Physiology & Behavior, Vol. 23, pp. 341-346. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Saccharin Aversion Memory in Rats: Inhibition of Cycloheximide-Resistant Memory by Ouabain A. R. T U C K E R A N D M. E. G I B B S
Department o f Psychology, La Trobe University, Bundoora, Victoria, Australia 3083 Received 3 March 1979 TUCKER, A. R. AND M. E. GIBBS. Saccharin aversion memory in rats: inhibition of cycloheximide-resistant memory by ouabain. PHYSIOL. BEHAV. 23(2)341-346, 1979.--Memory for saccharin aversion learning in rats was shown to persist for at least one hour when cerebral protein synthesis was inhibited by cyeiobeximide. By 4 hours after learning the level of retention had fallen to the amnesic levels seen 24 hours later. The anmesia seen at 24 hours was found to be dose dependent and a mass of 300/~g, administered intraventricularly, was adequate to produce maximum amnesia. When the labile memory inhibitor ouabain was administered 15 rain before saccharin and LiCl, amnesia was seen in some rats at I hr, at the time when cyclobeximide-insusceptible memory was still present. These results compare favorably to those seen in day-old-chickens where a sequentially dependent 3 phase model of memory has been formulated. Cyeloheximide
Ouabain
Memoryinhibition
Rats
THERE is considerable experimental evidence for the involvement of protein synthesis in the establishment of longterm memories [1, 2, 10, 17]. This evidence comes from a variety of species and learning tasks and from an increasing number of protein synthesis inhibitors. The amnesia which occurs as a resuR of protein synthesis inhibition before or soon after learning, does not develop immediately. There is a period of half an hour to 3 hr [3, 6, 9] during which time memory retention appears normal despite the absence of brain protein synthesis. The variation in the duration of this protein synthesis-independent memory may reflect differences in species and/or training procedures. In the young chicken this protein synthesis-indepondent memory has been separated pharmacologically into two phases [10]. The memory for a discriminated avoidance task which is not interfered with by protein synthesis inhibitors lasts for 30 rain and can be interrupted by two different classes of drugs. Memory decay occurs after 10 min when sodium pump activity (Na/K ATPase) is inhibited by ouabain or ethacrynic acid at the time of learning, implying a phase of memory which is not interfered with by these drugs. This 10 rain memory, in turn, can be abolished by drugs which alter neuronal membrane potentials, for example by monosodium glutamate or potassium chloride [10]. This information, along with data from the effective time of injection of the different drugs, provided the basis for a model of memory formation in which it is suggested that there are three different stales in memory development: short-term memory inhibited by glutamate; labile memory, inhibited by ouabaln; and long-term memory (LTM) inhibited by protein synthesis inhibitors. Labile memory represents a phase which can be modulated negatively or positively by manipulating sodium pump activity.
Saccharinaversion memory
The work reported in this paper was addressed to the question of the existence of a protein synthesis-independent phase of memory in rats and whether such a memory phase can be disrupted by inhibiting sodium pump activity. We also report the effect of systematic variation in dose of the protein synthesis inhibitor cycloheximide (CXM) on the degree of amnesia seen 24 hours after training. Memory was assessed in a discrimination test for a learned taste aversion [19]. In this task rats learn to avoid a novel and preferred tasting fluid (saccharin) after one pairing with a toxin (lithium chloride). GENERAL METHOD The apparatus and the behavioural and surgical procedures adopted have been described in detail previously
[18,19]. Animals Experimentally naive male and female hooded rats and male albino rats, aged 90-110 days were used. These animals were bred in the Department of Psychology, La Trobe University, and were maintained on ad lib food and water until 24 hr before the first pretraining day when water was removed. Apparatus
Rats were pretrained, trained and tested in a small clear plastic box on one wall of which were attached two graduated cylinders fitted with stainless steel drinking spouts. The wire gauze floor of the box and the spouts were connected to an electronic circuit which counted tongue con-
Copyright © 1979 Brain Research Publications Inc.--0031-9384/79/080341-06501.10/1
342
T U C K E R AND GIBBS
tacts with the spouts. The clear plastic box was housed in a larger sound-attenuating box, fitted with a one-way mirror door.
Drugs Cycloheximide (Actidione, Upjohn Co.) and ouabain octahydrate (Sigma) were dissolved in sterile 0.15 M NaCI just prior to use.
Behavioural Procedure In all experiments there were three stages: pretraining, training and testing. The pretraining stage (Days 1-3) consisted of placing the rats in the experimental box for 10 rain each day and training them to drink water from both cylinders. In the training trial (Day 4) all rats were offered a 0.1% (w/v) solution of sodium saccharin instead of water in both cylinders. Immediately after this saccharin drinking session each rat was given an intragastric (IG) injection of 0.15 M LiC1. Rats in Experiment 1 were allowed to drink saccharin for 10 rain (approx. 10 ml) and were given a 10 ml/kg dose of LiCI. Rats in Experiments 2 and 3 were allowed only a 5 rain (approx.) drink of saccharin (approximately 5 ml) and were given a 5 ml/kg dose of LiC1. Care was taken to ensure that the volume of saccharin drunk in training trials was comparable between treatment groups within any given experiment. In the test trial all rats were returned to the experimental box and offered saccharin in one cylinder and water in the other. The positions of the water and saccharin cylinders were varied randomly. In Experiment 1 the test trials were of 10 rain duration and were conducted 24 hours after training, but in Experiment 2 and 3 test trials were of 5 rain duration and were conducted 1, 2 or 4 hours after training on Day 4. At the conclusion of the test trial a saccharin aversion (SA) score was computed for each rat by the formula: SA score = number of water ticks × 100 total number of licks Hence a SA score approaching 100 indicates excellent memory whilst a SA score near zero indicates amnesia/no learning. In order to ensure that SA scores actually reflected taste discriminations no data were included from any rat which did not lick at both spouts. (Approximately 5% of all rats trained were rejected on this a priori criterion).
Surgical Procedure lntraventricular administration of drug or isotonic saline was accomplished by freehand injection [18,19]. Briefly, the rat was anaesthetized with ether and placed on a small animal rack. After incising the scalp a small hole was bored through the cranial bone over the left lateral ventricle. Thirty microlitres of solution were then expelled into the ventricle using a Hamilton dispenser and a 500 t~l syringe fitted with a stopped 26 ga needle. The hole was plugged with sterile bone wax and the wound sutured with skin clips. The entire operation usually took 3-4 min and the animals were fully recovered from the anaesthetic within 15 min. E X P E R I M E N T 1: T H E E F F E C T O F V A R I A T I O N O F CXM DOSE ON 24 H O U R A M N E S I A This experiment sought to determine the effect of systematic variation in dose of intraventricular CXM on saccharin aversion memory tested at 24 hr.
METHOD
Seventy male hooded rats were randomly assigned to one of 5 equal-sized groups at the end ofpretraining. Seven hours prior to the training trial all rats received an intraventricular injection of either 400, 300, 200, 100 or 0/~g of CXM in 30/zl of 0.15 M NaCI. A coding procedure ensured that these treatments were administered blind and the experimenter did not know the treatment condition of the rats during training or testing. Retention tests were conducted 24 hours after training. RESULTS AND DISCUSSION
A Kruskal-Wallis H test showed that there was a highly significant dose effect of CXM on 24 hr SA score (H=29.77, df=4, p
Saccharin Aversion Memory in Rats: Inhibition of Cycloheximide-Resistant Memory by Ouabain A. R. T U C K E R A N D M. E. G I B B S
Department o f Psychology, La Trobe University, Bundoora, Victoria, Australia 3083 Received 3 March 1979 TUCKER, A. R. AND M. E. GIBBS. Saccharin aversion memory in rats: inhibition of cycloheximide-resistant memory by ouabain. PHYSIOL. BEHAV. 23(2)341-346, 1979.--Memory for saccharin aversion learning in rats was shown to persist for at least one hour when cerebral protein synthesis was inhibited by cyeiobeximide. By 4 hours after learning the level of retention had fallen to the amnesic levels seen 24 hours later. The anmesia seen at 24 hours was found to be dose dependent and a mass of 300/~g, administered intraventricularly, was adequate to produce maximum amnesia. When the labile memory inhibitor ouabain was administered 15 rain before saccharin and LiCl, amnesia was seen in some rats at I hr, at the time when cyclobeximide-insusceptible memory was still present. These results compare favorably to those seen in day-old-chickens where a sequentially dependent 3 phase model of memory has been formulated. Cyeloheximide
Ouabain
Memoryinhibition
Rats
THERE is considerable experimental evidence for the involvement of protein synthesis in the establishment of longterm memories [1, 2, 10, 17]. This evidence comes from a variety of species and learning tasks and from an increasing number of protein synthesis inhibitors. The amnesia which occurs as a resuR of protein synthesis inhibition before or soon after learning, does not develop immediately. There is a period of half an hour to 3 hr [3, 6, 9] during which time memory retention appears normal despite the absence of brain protein synthesis. The variation in the duration of this protein synthesis-independent memory may reflect differences in species and/or training procedures. In the young chicken this protein synthesis-indepondent memory has been separated pharmacologically into two phases [10]. The memory for a discriminated avoidance task which is not interfered with by protein synthesis inhibitors lasts for 30 rain and can be interrupted by two different classes of drugs. Memory decay occurs after 10 min when sodium pump activity (Na/K ATPase) is inhibited by ouabain or ethacrynic acid at the time of learning, implying a phase of memory which is not interfered with by these drugs. This 10 rain memory, in turn, can be abolished by drugs which alter neuronal membrane potentials, for example by monosodium glutamate or potassium chloride [10]. This information, along with data from the effective time of injection of the different drugs, provided the basis for a model of memory formation in which it is suggested that there are three different stales in memory development: short-term memory inhibited by glutamate; labile memory, inhibited by ouabaln; and long-term memory (LTM) inhibited by protein synthesis inhibitors. Labile memory represents a phase which can be modulated negatively or positively by manipulating sodium pump activity.
Saccharinaversion memory
The work reported in this paper was addressed to the question of the existence of a protein synthesis-independent phase of memory in rats and whether such a memory phase can be disrupted by inhibiting sodium pump activity. We also report the effect of systematic variation in dose of the protein synthesis inhibitor cycloheximide (CXM) on the degree of amnesia seen 24 hours after training. Memory was assessed in a discrimination test for a learned taste aversion [19]. In this task rats learn to avoid a novel and preferred tasting fluid (saccharin) after one pairing with a toxin (lithium chloride). GENERAL METHOD The apparatus and the behavioural and surgical procedures adopted have been described in detail previously
[18,19]. Animals Experimentally naive male and female hooded rats and male albino rats, aged 90-110 days were used. These animals were bred in the Department of Psychology, La Trobe University, and were maintained on ad lib food and water until 24 hr before the first pretraining day when water was removed. Apparatus
Rats were pretrained, trained and tested in a small clear plastic box on one wall of which were attached two graduated cylinders fitted with stainless steel drinking spouts. The wire gauze floor of the box and the spouts were connected to an electronic circuit which counted tongue con-
Copyright © 1979 Brain Research Publications Inc.--0031-9384/79/080341-06501.10/1
342
T U C K E R AND GIBBS
tacts with the spouts. The clear plastic box was housed in a larger sound-attenuating box, fitted with a one-way mirror door.
Drugs Cycloheximide (Actidione, Upjohn Co.) and ouabain octahydrate (Sigma) were dissolved in sterile 0.15 M NaCI just prior to use.
Behavioural Procedure In all experiments there were three stages: pretraining, training and testing. The pretraining stage (Days 1-3) consisted of placing the rats in the experimental box for 10 rain each day and training them to drink water from both cylinders. In the training trial (Day 4) all rats were offered a 0.1% (w/v) solution of sodium saccharin instead of water in both cylinders. Immediately after this saccharin drinking session each rat was given an intragastric (IG) injection of 0.15 M LiC1. Rats in Experiment 1 were allowed to drink saccharin for 10 rain (approx. 10 ml) and were given a 10 ml/kg dose of LiCI. Rats in Experiments 2 and 3 were allowed only a 5 rain (approx.) drink of saccharin (approximately 5 ml) and were given a 5 ml/kg dose of LiC1. Care was taken to ensure that the volume of saccharin drunk in training trials was comparable between treatment groups within any given experiment. In the test trial all rats were returned to the experimental box and offered saccharin in one cylinder and water in the other. The positions of the water and saccharin cylinders were varied randomly. In Experiment 1 the test trials were of 10 rain duration and were conducted 24 hours after training, but in Experiment 2 and 3 test trials were of 5 rain duration and were conducted 1, 2 or 4 hours after training on Day 4. At the conclusion of the test trial a saccharin aversion (SA) score was computed for each rat by the formula: SA score = number of water ticks × 100 total number of licks Hence a SA score approaching 100 indicates excellent memory whilst a SA score near zero indicates amnesia/no learning. In order to ensure that SA scores actually reflected taste discriminations no data were included from any rat which did not lick at both spouts. (Approximately 5% of all rats trained were rejected on this a priori criterion).
Surgical Procedure lntraventricular administration of drug or isotonic saline was accomplished by freehand injection [18,19]. Briefly, the rat was anaesthetized with ether and placed on a small animal rack. After incising the scalp a small hole was bored through the cranial bone over the left lateral ventricle. Thirty microlitres of solution were then expelled into the ventricle using a Hamilton dispenser and a 500 t~l syringe fitted with a stopped 26 ga needle. The hole was plugged with sterile bone wax and the wound sutured with skin clips. The entire operation usually took 3-4 min and the animals were fully recovered from the anaesthetic within 15 min. E X P E R I M E N T 1: T H E E F F E C T O F V A R I A T I O N O F CXM DOSE ON 24 H O U R A M N E S I A This experiment sought to determine the effect of systematic variation in dose of intraventricular CXM on saccharin aversion memory tested at 24 hr.
METHOD
Seventy male hooded rats were randomly assigned to one of 5 equal-sized groups at the end ofpretraining. Seven hours prior to the training trial all rats received an intraventricular injection of either 400, 300, 200, 100 or 0/~g of CXM in 30/zl of 0.15 M NaCI. A coding procedure ensured that these treatments were administered blind and the experimenter did not know the treatment condition of the rats during training or testing. Retention tests were conducted 24 hours after training. RESULTS AND DISCUSSION
A Kruskal-Wallis H test showed that there was a highly significant dose effect of CXM on 24 hr SA score (H=29.77, df=4, p
