner walls and floor were constructed of two pairs of stainless steel plates each 3 1.2 cm long. The plates were separated by a 1.Ocm slit running lengthwise along the floor of the alleyway. The end wall of each arm was made of translucent white plexiglass behind which there was a dim light that could be illuminated. Each animal received 8 training trials. The rat was placed in an unlit (start) arm of the maze and after a 5 set interval, a 1mA footshock was delivered until the rat entered the (safe) lighted arm. If the animal did not enter the safe arm within 60 set, the shock was turned off. Animals that failed to reach the safe arm on two or more training trials were eliminated from the study. At the end of each trial, the rat was immediately removed from the apparatus (intertrial interval = 15 set). The safe (lighted) arm was alternated according to a set sequence that was random except that the same arm could not be correct on more than two consecutive trials. The turn at the first choice point was recorded as correct or incorrect for entry (four paws) into the lit or unlit area, respectively. Following training, the animal was connected to a stimulation and recording cable and was placed into a shielded recording chamber. Thirty seconds after the termination of the last training trial the animal received brain stimulation (30 PA/side, 100 Hz, 0.1 msec monophasic pulses, 10 set duration, monitored on an oscilloscope). Half of the rats served as implanted non-stimulated controls; they were treated exactly as animals in other groups except no stimulation was administered. Immediately after brain stimulation, the electrode leads were switched to a 4-channel Grass model 7 polygraph and electrographic activity was monitored for 60 sec. Following recording, the animal was returned to its home cage. Retention performance was assessed on a set of 8 training trials administered 24 h after completion of training. The difference between the number of correct responses to retraining (Day 2) and training (Day 1) was used as the measure of retention. Thus, high difference scores indicate good retention performance and low difference scores indicate poor retention performance (amnesia). All statistical comparisons of retention performance were made using two-tailed t-tests. At the conclusion of the experiment, animals with implanted electrodes were given an overdose of Nembutal and perfused intracardially with saline followed by 10% formalin solution. Their brains were removed and sectioned (40 pm thickness) to determine electrode tip placements.
Amygdala
UNOP
Stlmulatloii
SAL
PBZ
130 pAislde.
PROP
SAL
~2mg/kg1’05m-g/kgl
Pretralrltrlg
100Hz
PBZ ‘2vg/kg
li)sec
1
PROP .Jirnq/r
i
Treatmellt
Fig. 1. Mean ( f S.E.M.) difference scores (Day 2 - Day 1) for correct responses made by rats trained and tested (8 trials/day) in a visual discrimination shock-motivated task. Note that pretrial injections of either phenoxybenzamine (PBZ) or propranolol (PROP) did not themselves enhance retention performance but did attenuate the amnesia seen in the group that received pretrial saline injections and post-trial electrical stimulation of the amygdala.
RESULTS
Electrographic records indicated that no brain seizures occurred after stimulation. There were no apparent differences in the records of saline or drug-pretreated animals. The adrenergic antagonists, administered 30 min before training, had no effect on the number of correct choices during acquisition training (Day 1 means ranged from 3.61 $- 0.27 to 4.04 & 0.53). Retention performance, expressed in mean avoidance difference scores, is shown in Fig. 1. First, it should be noted that the avoidance difference scores of the saline + stimulation animals (mean = 0.29 f 0.29) were significantly lower than the scores of unimplanted and implanted, saline + no stimulation animals (means = 1.83 * 0.77 and 2.36 j, 0.39, respectively; Ps < 0.01). Second, phenoxybenzamine + no stimulation and propranolol +- no stimulation animals (means = 2.83 + 0.7 and 1.25 f 0.71, respectively) had scores which were not statistically different from those of saline + no stimulation animals. In contrast to the results obtained with the saline + stimulation group, the difference scores of propranolol -t stimulation and phenoxybenzamine + stimulation (means = 2.56 & 0.29 and 2.0 i 0.78, respectively) were not significantly different from the scores obtained with saline + no stimulation or drug + no stimulation animals. Furthermore, the propranolol f stimulation and phenoxybenzamine + stimulation difference scores were significantly higher than those of saline + stimulation animals (Ps < 0.01, for both comparisons). The findings of previous experiments indicate that amnesia is produced with bilateral amygdala stimulation without regard to the specific localization of the electrode
63 within the amugdalarsJ5. Similarly, in the present experiment, the electrode tip placements were not related to the extent of the deficits observed after stimulation. Also, in the implanted no-stimulation groups, populations of animals with electrodes in any amygdala region performed well during the retention testing. DISCUSSION
As reported previouslyls, post-trial subseizure electrical stimulation of the amygdala produced amnesia for visual discrimination training. The results presented here indicate that pretraining injections of either phenoxybenzamine or propranolol attenuates the amnesia produced by amygdala stimulation in this discrimination task. It is important to note that the attenuation does not appear to be the result of direct enhancement of retention performance by the adrenergic antagonists; in non-stimulated animals pretreated with the drugs, both acquisition and retention performance were comparable to that seen in saline-pretreated control animals. The present results are thus consistent with previous findings obtained with a one-trial inhibitory (passive) avoidance training 1s. With this task as well, phenoxybenzamine attenuated the retrograde amnesia produced by amygdala stimulation. Furthermore, phenoxybenzamine attenuated the amnesias produced by a convulsant drug, supraseizure electrical stimulation of frontal cortex, and norephinephrine and protein synthesis inhibitors. The observation that adrenergic antagonists attenuate retrograde amnesia for tasks that require either active or inhibitory responses suggest that the attenuation is not mediated by drug-induced alterations in motor performance at the time of training or testing. In the present study, both a- and /?-adrenergic antagonists were effective in blocking the amnesia produced by amygdala stimulation. Similarly, we recently found that amnesia produced by supraseizure electrical stimulation of frontal cortex is attenuated by several adrenergic antagonists (i.e. the a-antagonists phentolamine, piperoxane, and phenoxybenzamine, and the B-antagonist propranolol)33. Thus, it is evident that several adrenergic antagonists can attenuate the amnesias produced by several treatments. These findings therefore support the view that the amnesias produced by many classes of treatments may be mediated by common neurobiological mechanisms that include adrenergic involvement. With specific regard to amnesia produced by amygdala stimulation, there are two major interpretations of these findings. First, it is possible that the amygdalaitself plays an important role in memory storage processing, and it is rather easy to imagine that localized electrical stimulation disrupts patterned activity within this brain region. In its most general form, this view would include storage of specific information within the amygdala or amygdala-modulation of information stored elsewhere. The results obtained here with adrenergic antagonists seem consistent only with the latter view and then only if amygdala-modulation of memory processing is mediated by adrenergic systems. A second major interpretation of amygdala stimulation effects on memory is that this brain area is a particularly effective region from which to elicit autonomic responses and activity in whole brain noradrenergic systems ‘Jr. Treatments that result in altera-
64 tions in the post-trial
release of peripheral
and central epinephrine
and norepinephrine
have the capacity to modulate (enhance or impair) memory processing13J7-21. Thus, amygdala stimulation may elicit changes in the activity of peripheral and/or central adrenergic systems that result in amnesia. According to this view, the adrenergic antagonists would then act to attenuate the neurobiological consequences of this activity and thus attenuate
amnesia.
To examine
this possibility
further,
it would be useful to
study in detail several endogenous responses to training and to amygdala stimulation (as well as other amnestic agents) in the presence of adrenergic antagonists. Using such methods, it may be possible to define a relatively restricted set of neurobiological systems that are involved in retrograde amnesia. ACKNOWLEDGEMENT
This research
was supported
by USPHS
(NIMH)
Research
Grant
MH-31141.
REFERENCES 1 Arthur, J. B., Taste aversion learning is impaired by interpolated amygdaloid stimulation but not by posttraining amygdaloid stimulation, Behav. Biol., 13 (1975) 369-376. 2 Berman, R. F., Kesner, R. P. and Partlow, L. M., Passive avoidance impairment in rats following cycloheximide injection into the amygdala, Brain Research, 158 (1978) 171-188. 3 Bresnahan, E. and Routtenberg, A., Memory disruption by unilateral low level, sub-seizure stimulation of the medial amygdaloid nucleus, Physiol. Behav., 9 (1972) 513-525. 4 Brodie, B. B., Aronow, L. and Axelrod, J., The rate of dibenzyline in the body and the role of fat in its duration of action, J. Phnrmacol. exp. Ther., 201 (1954) 644-653. 5 Bylund, D. B., Charness, M. E. and Snyder, S. H., Beta adrenergic receptor labeling in intact animals with l~SI-hydroxybenzylpindolol, J. Pharmacol. exp. They., 201 (1977) 644-653. 6 Ellis, M. E. and Kesner, R. P., Norepinephrine injections into amygdala impair passive avoidance learning, Neurosci. Abstr., 4 (1978) 797. 7 Fuxe, K. and Gunne, L.-M., Depletion of the amine stores in brain catecholamine terminals on amygdaloid stimulation, Acta physiol. scund., 62 (1964) 493494. 8 Gallagher, M. and Kapp, B. S., Manipulation of opiate activity in the amygdala alters memory processes, tife Sci., 23 (1978) 1973-1977. 9 Gallagher, M., Kapp, B. S., Musty, R. E. and Driscoll, P. A., Memory formation: evidence for a specific neurochemical system in the amygdala, Science, 198 (1977) 4233425. 10 Glickman, S. E., Perserverative neural processes and consolidation of the memory trace, Psychol. Bull., 58 (1961) 218-233. 11 Goddard, G. V., Amygdaloid stimulation and learning in the rat, J. co,np. physiol. Psychol., 58 (1964) 23-30. 12 Gold, P. E., Macri, J. and McGaugh, J. L., Retrograde stimulation, Science, 179 (1973) 1343-1345.
13 Gold, P. E. and McGaugh, J. L., Endogenous Psychoneuroendocr.
in Reprod.,
amnesia gradients: Effects of direct cortical
modulators
of memory storage processes, Clin.
22 (1978) 25-46.
14 Gold, P. E., Rose, R. P. and Hankins, L. L., Retention impairment produced by unilateral amygdala implantation: reduction by posttrial amygdala stimulation, Behav. Biol., 22 (1978) 515-523.
15 Gold, P. E., Rose, R. P., Hankins, L. L. and Spanis, C. W., Impaired retention ofvisual discriminated escape training produced by subseizure amygdala stimulation, Bruin Research, 118 (1976) 73-85. 16 Gold, P. E. and Sternberg, D. B., Retrograde amnesia produced by several treatments: Evidence for a common neurobiological mechanism, Science, 201(1978) 367-369. 17 Gold, P. E. and van Buskirk, R. B., Enhancement of time-dependent memory processes with posttrial epinephrine injections, Behuv. Biol., 13 (1975) 145-153. 18 Gold, P. E. and van Buskirk, R. B., Effects of posttrial hormone injections on memory processes, Horm. Behuv., 7 (1976) 509-517.
65 19 Gold, P. E. and van Buskirk, R. B., Enhancement and impairment of memory processes with posttrial injections of adrenocorticotrophic hormone, Behav. Eiol., 16 (1976) 387-400. 20 Gold, P. E. and van Buskirk, R., Posttraining brain norepinephrine concentrations: correlation with retention performance of avoidance training and with peripheral epinephrine modulation of memory processing, Behav. Biol., 23 (1978) 509-520. 21 Gold, P. E. and van Buskirk, R. B., Effects of alpha- and beta-adrenergic receptor antagonists on posttrial epinephrine modulation of memory: relationship to posttraining brain norepinephrine concentrations, Behav. Biol., 24 (1978) 168-184. 22 Gold, P. E., Zornetzer, S. F. and McGaugh, J. L., Electrical stimulation of the brain: Effects on memory storage. In G. Newton and A. Riesen (Eds.), Advunces in Psychobiology, Vol. 2, Wiley Interscience, New York, 1974, pp. 64-75. 23 Handwerker, M. J., Gold, P. E. and McGaugb, J. L., Impairment of active avoidance learning with posttraining amygdala stimulation, Bruin Research, 75 (1974) 324-327. 24 Kesner, R. P., Berman, R. F., Burton, B. and Hankins, W. G., Effects of electrical stimulation of amygdala upon neophobia and taste aversion, Behav. Biol., 13 (1975) 349-358. 25 Kesner, R. P. and Conner, H. S., Effects of electrical stimulation of rat limbic system and midbrain reticular formation upon short- and long-term memory, Physiol. Behav., 12 (1974) 5-12. 26 Kesner, R. P. and Wilburn, M. W., A review of electrical stimulation of the brain in the context of learning and retention, Behuv. Biol., 10 (1974) 259-293. 27 Masouka, D., Appelgren, L. E. and Hansson, E., Autoradiographic distribution studies of adrenergic blocking agents. I. r4C-phenoxybenzamine (bensylyt NFN), an a-receptor blocking agent, Actuphurmucol. (Kbh.), 25 (1967) 113-122. 28 McGaugh, J. L. and Gold, P. E., Modulation of memory processes with electrical stimulation of the brain. In M. R. Rosenzweig and E. L. Bennett (Eds.), Neural Mechanisms ofLearning and Memory, MIT Press, Cambridge, Mass., 1976, pp. 549-560. 29 McGaugh, J. L. and Herz, M. J., Memory Consolidution, Albion, San Francsico, 1972. 30 Pellegrino, L. J. and Cushman, A. J., A Stereotuxic Atlas of the Rut Bruin, Meredith, New York, 1967. 31 Reis, D. J. and Gunne, L.-M., Brain catecholamines: relation to the defense reaction evoked by amygdaloid stimulation in the cat, Science, 149 (1965) 450451. 32 Routtenberg, A. and Holzman, N., Memory disruption by electrical stimulation of substantia nigra, pars compacta, Science, 181 (1973) 83-86. 33 Sternberg, D. B. and Gold, P. E., Effects of a- and p-adrenergic receptor antagonists on retrograde amnesia produced by frontal cortex stimulation, Behuv. neural Biol., 29 (1980) 289-302. 34 Todd, J. W. and Kesner, R. P., Effects of posttraining injection of cholinergic agonists and antagonists into the amygdala on retention of passive avoidance training in rats, J. comp.physiol. Psycho& Psychol., 92 (1978) 958-968. 35 U’Prichard, D. C., Greenberg,
D. A. and Snyder, S. H., Binding characteristics of a radiolabeled agonist and antagonist at central nervous system alpha noradrenergic receptors, Molec. Phurmucol.,
13 (1977) 454-473. 36 Zetumer, H. G., Musty, R. E., Kapp, B. S. and Driscoll, P. A., Effect of noradrenergic blockade in the amygdala on retention for passive avoidance conditioning, Neurosci. Abstr., 2 (1976) 653.
RETROGRADE AMNESIA PRODUCED OF THE AMYGDALA: ATTENUATION ANTAGONISTS
DEBRA B. STERNBERG*
59
BY ELECTRICAL STIMULATION WITH ADRENERGIC
and PAUL E. GOLD**
Department of Psychology Gilmer Hall University of Virginia Charlottesville, Vu. 22901 (U.S.A.) (Accepted September l&h, 1980) Key words: retrograde amnesia - memory storage - amygdala ance training - catecholamines - memory
adrenergic antagonists -
avoid-
SUMMARY
Subseizure electrical stimulation of the amygdala produced retrograde amnesia for a visual discrimination shock-motivated task. Animals pretreated with the aadrenergic antagonist phenoxybenzamine, or the p-adrenergic antagonist propranolol, did not develop amnesia. The findings indicate that adrenergic antagonists attenuate amnesia produced by amygdala stimulation for visual discrimination training. These results are consistent with previous evidence indicating that adrenergic antagonists attenuate the amnesias produced by a variety of agents, and thus, suggest that adrenergic mechanisms may be involved in the production of retrograde amnesia.
INTRODUCTION
Many treatments, including electroconvulsive shock, convulsant drugs and direct electrical stimulation of specific brain regions, produce retrograde amnesiarO>ss. The findings of several studies indicate that subseizure electrical stimulation of discrete brain sites may be particularly effective in impairing later retentionsa~se~s*.In particular, post-training, subseizure amygdala stimulation can produce amnesia for many learned responses - i.e. for inhibitory (passive) avoidancesJrJ4*25, active avoidancess, visual discriminationr5 and taste aversion 1924training. Others have found that pharmacological manipulation of cholinergics4, adrenergicsJ@rJs, opiate* and protein synthesis2 systems in the amygdala also disrupt memory formation. * Present address: Department of Psychobiology, University of California, Irvine, Calif. 92717, U.S.A. ** To whom correspondence and reprint requests should be addressed.
60 A common interpretation of such findings is that the amygdala is one of the neuroanatomical substrates of learning and memory 2s,2s,32. According to this view, localized chemical or electrical stimulation impairs memory processing occurring at the electrode site or at a brain area anatomically coupled to the stimulation site. An alternative explanation
of the above data is that the amygdala
manipulations
may elicit widespread
brain changes that alter memory storage processing in many brain areas. For example, manipulations ofthe amygdala may elicit central or peripheral nervous system responses that contribute view, electrical
to the mechanisms that produce amnesia. Of relevance to this point of (or chemical) stimulation of the amygdala produces many alterations
in the autonomic nervous system. For example, amygdala stimulation appears to cause central and peripheral release of norepinephrine 7,s1 which may interfere with the processes involved in memory formation. If one pharmacologically attenuates the effect of excessive norepinephrine release, then one may attenuate the production of retrograde amnesia. Recently we demonstrated that phenoxybenzamine (PBZ), an a-adrenergic antagonist, attenuates amnesia produced by various treatments, including amygdala stimulationls. Since a single drug can block the memory-impairing actions of these various treatments, the results suggest that many, if not all, amnestic treatments may act through a common neurobiological mechanism. Thus, many treatments, including amygdala stimulation, may impair memory not through their putative ‘major’ action but by altering the activity of neuroendocrine systems shortly after training. The present experiment focusses on the use of adrenergic antagonists to attenuate retrograde amnesia produced by subseizure electrical stimulation of the amygdala. The findings indicate that peripheral injections of either an a-adrenergic antagonist, phenoxybenzamine, or a /3-adrenergic antagonist, propranolol, can attenuate amygdala stimulation-produced retrograde amnesia for visual discrimination escape training. METHODS
Male Sprague-Dawley rats (70-90 days old) weighing 250-300 g were used. Animals were individually housed upon arrival from the supplier (Flow Laboratories). The animals were maintained on a 12 h light-dark cycle (08.00 h on-20.00 h off) with ad libitum access to food and water. Surgery was performed under Nembutal anesthesia (45-50 mg/kg). Bipolar amygdala electrodes made of twisted stainless steel wire (250 pm) were implanted bilaterally in 62 rats. The wire was coated with enamel except at the tips. Stereotaxic coordinates were 0.8 mm posterior to bregma, +4.5 mm lateral to the midline, and 8.9 mm below the surface of the cortex 30. In addition, 4 stainless steel screws were placed in the skull over frontal (2.0 mm anterior to bregma, *2.0 mm lateral to the midline) and posterior (7.0 mm posterior to bregma, 12.0 mm lateral to the midline) cortex. The left frontal and right posterior screws and the bipolar electrodes were connected to leads from an Amphenol microminiature connector strip. The entire assembly was affixed to the skull with dental acrylic cement. Animals received i.p. injections of either saline, phenoxybenzamine (2 mg/kg,
61 0.2 mg/lOO g) or propranolol (0.5 mg/kg, 0.1 ml/100 g) 30 min prior to training. All drugs were dissolved in isotonic saline. In pilot experiments, we examined the doseresponse curves for the effects of these pre-trial injections on retention. For each drug in this experiment, we chose a dose known to block adrenergic function*,5*27Js that does not itself affect retention performancess. The groups of animals used were as follows: saline + stimulation (n = 14); saline + no stimulation (n = 11); phenoxybenzamine + stimulation (n = 9); phenoxybenzamine + no stimulation (n = 6); propranolol + stimulation (n = 8); propranolol + no stimulation (n = 8); and unoperated controls - saline + no stimulation (n = 6). Training began l-2 weeks following the completion of surgery. The animals were trained to escape footshock in a visual discrimination Y-maze. Each arm was a troughshaped alleyway 63 cm long x 13.5 cm high, and was covered with a dark translucent plexiglass roof. The width was 4.5 cm at floor level and 7.5 cm at the top. The inner walls and floor were constructed of two pairs of stainless steel plates each 3 1.2 cm long. The plates were separated by a 1.Ocm slit running lengthwise along the floor of the alleyway. The end wall of each arm was made of translucent white plexiglass behind which there was a dim light that could be illuminated. Each animal received 8 training trials. The rat was placed in an unlit (start) arm of the maze and after a 5 set interval, a 1mA footshock was delivered until the rat entered the (safe) lighted arm. If the animal did not enter the safe arm within 60 set, the shock was turned off. Animals that failed to reach the safe arm on two or more training trials were eliminated from the study. At the end of each trial, the rat was immediately removed from the apparatus (intertrial interval = 15 set). The safe (lighted) arm was alternated according to a set sequence that was random except that the same arm could not be correct on more than two consecutive trials. The turn at the first choice point was recorded as correct or incorrect for entry (four paws) into the lit or unlit area, respectively. Following training, the animal was connected to a stimulation and recording cable and was placed into a shielded recording chamber. Thirty seconds after the termination of the last training trial the animal received brain stimulation (30 PA/side, 100 Hz, 0.1 msec monophasic pulses, 10 set duration, monitored on an oscilloscope). Half of the rats served as implanted non-stimulated controls; they were treated exactly as animals in other groups except no stimulation was administered. Immediately after brain stimulation, the electrode leads were switched to a 4-channel Grass model 7 polygraph and electrographic activity was monitored for 60 sec. Following recording, the animal was returned to its home cage. Retention performance was assessed on a set of 8 training trials administered 24 h after completion of training. The difference between the number of correct responses to retraining (Day 2) and training (Day 1) was used as the measure of retention. Thus, high difference scores indicate good retention performance and low difference scores indicate poor retention performance (amnesia). All statistical comparisons of retention performance were made using two-tailed t-tests. At the conclusion of the experiment, animals with implanted electrodes were given an overdose of Nembutal and perfused intracardially with saline followed by 10% formalin solution. Their brains were removed and sectioned (40 pm thickness) to determine electrode tip placements.
Amygdala
UNOP
Stlmulatloii
SAL
PBZ
130 pAislde.
PROP
SAL
~2mg/kg1’05m-g/kgl
Pretralrltrlg
100Hz
PBZ ‘2vg/kg
li)sec
1
PROP .Jirnq/r
i
Treatmellt
Fig. 1. Mean ( f S.E.M.) difference scores (Day 2 - Day 1) for correct responses made by rats trained and tested (8 trials/day) in a visual discrimination shock-motivated task. Note that pretrial injections of either phenoxybenzamine (PBZ) or propranolol (PROP) did not themselves enhance retention performance but did attenuate the amnesia seen in the group that received pretrial saline injections and post-trial electrical stimulation of the amygdala.
RESULTS
Electrographic records indicated that no brain seizures occurred after stimulation. There were no apparent differences in the records of saline or drug-pretreated animals. The adrenergic antagonists, administered 30 min before training, had no effect on the number of correct choices during acquisition training (Day 1 means ranged from 3.61 $- 0.27 to 4.04 & 0.53). Retention performance, expressed in mean avoidance difference scores, is shown in Fig. 1. First, it should be noted that the avoidance difference scores of the saline + stimulation animals (mean = 0.29 f 0.29) were significantly lower than the scores of unimplanted and implanted, saline + no stimulation animals (means = 1.83 * 0.77 and 2.36 j, 0.39, respectively; Ps < 0.01). Second, phenoxybenzamine + no stimulation and propranolol +- no stimulation animals (means = 2.83 + 0.7 and 1.25 f 0.71, respectively) had scores which were not statistically different from those of saline + no stimulation animals. In contrast to the results obtained with the saline + stimulation group, the difference scores of propranolol -t stimulation and phenoxybenzamine + stimulation (means = 2.56 & 0.29 and 2.0 i 0.78, respectively) were not significantly different from the scores obtained with saline + no stimulation or drug + no stimulation animals. Furthermore, the propranolol f stimulation and phenoxybenzamine + stimulation difference scores were significantly higher than those of saline + stimulation animals (Ps < 0.01, for both comparisons). The findings of previous experiments indicate that amnesia is produced with bilateral amygdala stimulation without regard to the specific localization of the electrode
63 within the amugdalarsJ5. Similarly, in the present experiment, the electrode tip placements were not related to the extent of the deficits observed after stimulation. Also, in the implanted no-stimulation groups, populations of animals with electrodes in any amygdala region performed well during the retention testing. DISCUSSION
As reported previouslyls, post-trial subseizure electrical stimulation of the amygdala produced amnesia for visual discrimination training. The results presented here indicate that pretraining injections of either phenoxybenzamine or propranolol attenuates the amnesia produced by amygdala stimulation in this discrimination task. It is important to note that the attenuation does not appear to be the result of direct enhancement of retention performance by the adrenergic antagonists; in non-stimulated animals pretreated with the drugs, both acquisition and retention performance were comparable to that seen in saline-pretreated control animals. The present results are thus consistent with previous findings obtained with a one-trial inhibitory (passive) avoidance training 1s. With this task as well, phenoxybenzamine attenuated the retrograde amnesia produced by amygdala stimulation. Furthermore, phenoxybenzamine attenuated the amnesias produced by a convulsant drug, supraseizure electrical stimulation of frontal cortex, and norephinephrine and protein synthesis inhibitors. The observation that adrenergic antagonists attenuate retrograde amnesia for tasks that require either active or inhibitory responses suggest that the attenuation is not mediated by drug-induced alterations in motor performance at the time of training or testing. In the present study, both a- and /?-adrenergic antagonists were effective in blocking the amnesia produced by amygdala stimulation. Similarly, we recently found that amnesia produced by supraseizure electrical stimulation of frontal cortex is attenuated by several adrenergic antagonists (i.e. the a-antagonists phentolamine, piperoxane, and phenoxybenzamine, and the B-antagonist propranolol)33. Thus, it is evident that several adrenergic antagonists can attenuate the amnesias produced by several treatments. These findings therefore support the view that the amnesias produced by many classes of treatments may be mediated by common neurobiological mechanisms that include adrenergic involvement. With specific regard to amnesia produced by amygdala stimulation, there are two major interpretations of these findings. First, it is possible that the amygdalaitself plays an important role in memory storage processing, and it is rather easy to imagine that localized electrical stimulation disrupts patterned activity within this brain region. In its most general form, this view would include storage of specific information within the amygdala or amygdala-modulation of information stored elsewhere. The results obtained here with adrenergic antagonists seem consistent only with the latter view and then only if amygdala-modulation of memory processing is mediated by adrenergic systems. A second major interpretation of amygdala stimulation effects on memory is that this brain area is a particularly effective region from which to elicit autonomic responses and activity in whole brain noradrenergic systems ‘Jr. Treatments that result in altera-
64 tions in the post-trial
release of peripheral
and central epinephrine
and norepinephrine
have the capacity to modulate (enhance or impair) memory processing13J7-21. Thus, amygdala stimulation may elicit changes in the activity of peripheral and/or central adrenergic systems that result in amnesia. According to this view, the adrenergic antagonists would then act to attenuate the neurobiological consequences of this activity and thus attenuate
amnesia.
To examine
this possibility
further,
it would be useful to
study in detail several endogenous responses to training and to amygdala stimulation (as well as other amnestic agents) in the presence of adrenergic antagonists. Using such methods, it may be possible to define a relatively restricted set of neurobiological systems that are involved in retrograde amnesia. ACKNOWLEDGEMENT
This research
was supported
by USPHS
(NIMH)
Research
Grant
MH-31141.
REFERENCES 1 Arthur, J. B., Taste aversion learning is impaired by interpolated amygdaloid stimulation but not by posttraining amygdaloid stimulation, Behav. Biol., 13 (1975) 369-376. 2 Berman, R. F., Kesner, R. P. and Partlow, L. M., Passive avoidance impairment in rats following cycloheximide injection into the amygdala, Brain Research, 158 (1978) 171-188. 3 Bresnahan, E. and Routtenberg, A., Memory disruption by unilateral low level, sub-seizure stimulation of the medial amygdaloid nucleus, Physiol. Behav., 9 (1972) 513-525. 4 Brodie, B. B., Aronow, L. and Axelrod, J., The rate of dibenzyline in the body and the role of fat in its duration of action, J. Phnrmacol. exp. Ther., 201 (1954) 644-653. 5 Bylund, D. B., Charness, M. E. and Snyder, S. H., Beta adrenergic receptor labeling in intact animals with l~SI-hydroxybenzylpindolol, J. Pharmacol. exp. They., 201 (1977) 644-653. 6 Ellis, M. E. and Kesner, R. P., Norepinephrine injections into amygdala impair passive avoidance learning, Neurosci. Abstr., 4 (1978) 797. 7 Fuxe, K. and Gunne, L.-M., Depletion of the amine stores in brain catecholamine terminals on amygdaloid stimulation, Acta physiol. scund., 62 (1964) 493494. 8 Gallagher, M. and Kapp, B. S., Manipulation of opiate activity in the amygdala alters memory processes, tife Sci., 23 (1978) 1973-1977. 9 Gallagher, M., Kapp, B. S., Musty, R. E. and Driscoll, P. A., Memory formation: evidence for a specific neurochemical system in the amygdala, Science, 198 (1977) 4233425. 10 Glickman, S. E., Perserverative neural processes and consolidation of the memory trace, Psychol. Bull., 58 (1961) 218-233. 11 Goddard, G. V., Amygdaloid stimulation and learning in the rat, J. co,np. physiol. Psychol., 58 (1964) 23-30. 12 Gold, P. E., Macri, J. and McGaugh, J. L., Retrograde stimulation, Science, 179 (1973) 1343-1345.
13 Gold, P. E. and McGaugh, J. L., Endogenous Psychoneuroendocr.
in Reprod.,
amnesia gradients: Effects of direct cortical
modulators
of memory storage processes, Clin.
22 (1978) 25-46.
14 Gold, P. E., Rose, R. P. and Hankins, L. L., Retention impairment produced by unilateral amygdala implantation: reduction by posttrial amygdala stimulation, Behav. Biol., 22 (1978) 515-523.
15 Gold, P. E., Rose, R. P., Hankins, L. L. and Spanis, C. W., Impaired retention ofvisual discriminated escape training produced by subseizure amygdala stimulation, Bruin Research, 118 (1976) 73-85. 16 Gold, P. E. and Sternberg, D. B., Retrograde amnesia produced by several treatments: Evidence for a common neurobiological mechanism, Science, 201(1978) 367-369. 17 Gold, P. E. and van Buskirk, R. B., Enhancement of time-dependent memory processes with posttrial epinephrine injections, Behuv. Biol., 13 (1975) 145-153. 18 Gold, P. E. and van Buskirk, R. B., Effects of posttrial hormone injections on memory processes, Horm. Behuv., 7 (1976) 509-517.
65 19 Gold, P. E. and van Buskirk, R. B., Enhancement and impairment of memory processes with posttrial injections of adrenocorticotrophic hormone, Behav. Eiol., 16 (1976) 387-400. 20 Gold, P. E. and van Buskirk, R., Posttraining brain norepinephrine concentrations: correlation with retention performance of avoidance training and with peripheral epinephrine modulation of memory processing, Behav. Biol., 23 (1978) 509-520. 21 Gold, P. E. and van Buskirk, R. B., Effects of alpha- and beta-adrenergic receptor antagonists on posttrial epinephrine modulation of memory: relationship to posttraining brain norepinephrine concentrations, Behav. Biol., 24 (1978) 168-184. 22 Gold, P. E., Zornetzer, S. F. and McGaugh, J. L., Electrical stimulation of the brain: Effects on memory storage. In G. Newton and A. Riesen (Eds.), Advunces in Psychobiology, Vol. 2, Wiley Interscience, New York, 1974, pp. 64-75. 23 Handwerker, M. J., Gold, P. E. and McGaugb, J. L., Impairment of active avoidance learning with posttraining amygdala stimulation, Bruin Research, 75 (1974) 324-327. 24 Kesner, R. P., Berman, R. F., Burton, B. and Hankins, W. G., Effects of electrical stimulation of amygdala upon neophobia and taste aversion, Behav. Biol., 13 (1975) 349-358. 25 Kesner, R. P. and Conner, H. S., Effects of electrical stimulation of rat limbic system and midbrain reticular formation upon short- and long-term memory, Physiol. Behav., 12 (1974) 5-12. 26 Kesner, R. P. and Wilburn, M. W., A review of electrical stimulation of the brain in the context of learning and retention, Behuv. Biol., 10 (1974) 259-293. 27 Masouka, D., Appelgren, L. E. and Hansson, E., Autoradiographic distribution studies of adrenergic blocking agents. I. r4C-phenoxybenzamine (bensylyt NFN), an a-receptor blocking agent, Actuphurmucol. (Kbh.), 25 (1967) 113-122. 28 McGaugh, J. L. and Gold, P. E., Modulation of memory processes with electrical stimulation of the brain. In M. R. Rosenzweig and E. L. Bennett (Eds.), Neural Mechanisms ofLearning and Memory, MIT Press, Cambridge, Mass., 1976, pp. 549-560. 29 McGaugh, J. L. and Herz, M. J., Memory Consolidution, Albion, San Francsico, 1972. 30 Pellegrino, L. J. and Cushman, A. J., A Stereotuxic Atlas of the Rut Bruin, Meredith, New York, 1967. 31 Reis, D. J. and Gunne, L.-M., Brain catecholamines: relation to the defense reaction evoked by amygdaloid stimulation in the cat, Science, 149 (1965) 450451. 32 Routtenberg, A. and Holzman, N., Memory disruption by electrical stimulation of substantia nigra, pars compacta, Science, 181 (1973) 83-86. 33 Sternberg, D. B. and Gold, P. E., Effects of a- and p-adrenergic receptor antagonists on retrograde amnesia produced by frontal cortex stimulation, Behuv. neural Biol., 29 (1980) 289-302. 34 Todd, J. W. and Kesner, R. P., Effects of posttraining injection of cholinergic agonists and antagonists into the amygdala on retention of passive avoidance training in rats, J. comp.physiol. Psycho& Psychol., 92 (1978) 958-968. 35 U’Prichard, D. C., Greenberg,
D. A. and Snyder, S. H., Binding characteristics of a radiolabeled agonist and antagonist at central nervous system alpha noradrenergic receptors, Molec. Phurmucol.,
13 (1977) 454-473. 36 Zetumer, H. G., Musty, R. E., Kapp, B. S. and Driscoll, P. A., Effect of noradrenergic blockade in the amygdala on retention for passive avoidance conditioning, Neurosci. Abstr., 2 (1976) 653.