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The Effects of Pretraining and Reminder Treatments on Retrograde Amnesia in Rats: Comparison of Lesions to the Fornix or Perirhinal and Entorhinal Cortices
Neurobiology of Learning and Memory 78, 365–378 (2002) doi:10.1006/nlme.2002.4070
The Effects of Pretraining and Reminder Treatments on Retrograde Amnesia in Rats: Comparison of Lesions to the Fornix or Perirhinal and Entorhinal Cortices Gretchen R. Hanson, Michael D. Bunsey, and David C. Riccio Department of Psychology, Kent State University, Kent, Ohio 44242-0001
The present experiment examined the effects of pretraining and reminder treatments on the retention of a nonrelational odor-guided digging task following lesions to the hippocampal formation (i.e., fornix) or parahippocampal region (i.e., perirhinal and entorhinal cortices). The results showed that fornix-lesioned rats and control rats had good retention of the task and did not differ from each other; however, perirhinal- and entorhinal-lesioned rats were severely impaired and differed from fornix and control rats. The present experiment found no attenuation of amnesia following pretraining, which may be due to the lesion technique employed and the size of the resulting lesions. However, the experiment found a significant difference in performance following a reminder treatment, even in the severely impaired perirhinal- and entorhinal-lesioned group. 䉷 2002 Elsevier Science (USA)
The hippocampal memory system has been a major source of investigation for the past few decades, and much work has been conducted in an attempt to isolate the different components of this system (e.g., Eichenbaum, Otto, & Cohen, 1994, 1992). For years, the focus has been on the hippocampal formation (i.e., hippocampus proper, dentate gyrus, subicular complex, and fornix); recently, however, it has become clear that the hippocampal formation is not the only important neural structure with regard to memory. Other neural structures, specifically, the parahippocampal region (i.e., perirhinal and entorhinal cortices), appear to serve distinct and important memory functions (e.g., Eichenbaum, Otto, & Cohen, 1994, 1992; Vargha-Khadem et al., 1997). The hippocampal formation seems to be essential for relational memory (i.e., spatial and episodic memory), whereas the This article is dedicated to the memory of Dr. Michael D. Bunsey, former advisor of the first author. This research was supported by NIMH MERIT Grant 37535 to D.C.R. and NIMH FIRST Award 1R29NS36962-01 to M.D.B. This research was conducted in accordance with the guidelines established by the Kent State University Animal Care and Use Committee. Address correspondence and reprint requests to Gretchen R. Hanson. E-mail: [email protected] 365
1074-7427/02 $35.00 䉷 2002 Elsevier Science (USA) All rights reserved.
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parahippocampal region seems to be important for nonrelational memory (i.e., stimulus– stimulus associations and stimulus recognition). The relational/nonrelational distinction is only one aspect of hippocampal system function. Another aspect of this memory system is the temporal gradient of retrograde amnesia, which shows that retrieval of recently acquired information is impaired, whereas retrieval of remotely acquired information is spared following an amnestic insult. This gradual strengthening of memories over time can be explained by assuming the hippocampus has a time-limited role in the acquisition of memories (e.g., Kim & Fanselow, 1992; Nadel & Moscovitch, 1997; Wiig, Cooper, & Bear, 1996; Zola-Morgan & Squire, 1990). Presumably, once information has been processed by the hippocampus, it is transferred to neocortical association areas for further processing and permanent storage (e.g., Nadel & Moscovitch, 1998; Teyler & DiScenna, 1986). This would explain why lesions of the hippocampus disrupt recent information (i.e., information that is still being processed by the hippocampus), but do not ordinarily disrupt remote information (i.e., information that has already been processed by the hippocampus). Perhaps information that has already been processed by the hippocampus and has been transferred to other brain regions (e.g., neocortex) may be less vulnerable to the effects of a hippocampal lesion. To examine these aspects of the hippocampal memory system, pretraining and reminder treatments were employed in the present experiment. Pretraining or familiarization experiments have shown that pretraining (e.g., exposure to context or cues) can attenuate the effects of amnestic treatments (e.g., Riccio & Richardson, 1984). In addition, numerous studies have shown that presenting a reminder treatment (e.g., brief exposure to one component of the training conditions) can lead to reactivation of a presumably inaccessible memory (e.g., Gordon & Mowrer, 1980; Miller & Springer, 1973; Spear & Riccio, 1994), although the reminder does not produce new learning in previously untrained controls. The effects of pretraining have been studied rather extensively with a wide range of amnestic treatments that have included direct electrical stimulation of the cortex, hypothermia, electroconvulsive shock (ECS), and intrahippocampal injections of AP5 (an NMDA antagonist) (e.g., Gold, Bueno, & McGaugh, 1973; Jensen & Riccio, 1970; Lewis, Miller, & Misanin, 1968; Roesler et al., 1998; Sara & David-Remacle, 1974). However, studies involving pretraining are limited with regard to amnesia produced by brain lesions, and the experiments that have been conducted have led to inconsistent findings (for review, see Olton & Markowska, 1989). The effects of reminder treatments have been examined in experiments involving hippocampal lesions (e.g., Gisquet-Verrier & Schenk, 1994; Land, Bunsey, & Riccio, 2000); however, no studies have examined the effect of reminder treatments on perirhinal- and entorhinal- (PRER) lesioned rats. The present experiment examined the effects of pretraining and reminder treatments by first pretraining and then training rats on an odor-guided digging task followed by lesions to the hippocampal formation (i.e., fornix), the parahippocampal region (i.e., PRER), or no lesion at all. One week later, rats were given two nonreinforced probe trials to test for their retention of the previously learned discriminations. The first probe trial served as a reminder treatment for the second probe trial. In essence, if pretraining attenuates amnesia and the temporal gradient of retrograde amnesia shows that more remotely learned items are spared with hippocampal system damage, then perhaps familiarizing rats by pretraining them several weeks prior to receiving a lesion would protect subsequently learned discriminations from the effects of the lesion. Theoretically, this
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attenuation of amnesia may occur because newly acquired information would be readily incorporated into an already existing network of information that is no longer dependent on the hippocampal system. In addition, the reminder treatment should lead to better performance on the retention test. METHOD Subjects Forty-one naı¨ve male Long–Evans rats (Charles River Laboratories, Wilmington, MA) were used in this experiment. They were reduced to 85% of their free-feeding weights prior to and during experimentation; water was available ad libitum. Rats were housed singly in Plexiglas cages in a colony room, which was maintained on a 15/9-h light/dark cycle with the lights coming on at 7:00 AM. Procedure All shaping, training, and testing took place in the rat’s home cage. Handling and Days 1 and 2 of shaping were conducted in the colony room where the rats were housed. However, Day 3 of shaping and all training and testing took place in an experimental room separate from the colony room. Rats dug in clear plastic cups (6.5 cm height by 6.5 cm width; Nalgene, Rochester, NY) containing sand that were placed in the rats’ home cages. Rats were required to retrieve buried sweet cereal rewards (i.e., Froot Loops cereal that was broken into thirds). The odor pairs used in discrimination training were obtained by mixing a percentage of a particular dry spice with sand (i.e., 150 g total scent/sand mixture) and consisted of the following odors: coffee (1%), thyme (1%), cocoa (1%), cinnamon (1%), cumin (0.5%), and garlic (1%). Shaping was given by burying Froot Loops (FL) pieces (broken into one-third sized pieces) in the sand and allowing the rat to retrieve the FL before termination of each trial. Days 1 and 2 of shaping took place in the rat’s home cage in the colony room. On Day 1, rats received two trials in which 12 FL pieces were buried in one cup of unscented sand (24 FL pieces total). Day 2 of shaping was identical to Day 1, except rats received two trials in which they were presented with 2 cups of unscented sand (1 of which contained 12 FL pieces). On Day 3, rats were taken to an experimental room where they received six trials in which they were presented with 2 cups of unscented sand, 1 of which contained 12 FL pieces. Rats were only allowed to retrieve 2 FL pieces per trial on Day 3 (i.e., 12 FL pieces total). One day following shaping (i.e., Day 4), rats were trained on Discrimination 1 (i.e., coffee versus thyme). During training, all rats received eight training trials in which they were presented with 2 cups of scented sand (1 of which contained three one-third sized FL pieces). Each rat was allowed to retrieve two FL pieces from the “correct” cup on each training trial. One particular odor was arbitrarily designated as correct for each particular rat. Presentation of the cups on the left or the right was counterbalanced from trial to trial. The only difference between the Recent and Remote Pretraining groups was that rats in the Recent Pretraining group (n ⫽ 22) were trained on Discriminations 2 (i.e., cocoa
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versus cinnamon) and 3 (i.e., cumin versus garlic) 1 day after Discrimination 1 training (Day 5), whereas rats in the Remote Pretraining group (n ⫽ 19) were trained on Discriminations 2 and 3 21 days after Discrimination 1 training (Day 25). One day following training on Discriminations 2 and 3, rats received a fornix lesion (Recent: n ⫽ 5; Remote: n ⫽ 5), a PRER lesion (Recent: n ⫽ 7; Remote: n ⫽ 5), a fornix sham lesion (Recent: n ⫽ 4; Remote: n ⫽ 3), or no lesion at all (Recent: n ⫽ 6; Remote: n ⫽ 6). Surgery All surgeries were performed under aseptic conditions. Rats were anesthetized with a 65 mg/kg intraperitoneal (iP) injection of nembutal (i.e., sodium pentobarbital). Once anesthetized, the rat’s head was shaved and the scalp cleaned with betadine disinfectant. Following surgery, the incision was sutured and then cleaned with betadine disinfectant. Rats were placed in a recovery room where they were placed on a heating source and monitored for 24 h prior to returning to the colony room. Rats were allowed a 7-day recovery period prior to testing. Fornix lesions. The rat was positioned in a stereotaxic instrument (Stoelting Instruments) using ear bars with the incisor bar positioned at 3.3 mm below the interaural line. A midline incision was made, and the scalp was retracted using hemostats. The surface of the skull was cleaned and bregma was obtained. Bilateral portions of the skull were drilled using a precision drill (Foredom Electronics). The electrolytic lesions were created by delivering 3 mA of DC cathodal current for 10 s through a stainless steel electrode (00-gauge) that was insulated except for the tip. The electrode was lowered into the brain at four sites bilaterally and one site that was centrally located (i.e., nine sites total). Coordinates for the fornix lesion were as follows (Paxinos & Watson, 1986) from bregma: AP ⫽ ⫺0.9 mm, ML ⫽ ⫹ 0.9 mm, DV ⫽ ⫺3.9 mm and ⫺4.3 mm; AP ⫽ ⫺1.4 mm, ML ⫽ ⫹ 1.6 mm, DV ⫽ ⫺3.9 mm and ⫺4.1 mm; AP ⫽ ⫺1.4, ML⫽ ⫺1.0, DV ⫽ ⫺3.9 (at a 10⬚ tilt). Perirhinal and entorhinal lesions. The rat was positioned in an adjustable head holder that secured the rat’s nose in an incisor bar. This apparatus allowed the rat’s head to be moved and repositioned in the medial–lateral plane. Following a midline incision, the scalp and temporal muscle were carefully removed through the use of a surgical probe and were then retracted using hemostats. Bilateral portions of the temporal bone were drilled exposing the rhinal sulcus. An aspirator with a 20-gauge needle attached to a vacuum pump was inserted into the opening and was moved by hand in an anterior to posterior direction, essentially removing the PRER. The resultant cavity was packed with Gelfoam prior to repositioning the muscle and scalp and suturing the midline incision. Sham lesions. All procedures for the sham lesions were identical to the fornix lesions described above, except no current was delivered when the electrode was lowered. No sham surgeries were explicitly carried out for the PRER lesion group. The fornix sham surgeries allowed for the control of exposure to anesthesia, midline incision, drilling, suturing, and recovery from surgery, which is essentially identical for both fornix and PRER lesion surgeries.
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Testing After a 1-week recovery period, rats were tested with two probe trials to measure their retention of Discriminations 2 and 3 (Discrimination 1 was used simply as a pretraining manipulation and rats were not tested for their retention of Discrimination 1). Probe trials consisted of presenting the scented cups without the buried reward. Rats received a probe trial for Discrimination 2 followed 5 min later by a test of Discrimination 3. The Discrimination 2 probe trial was also intended to serve as the reminder treatment for the Discrimination 3 probe trial. In addition to measuring retention of discriminations, probe trials helped to rule out the possibility that rats were solving the task simply by smelling the FL buried in the sand. Probe trials were videotaped and latency to dig and first choice were measured. Probe trials were terminated when the rat had dug for a total of 10 s or had ceased digging for 10 consecutive s. Histology Following testing, rats were euthanized with a 120-mg/kg dose of nembutal (i.e., sodium pentobarbital) and perfused transcardially with physiological saline followed by a 10% formalin solution. The rats were decapitated and their brains removed. The brains were placed in a 10% formalin solution (20 ml total); sucrose (10–13 g) was added to the formalin solution after several hours. Brains remained in the formalin/sucrose solution for at least 24 h. Brains were then embedded in gelatin (Sigma Type-B Bovine) and returned to the 10% formalin solution. Embedded brains remained in the 10% formalin solution for at least 1 week prior to slicing. Brains were sliced on a dry-ice microtome in 60 coronal slices. Every fourth slice was slide mounted and then stained with Cresyl violet. Lesion Analysis Brain slices were analyzed using the Paxinos and Watson (1986) atlas of the rat brain in stereotaxic coordinates. Plates representative of the full extent of a fornix lesion and a PRER lesion were selected from the atlas. Analyses were conducted on the largest and smallest lesions of the fornix and the PRER for both the Recent and Remote Pretraining groups. Largest and smallest lesions were selected based on the amount of damage to areas other than the targeted region. RESULTS Pretraining did not enhance performance; rats performed equally well in the Recent and Remote Pretraining groups. The performance of shams and controls did not differ from each other in either the Recent or the Remote conditions so their scores were combined for subsequent analyses. The latency to dig and choice measures showed that fornix and control rats performed significantly better than PRER rats on Discrimination 2. In fact, most PRER rats did not dig on the Discrimination 2 probe trial. However, PRER rats appeared to have normal motor abilities. They would approach the cups, smell them, and walk on them; however, they simply would not dig in the cups on Discrimination
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2. Moreover, the choice data showed that PRER rats chose the correct cup significantly more after the reminder treatment (i.e., Discrimination 2 probe trial). In fact, the performance of PRER rats on the choice measure did not differ from fornix and control rats on Discrimination 3. So it appears that the deficit seen in this group was due to a memory impairment and not due to an inability to perform the task. These conclusions are borne out by statistical analyses that are discussed in the following sections. Discrimination 2 The latency to dig and choice measures both showed a main effect of lesion, latency to dig: F (2, 35) ⫽ 11.96, p ⬍ 0.0001; choice: F(2, 38) ⫽ 12.84, p ⬍ 0.0001. However, there was no effect of group (i.e., Recent versus Remote Pretraining), latency to dig: F(1, 35) ⫽ 0.25, p ⬎ 0.05; choice: F(1, 39) ⫽ 0.0001, p ⬎ 0.05; and the interaction of group by lesion was not significant, latency to dig: F(2, 35) ⫽ 0.08, p ⬎ 0.05. A Tukey HSD post hoc analysis of latency to dig and choice showed that both fornix rats (latency: M ⫽ 48.2; choice: M ⫽ 0.7) and control rats (latency: M ⫽ 44.51; choice: M ⫽ 0.94) differed significantly from PRER rats (latency: M ⫽ 132.61; choice: M ⫽ 0.24), latency: p ⬍ 0.01; choice: p ⬍ 0.05; and fornix and control rats were not significantly different from each other, latency and choice: p ⬎ 0.05. Discrimination 3 The latency measure shows a main effect of lesion, F(2, 35) ⫽ 5.33, p ⬍ 0.01. Tukey HSD post hoc analyses showed that latency to dig in fornix rats (M ⫽ 16.9) and control rats (M ⫽ 19.11) was significantly different from PRER rats (M ⫽ 61.83), p ⬍ 0.05. Fornix and control rats did not significantly differ from each other, p ⬎ 0.05. However, the choice measure indicated no differences between lesion groups, F(2, 38) ⫽ 1.65, p ⬎ 0.05. Tukey HSD post hoc analyses of choice indicated no differences between fornix (M ⫽ 1.0), PRER (M ⫽ 0.68), and controls (M ⫽ 0.89), p ⬎ 0.05. Once again, there was no effect of group (i.e., Recent versus Remote Pretraining), latency to dig: F(1, 35) ⫽ 0.97, p ⬎ 0.05; choice: F(1, 39) ⫽ 0.09, p ⬎ 0.05, and the interaction between lesion and group was not significant, latency to dig: F(2, 35) ⫽ 0.65, p ⬎ 0.05. This discrepancy between latency and choice measures points to a fundamental difference between these two measures. Latency to dig scores may focus more on memory for task requirements (e.g., dig to retrieve FL), while choice measures may focus more on memory for the discrimination itself (e.g., dig in cocoa to retrieve FL). Discrimination 2 versus Discrimination 3 Since there was no significant difference between the Remote and Recent Pretraining groups, these group were combined for further analyses. As can be seen in Fig. 1, latency to dig scores decreased significantly from Discrimination 2 to Discrimination 3 for all groups; fornix: F(1, 18) ⫽ 4.88, p ⬍ 0.05, PRER: F(1, 22) ⫽ 7.41, p ⬍ 0.05, control: F(1, 36) ⫽ 6.95, p ⬍ 0.05. Additionally, choice performance significantly improved in the PRER group following the Discrimination 2 reminder treatment, F(5, 76) ⫽ 2.94, p ⬍ 0.05 (see Fig. 2).
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FIG. 1. Latency to dig on Discriminations 2 and 3 for fornix, PRER, and control rats. PRER rats had significantly slower latencies to dig than fornix and control rats on Discriminations 2 and 3. In addition, all groups had significantly faster latencies to dig on Discrimination 3 than on Discrimination 2.
Fornix Lesion Analysis Brain slices were analyzed and the largest and smallest lesions of the fornix were determined for both the Recent and Remote Pretraining groups. See Figs. 3 and 4 for representative sketches of these lesions. In addition to fornix damage, the brains with larger lesions showed damage to lateral and medial septal nuclei, caudate putamen, corpus callosum, and dorsal hippocampus. PRER Lesion Analysis Brain slices were analyzed and the largest and smallest lesions of the PRER were determined for both the Recent and Remote Pretraining groups. See Figs. 5 and 6 for representative sketches of these lesions. In addition to perirhinal and entorhinal damage, the larger lesions showed damage to ventral hippocampus, amygdala, and piriform cortex. Damage to piriform cortex may produce deficits in odor discrimination performance. However, all PRER rats improved significantly following the reminder treatment, indicating they could discriminate between the odors, so the probability of the observed impairments being due to a nonmemory interpretation (like piriform cortex damage) are unlikely. DISCUSSION To summarize, three findings were obtained in this experiment. First, pretraining failed to attenuate the retrograde amnesia produced by parahippocampal region damage (i.e.,
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FIG. 2. Choice scores on Discriminations 2 and 3 for fornix, PRER, and control rats. Choice scores ranged from 0 (incorrect choice) to 1 (correct choice). PRER rats made significantly more incorrect choices than fornix and control rats on Discrimination 2. However, PRER rats performed significantly better on Discrimination 3 and did not differ from fornix and control rats.
PRER lesions). Second, PRER rats exhibited severe memory impairments, whereas fornix lesioned rats performed as well as controls. Third, the reminder treatment (Discrimination 2) lead to improved performance for all groups on Discrimination 3. Pretraining The pretraining manipulation did not attenuate the retrograde amnesia produced by PRER damage. Previous research has suggested that prior experience with the training apparatus or actual training trials prior to an amnestic event may attenuate retrograde amnesia (e.g., Jensen & Riccio, 1970; Lewis, Miller, & Misanin, 1968; Riccio & Richardson, 1984; Roesler et al., 1998). However, few pretraining studies have been conducted involving lesions to the hippocampal system, so it is difficult to say what the optimal conditions for attenuation of amnesia are when using pretraining manipulations. Olton and Markowska (1989) suggest that optimal conditions are spatial rather than nonspatial tasks and neurotoxic rather than electrolytic or aspiration lesions. Lesion When comparing lesion groups using the latency to dig measure the results are consistent with previous research (e.g., Eichenbaum et al., 1992, 1994). Rats with lesions of the
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FIG. 3. Representative small fornix lesion appears on the left, while the corresponding normal brain slice appears on the right. This brain had damage to the fornix, along with damage to the lateral and medial septal nuclei and the dorsal hippocampus. Taken from The Rat Brain in Stereotaxic Coordinates (Figs. 21, 23, and 25) by Paxinos and Watson (1986).
fornix showed latency to dig scores that did not differ from control rats, but did differ from PRER rats on a nonspatial, nonrelational task. The PRER appears to be essential for the solution of nonspatial tasks that require the learning and storage of specific stimuli (e.g., specific odors).
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FIG. 4. Representative large fornix lesion appears on the left, while the corresponding normal brain slice appears on the right. This brain had damage to the fornix, along with damage to the lateral and medial septal nuclei, thalamus, and unilateral damage to the dorsal hippocampus. Taken from The Rat Brain in Stereotaxic Coordinates (Figs. 21, 23, and 25) by Paxinos and Watson (1986).
Reminder Treatment All lesion groups had significantly faster latencies to dig and better choice performance on the Discrimination 3 probe trial than on the Discrimination 2 probe trial. Numerous studies have shown that presenting a reminder treatment prior to testing (e.g., brief exposure to the conditioned stimulus or the apparatus) can lead to reactivation of a presumably inaccessible memory (e.g., Riccio & Richardson, 1984; Spear, 1973). Gisquet-Verrier and Schenk (1994) examined the effects of reminder treatments on rats that received hippocampal lesions prior to training and found that both the hippocampal lesioned rats and the controls that had received the reminder treatment prior to testing performed significantly better than the hippocampal lesioned rats and the controls that had not received the reminder treatment. In addition, Land et al. (2000) examined the
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FIG. 5. Representative small PRER lesion appears on the left, while the corresponding normal brain slice appears on the right. This brain had damage to the perirhinal and entorhinal cortices, along with damage to the caudate and putamen and the ventral hippocampus. Taken from The Rat Brain in Stereotaxic Coordinates (Figs. 26, 35, 43, and 46) by Paxinos and Watson (1986).
effectiveness of a reminder treatment on rats with posttraining hippocampal lesions and found that hippocampal-lesioned rats that received a reminder treatment prior to testing performed significantly better than lesioned rats not receiving a reminder treatment. The reminder treatment phenomenon can be used to explain the decreased latencies and improved choice behavior found in the present experiment in all groups on probe
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FIG. 6. Representative large PRER lesion appears on the left, while the corresponding normal brain slice appears on the right. This brain had damage to the perirhinal and entorhinal cortices, along with damage to the ventral hippocampus. Taken from The Rat Brain in Stereotaxic Coordinates (Figs. 26, 35, 43, and 46) by Paxinos and Watson (1986).
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trials from Discrimination 2 to Discrimination 3. Presumably, the presentation of the probe trial for Discrimination 2 served as a reminder treatment and reactivated the memory for Discrimination 3, which led to better performance when rats were tested on Discrimination 3. The Discrimination 2 probe trial did not allow for new learning to take place because no reinforcement was provided (i.e., no FL were buried in the sand). In fact, because the Discrimination 2 probe trial was not reinforced, it served essentially as an extinction trial. So if rats learned anything from the Discrimination 2 probe trial it should have been that food was no longer available and that they should stop digging. In addition, previous pilot data from our lab suggests that Discrimination 3 (i.e., cumin versus garlic) is not easier to learn or retain than is Discrimination 2 (i.e., cocoa versus cinnamon). This reminder treatment improvement was seen in all groups and lends support to GisquetVerrier and Schenk (1994) and Land et al. (2000). In addition, the present finding of improved test performance in PRER rats extends the phenomenon of reminder treatmentinduced improvements in lesioned rats. GENERAL DISCUSSION The findings of the present experiment lend support to the Eichenbaum et al. theory of hippocampal system function. As expected, rats with lesions to the hippocampal formation performed similarly to controls on a nonspatial, odor-guided digging task. However, rats with lesions to the PRER were severely impaired on this simple discrimination task. The data from the pretraining manipulation were less clear. Based on the pretraining research described above, it would be expected that the pretraining manipulation would have resulted in improved performance for rats in those conditions. However, that appears not to be the case. It remains possible that alterations in training parameters (e.g., longer or shorter delay intervals between pretraining and surgery) or different lesion techniques (e.g., neurotoxic lesions rather than electrolytic or aspiration lesions) would enhance performance. The reminder treatment findings are interesting in that they show an improvement in performance for animals that are not impaired (i.e., fornix and control rats) as well as those exhibiting severe memory impairments (i.e., PRER-lesioned rats). REFERENCES Eichenbaum, H., Otto, T., & Cohen, N. J. (1994). Two functional components of the hippocampal memory system. Behavioral Brain Sciences, 17, 449–518. Eichenbaum, H., Otto, T., & Cohen, N. J. (1992). The hippocampus: What does it do? Behavioral and Neural Biology, 57, 2–36. Gisquet-Verrier, P., & Schenk, F. (1994). Selective hippocampal lesions in rats do not affect retrieval processes promoted by prior cuing with the conditioned stimulus or the context. Psychobiology, 22(4), 298–303. Gold, P. E., Bueno, O. F., & McGaugh, J. L. (1973). Training and task-related differences in retrograde amnesia thresholds determined by direct electrical stimulation of the cortex in rats. Physiology and Behavior, 11, 57–63. Gordon, W. C., & Mowrer, R. R. (1980). The use of an extinction trial as a reminder treatment following CS. Animal Learning and Behavior, 8, 363–367. Jensen, R. A., & Riccio, D. C. (1970). Effects of prior experience upon retrograde amnesia produced by hypothermia. Physiology and Behavior, 5, 1291–1294.
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Kim, J. J., & Fanselow, M. S. (1992). Modality-specific retrograde amnesia of fear. Science, 256, 675–677. Land, C., Bunsey, M., & Riccio, D. C. (2000). Anomalous properties of hippocampal lesion-induced retrograde amnesia. Psychobiology, 28(4), 476–485. Lewis, D. J., Miller, R. R., & Misanin, J. R. (1968). Control of retrograde amnesia. Journal of Comparative and Physiological Psychology, 66, 48–52. Miller, R. R., & Springer, A. D. (1973). Amnesia, consolidation, and retrieval. Psychological Review, 80(1), 69–79. Nadel, L., & Moscovitch, M. (1997). Memory consolidation, retrograde amnesia and the hippocampal complex. Current Opinion in Neurobiology, 7, 217–227. Nadel, L., & Moscovitch, M. (1998). Hippocampal contribution to cortical plasticity. Neuropharmacology, 37, 431–439. Olton, D. S., & Markowska, A. L. (1989). The effects of preoperative experience upon postoperative performance of rats following lesions of the hippocampal system. In J. Schulkin (Ed.), Preoperative events: Their effects on behavior following brain damage (pp. 151–173). Hillsdale, NJ: Erlbaum. Paxinos, G., & Watson, C. (1986). The rat brain in stereotaxic coordinates. New York: Academic Press. Riccio, D. C., & Richardson, R. (1984). The status of memory following experimentally induced amnesias: Gone, but not forgotten. Physiological Psychology, 12(2), 59–72. Roesler, R., Vianna, M., Sant’Anna, M. K., Kuyven, C. R., Kruel, A. V. S., Quevedo, J., and Ferreira, M. B. C. (1998). Intrahippocampal infusion of the NMDA receptor antagonist AP5 impairs retention of an inhibitory avoidance task: Protection from impairment by pretraining or preexposure to the task apparatus. Neurobiology of Learning and Memory, 69, 87–91. Sara, S. J., & David-Remacle, M. (1974). Recovery from electroconvulsive shock-induced amnesia by exposure to the training environment: Pharmacological enhancement by piracetam. Psychopharmacologia, 36(1), 59–66. Spear, N. E. (1973). Retrieval of memory in animals. Psychological Review, 80(3), 163–194. Spear, N. E., & Riccio, D. C. (1994). Memory: Phenomena and principles. Boston: Allyn & Bacon. Teyler, T. J., & DiScenna, P. (1986). The hippocampal memory indexing theory. Behavioral Neuroscience, 100(2), 147–154. Vargha-Khadem, F., Gadian, D. G., Watkins, K. E., Connelly, A., Van Paesschen, W., & Mishkin, M. (1997). Differential effects of early hippocampal pathology on episodic and semantic memory. Science, 277, 376–380. Wiig, K. A., Cooper, L. N., & Bear, M. F. (1996). Temporally graded retrograde amnesia following separate and combined lesions of the perirhinal cortex and fornix in the rat. Learning and Memory, 3, 313–325. Zola-Morgan, S. M. & Squire, L. R. (1990). The primate hippocampal formation: Evidence for a time-limited role in memory storage. Science, 250, 288–290.
The Effects of Pretraining and Reminder Treatments on Retrograde Amnesia in Rats: Comparison of Lesions to the Fornix or Perirhinal and Entorhinal Cortices Gretchen R. Hanson, Michael D. Bunsey, and David C. Riccio Department of Psychology, Kent State University, Kent, Ohio 44242-0001
The present experiment examined the effects of pretraining and reminder treatments on the retention of a nonrelational odor-guided digging task following lesions to the hippocampal formation (i.e., fornix) or parahippocampal region (i.e., perirhinal and entorhinal cortices). The results showed that fornix-lesioned rats and control rats had good retention of the task and did not differ from each other; however, perirhinal- and entorhinal-lesioned rats were severely impaired and differed from fornix and control rats. The present experiment found no attenuation of amnesia following pretraining, which may be due to the lesion technique employed and the size of the resulting lesions. However, the experiment found a significant difference in performance following a reminder treatment, even in the severely impaired perirhinal- and entorhinal-lesioned group. 䉷 2002 Elsevier Science (USA)
The hippocampal memory system has been a major source of investigation for the past few decades, and much work has been conducted in an attempt to isolate the different components of this system (e.g., Eichenbaum, Otto, & Cohen, 1994, 1992). For years, the focus has been on the hippocampal formation (i.e., hippocampus proper, dentate gyrus, subicular complex, and fornix); recently, however, it has become clear that the hippocampal formation is not the only important neural structure with regard to memory. Other neural structures, specifically, the parahippocampal region (i.e., perirhinal and entorhinal cortices), appear to serve distinct and important memory functions (e.g., Eichenbaum, Otto, & Cohen, 1994, 1992; Vargha-Khadem et al., 1997). The hippocampal formation seems to be essential for relational memory (i.e., spatial and episodic memory), whereas the This article is dedicated to the memory of Dr. Michael D. Bunsey, former advisor of the first author. This research was supported by NIMH MERIT Grant 37535 to D.C.R. and NIMH FIRST Award 1R29NS36962-01 to M.D.B. This research was conducted in accordance with the guidelines established by the Kent State University Animal Care and Use Committee. Address correspondence and reprint requests to Gretchen R. Hanson. E-mail: [email protected] 365
1074-7427/02 $35.00 䉷 2002 Elsevier Science (USA) All rights reserved.
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parahippocampal region seems to be important for nonrelational memory (i.e., stimulus– stimulus associations and stimulus recognition). The relational/nonrelational distinction is only one aspect of hippocampal system function. Another aspect of this memory system is the temporal gradient of retrograde amnesia, which shows that retrieval of recently acquired information is impaired, whereas retrieval of remotely acquired information is spared following an amnestic insult. This gradual strengthening of memories over time can be explained by assuming the hippocampus has a time-limited role in the acquisition of memories (e.g., Kim & Fanselow, 1992; Nadel & Moscovitch, 1997; Wiig, Cooper, & Bear, 1996; Zola-Morgan & Squire, 1990). Presumably, once information has been processed by the hippocampus, it is transferred to neocortical association areas for further processing and permanent storage (e.g., Nadel & Moscovitch, 1998; Teyler & DiScenna, 1986). This would explain why lesions of the hippocampus disrupt recent information (i.e., information that is still being processed by the hippocampus), but do not ordinarily disrupt remote information (i.e., information that has already been processed by the hippocampus). Perhaps information that has already been processed by the hippocampus and has been transferred to other brain regions (e.g., neocortex) may be less vulnerable to the effects of a hippocampal lesion. To examine these aspects of the hippocampal memory system, pretraining and reminder treatments were employed in the present experiment. Pretraining or familiarization experiments have shown that pretraining (e.g., exposure to context or cues) can attenuate the effects of amnestic treatments (e.g., Riccio & Richardson, 1984). In addition, numerous studies have shown that presenting a reminder treatment (e.g., brief exposure to one component of the training conditions) can lead to reactivation of a presumably inaccessible memory (e.g., Gordon & Mowrer, 1980; Miller & Springer, 1973; Spear & Riccio, 1994), although the reminder does not produce new learning in previously untrained controls. The effects of pretraining have been studied rather extensively with a wide range of amnestic treatments that have included direct electrical stimulation of the cortex, hypothermia, electroconvulsive shock (ECS), and intrahippocampal injections of AP5 (an NMDA antagonist) (e.g., Gold, Bueno, & McGaugh, 1973; Jensen & Riccio, 1970; Lewis, Miller, & Misanin, 1968; Roesler et al., 1998; Sara & David-Remacle, 1974). However, studies involving pretraining are limited with regard to amnesia produced by brain lesions, and the experiments that have been conducted have led to inconsistent findings (for review, see Olton & Markowska, 1989). The effects of reminder treatments have been examined in experiments involving hippocampal lesions (e.g., Gisquet-Verrier & Schenk, 1994; Land, Bunsey, & Riccio, 2000); however, no studies have examined the effect of reminder treatments on perirhinal- and entorhinal- (PRER) lesioned rats. The present experiment examined the effects of pretraining and reminder treatments by first pretraining and then training rats on an odor-guided digging task followed by lesions to the hippocampal formation (i.e., fornix), the parahippocampal region (i.e., PRER), or no lesion at all. One week later, rats were given two nonreinforced probe trials to test for their retention of the previously learned discriminations. The first probe trial served as a reminder treatment for the second probe trial. In essence, if pretraining attenuates amnesia and the temporal gradient of retrograde amnesia shows that more remotely learned items are spared with hippocampal system damage, then perhaps familiarizing rats by pretraining them several weeks prior to receiving a lesion would protect subsequently learned discriminations from the effects of the lesion. Theoretically, this
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attenuation of amnesia may occur because newly acquired information would be readily incorporated into an already existing network of information that is no longer dependent on the hippocampal system. In addition, the reminder treatment should lead to better performance on the retention test. METHOD Subjects Forty-one naı¨ve male Long–Evans rats (Charles River Laboratories, Wilmington, MA) were used in this experiment. They were reduced to 85% of their free-feeding weights prior to and during experimentation; water was available ad libitum. Rats were housed singly in Plexiglas cages in a colony room, which was maintained on a 15/9-h light/dark cycle with the lights coming on at 7:00 AM. Procedure All shaping, training, and testing took place in the rat’s home cage. Handling and Days 1 and 2 of shaping were conducted in the colony room where the rats were housed. However, Day 3 of shaping and all training and testing took place in an experimental room separate from the colony room. Rats dug in clear plastic cups (6.5 cm height by 6.5 cm width; Nalgene, Rochester, NY) containing sand that were placed in the rats’ home cages. Rats were required to retrieve buried sweet cereal rewards (i.e., Froot Loops cereal that was broken into thirds). The odor pairs used in discrimination training were obtained by mixing a percentage of a particular dry spice with sand (i.e., 150 g total scent/sand mixture) and consisted of the following odors: coffee (1%), thyme (1%), cocoa (1%), cinnamon (1%), cumin (0.5%), and garlic (1%). Shaping was given by burying Froot Loops (FL) pieces (broken into one-third sized pieces) in the sand and allowing the rat to retrieve the FL before termination of each trial. Days 1 and 2 of shaping took place in the rat’s home cage in the colony room. On Day 1, rats received two trials in which 12 FL pieces were buried in one cup of unscented sand (24 FL pieces total). Day 2 of shaping was identical to Day 1, except rats received two trials in which they were presented with 2 cups of unscented sand (1 of which contained 12 FL pieces). On Day 3, rats were taken to an experimental room where they received six trials in which they were presented with 2 cups of unscented sand, 1 of which contained 12 FL pieces. Rats were only allowed to retrieve 2 FL pieces per trial on Day 3 (i.e., 12 FL pieces total). One day following shaping (i.e., Day 4), rats were trained on Discrimination 1 (i.e., coffee versus thyme). During training, all rats received eight training trials in which they were presented with 2 cups of scented sand (1 of which contained three one-third sized FL pieces). Each rat was allowed to retrieve two FL pieces from the “correct” cup on each training trial. One particular odor was arbitrarily designated as correct for each particular rat. Presentation of the cups on the left or the right was counterbalanced from trial to trial. The only difference between the Recent and Remote Pretraining groups was that rats in the Recent Pretraining group (n ⫽ 22) were trained on Discriminations 2 (i.e., cocoa
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versus cinnamon) and 3 (i.e., cumin versus garlic) 1 day after Discrimination 1 training (Day 5), whereas rats in the Remote Pretraining group (n ⫽ 19) were trained on Discriminations 2 and 3 21 days after Discrimination 1 training (Day 25). One day following training on Discriminations 2 and 3, rats received a fornix lesion (Recent: n ⫽ 5; Remote: n ⫽ 5), a PRER lesion (Recent: n ⫽ 7; Remote: n ⫽ 5), a fornix sham lesion (Recent: n ⫽ 4; Remote: n ⫽ 3), or no lesion at all (Recent: n ⫽ 6; Remote: n ⫽ 6). Surgery All surgeries were performed under aseptic conditions. Rats were anesthetized with a 65 mg/kg intraperitoneal (iP) injection of nembutal (i.e., sodium pentobarbital). Once anesthetized, the rat’s head was shaved and the scalp cleaned with betadine disinfectant. Following surgery, the incision was sutured and then cleaned with betadine disinfectant. Rats were placed in a recovery room where they were placed on a heating source and monitored for 24 h prior to returning to the colony room. Rats were allowed a 7-day recovery period prior to testing. Fornix lesions. The rat was positioned in a stereotaxic instrument (Stoelting Instruments) using ear bars with the incisor bar positioned at 3.3 mm below the interaural line. A midline incision was made, and the scalp was retracted using hemostats. The surface of the skull was cleaned and bregma was obtained. Bilateral portions of the skull were drilled using a precision drill (Foredom Electronics). The electrolytic lesions were created by delivering 3 mA of DC cathodal current for 10 s through a stainless steel electrode (00-gauge) that was insulated except for the tip. The electrode was lowered into the brain at four sites bilaterally and one site that was centrally located (i.e., nine sites total). Coordinates for the fornix lesion were as follows (Paxinos & Watson, 1986) from bregma: AP ⫽ ⫺0.9 mm, ML ⫽ ⫹ 0.9 mm, DV ⫽ ⫺3.9 mm and ⫺4.3 mm; AP ⫽ ⫺1.4 mm, ML ⫽ ⫹ 1.6 mm, DV ⫽ ⫺3.9 mm and ⫺4.1 mm; AP ⫽ ⫺1.4, ML⫽ ⫺1.0, DV ⫽ ⫺3.9 (at a 10⬚ tilt). Perirhinal and entorhinal lesions. The rat was positioned in an adjustable head holder that secured the rat’s nose in an incisor bar. This apparatus allowed the rat’s head to be moved and repositioned in the medial–lateral plane. Following a midline incision, the scalp and temporal muscle were carefully removed through the use of a surgical probe and were then retracted using hemostats. Bilateral portions of the temporal bone were drilled exposing the rhinal sulcus. An aspirator with a 20-gauge needle attached to a vacuum pump was inserted into the opening and was moved by hand in an anterior to posterior direction, essentially removing the PRER. The resultant cavity was packed with Gelfoam prior to repositioning the muscle and scalp and suturing the midline incision. Sham lesions. All procedures for the sham lesions were identical to the fornix lesions described above, except no current was delivered when the electrode was lowered. No sham surgeries were explicitly carried out for the PRER lesion group. The fornix sham surgeries allowed for the control of exposure to anesthesia, midline incision, drilling, suturing, and recovery from surgery, which is essentially identical for both fornix and PRER lesion surgeries.
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Testing After a 1-week recovery period, rats were tested with two probe trials to measure their retention of Discriminations 2 and 3 (Discrimination 1 was used simply as a pretraining manipulation and rats were not tested for their retention of Discrimination 1). Probe trials consisted of presenting the scented cups without the buried reward. Rats received a probe trial for Discrimination 2 followed 5 min later by a test of Discrimination 3. The Discrimination 2 probe trial was also intended to serve as the reminder treatment for the Discrimination 3 probe trial. In addition to measuring retention of discriminations, probe trials helped to rule out the possibility that rats were solving the task simply by smelling the FL buried in the sand. Probe trials were videotaped and latency to dig and first choice were measured. Probe trials were terminated when the rat had dug for a total of 10 s or had ceased digging for 10 consecutive s. Histology Following testing, rats were euthanized with a 120-mg/kg dose of nembutal (i.e., sodium pentobarbital) and perfused transcardially with physiological saline followed by a 10% formalin solution. The rats were decapitated and their brains removed. The brains were placed in a 10% formalin solution (20 ml total); sucrose (10–13 g) was added to the formalin solution after several hours. Brains remained in the formalin/sucrose solution for at least 24 h. Brains were then embedded in gelatin (Sigma Type-B Bovine) and returned to the 10% formalin solution. Embedded brains remained in the 10% formalin solution for at least 1 week prior to slicing. Brains were sliced on a dry-ice microtome in 60 coronal slices. Every fourth slice was slide mounted and then stained with Cresyl violet. Lesion Analysis Brain slices were analyzed using the Paxinos and Watson (1986) atlas of the rat brain in stereotaxic coordinates. Plates representative of the full extent of a fornix lesion and a PRER lesion were selected from the atlas. Analyses were conducted on the largest and smallest lesions of the fornix and the PRER for both the Recent and Remote Pretraining groups. Largest and smallest lesions were selected based on the amount of damage to areas other than the targeted region. RESULTS Pretraining did not enhance performance; rats performed equally well in the Recent and Remote Pretraining groups. The performance of shams and controls did not differ from each other in either the Recent or the Remote conditions so their scores were combined for subsequent analyses. The latency to dig and choice measures showed that fornix and control rats performed significantly better than PRER rats on Discrimination 2. In fact, most PRER rats did not dig on the Discrimination 2 probe trial. However, PRER rats appeared to have normal motor abilities. They would approach the cups, smell them, and walk on them; however, they simply would not dig in the cups on Discrimination
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2. Moreover, the choice data showed that PRER rats chose the correct cup significantly more after the reminder treatment (i.e., Discrimination 2 probe trial). In fact, the performance of PRER rats on the choice measure did not differ from fornix and control rats on Discrimination 3. So it appears that the deficit seen in this group was due to a memory impairment and not due to an inability to perform the task. These conclusions are borne out by statistical analyses that are discussed in the following sections. Discrimination 2 The latency to dig and choice measures both showed a main effect of lesion, latency to dig: F (2, 35) ⫽ 11.96, p ⬍ 0.0001; choice: F(2, 38) ⫽ 12.84, p ⬍ 0.0001. However, there was no effect of group (i.e., Recent versus Remote Pretraining), latency to dig: F(1, 35) ⫽ 0.25, p ⬎ 0.05; choice: F(1, 39) ⫽ 0.0001, p ⬎ 0.05; and the interaction of group by lesion was not significant, latency to dig: F(2, 35) ⫽ 0.08, p ⬎ 0.05. A Tukey HSD post hoc analysis of latency to dig and choice showed that both fornix rats (latency: M ⫽ 48.2; choice: M ⫽ 0.7) and control rats (latency: M ⫽ 44.51; choice: M ⫽ 0.94) differed significantly from PRER rats (latency: M ⫽ 132.61; choice: M ⫽ 0.24), latency: p ⬍ 0.01; choice: p ⬍ 0.05; and fornix and control rats were not significantly different from each other, latency and choice: p ⬎ 0.05. Discrimination 3 The latency measure shows a main effect of lesion, F(2, 35) ⫽ 5.33, p ⬍ 0.01. Tukey HSD post hoc analyses showed that latency to dig in fornix rats (M ⫽ 16.9) and control rats (M ⫽ 19.11) was significantly different from PRER rats (M ⫽ 61.83), p ⬍ 0.05. Fornix and control rats did not significantly differ from each other, p ⬎ 0.05. However, the choice measure indicated no differences between lesion groups, F(2, 38) ⫽ 1.65, p ⬎ 0.05. Tukey HSD post hoc analyses of choice indicated no differences between fornix (M ⫽ 1.0), PRER (M ⫽ 0.68), and controls (M ⫽ 0.89), p ⬎ 0.05. Once again, there was no effect of group (i.e., Recent versus Remote Pretraining), latency to dig: F(1, 35) ⫽ 0.97, p ⬎ 0.05; choice: F(1, 39) ⫽ 0.09, p ⬎ 0.05, and the interaction between lesion and group was not significant, latency to dig: F(2, 35) ⫽ 0.65, p ⬎ 0.05. This discrepancy between latency and choice measures points to a fundamental difference between these two measures. Latency to dig scores may focus more on memory for task requirements (e.g., dig to retrieve FL), while choice measures may focus more on memory for the discrimination itself (e.g., dig in cocoa to retrieve FL). Discrimination 2 versus Discrimination 3 Since there was no significant difference between the Remote and Recent Pretraining groups, these group were combined for further analyses. As can be seen in Fig. 1, latency to dig scores decreased significantly from Discrimination 2 to Discrimination 3 for all groups; fornix: F(1, 18) ⫽ 4.88, p ⬍ 0.05, PRER: F(1, 22) ⫽ 7.41, p ⬍ 0.05, control: F(1, 36) ⫽ 6.95, p ⬍ 0.05. Additionally, choice performance significantly improved in the PRER group following the Discrimination 2 reminder treatment, F(5, 76) ⫽ 2.94, p ⬍ 0.05 (see Fig. 2).
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FIG. 1. Latency to dig on Discriminations 2 and 3 for fornix, PRER, and control rats. PRER rats had significantly slower latencies to dig than fornix and control rats on Discriminations 2 and 3. In addition, all groups had significantly faster latencies to dig on Discrimination 3 than on Discrimination 2.
Fornix Lesion Analysis Brain slices were analyzed and the largest and smallest lesions of the fornix were determined for both the Recent and Remote Pretraining groups. See Figs. 3 and 4 for representative sketches of these lesions. In addition to fornix damage, the brains with larger lesions showed damage to lateral and medial septal nuclei, caudate putamen, corpus callosum, and dorsal hippocampus. PRER Lesion Analysis Brain slices were analyzed and the largest and smallest lesions of the PRER were determined for both the Recent and Remote Pretraining groups. See Figs. 5 and 6 for representative sketches of these lesions. In addition to perirhinal and entorhinal damage, the larger lesions showed damage to ventral hippocampus, amygdala, and piriform cortex. Damage to piriform cortex may produce deficits in odor discrimination performance. However, all PRER rats improved significantly following the reminder treatment, indicating they could discriminate between the odors, so the probability of the observed impairments being due to a nonmemory interpretation (like piriform cortex damage) are unlikely. DISCUSSION To summarize, three findings were obtained in this experiment. First, pretraining failed to attenuate the retrograde amnesia produced by parahippocampal region damage (i.e.,
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FIG. 2. Choice scores on Discriminations 2 and 3 for fornix, PRER, and control rats. Choice scores ranged from 0 (incorrect choice) to 1 (correct choice). PRER rats made significantly more incorrect choices than fornix and control rats on Discrimination 2. However, PRER rats performed significantly better on Discrimination 3 and did not differ from fornix and control rats.
PRER lesions). Second, PRER rats exhibited severe memory impairments, whereas fornix lesioned rats performed as well as controls. Third, the reminder treatment (Discrimination 2) lead to improved performance for all groups on Discrimination 3. Pretraining The pretraining manipulation did not attenuate the retrograde amnesia produced by PRER damage. Previous research has suggested that prior experience with the training apparatus or actual training trials prior to an amnestic event may attenuate retrograde amnesia (e.g., Jensen & Riccio, 1970; Lewis, Miller, & Misanin, 1968; Riccio & Richardson, 1984; Roesler et al., 1998). However, few pretraining studies have been conducted involving lesions to the hippocampal system, so it is difficult to say what the optimal conditions for attenuation of amnesia are when using pretraining manipulations. Olton and Markowska (1989) suggest that optimal conditions are spatial rather than nonspatial tasks and neurotoxic rather than electrolytic or aspiration lesions. Lesion When comparing lesion groups using the latency to dig measure the results are consistent with previous research (e.g., Eichenbaum et al., 1992, 1994). Rats with lesions of the
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FIG. 3. Representative small fornix lesion appears on the left, while the corresponding normal brain slice appears on the right. This brain had damage to the fornix, along with damage to the lateral and medial septal nuclei and the dorsal hippocampus. Taken from The Rat Brain in Stereotaxic Coordinates (Figs. 21, 23, and 25) by Paxinos and Watson (1986).
fornix showed latency to dig scores that did not differ from control rats, but did differ from PRER rats on a nonspatial, nonrelational task. The PRER appears to be essential for the solution of nonspatial tasks that require the learning and storage of specific stimuli (e.g., specific odors).
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FIG. 4. Representative large fornix lesion appears on the left, while the corresponding normal brain slice appears on the right. This brain had damage to the fornix, along with damage to the lateral and medial septal nuclei, thalamus, and unilateral damage to the dorsal hippocampus. Taken from The Rat Brain in Stereotaxic Coordinates (Figs. 21, 23, and 25) by Paxinos and Watson (1986).
Reminder Treatment All lesion groups had significantly faster latencies to dig and better choice performance on the Discrimination 3 probe trial than on the Discrimination 2 probe trial. Numerous studies have shown that presenting a reminder treatment prior to testing (e.g., brief exposure to the conditioned stimulus or the apparatus) can lead to reactivation of a presumably inaccessible memory (e.g., Riccio & Richardson, 1984; Spear, 1973). Gisquet-Verrier and Schenk (1994) examined the effects of reminder treatments on rats that received hippocampal lesions prior to training and found that both the hippocampal lesioned rats and the controls that had received the reminder treatment prior to testing performed significantly better than the hippocampal lesioned rats and the controls that had not received the reminder treatment. In addition, Land et al. (2000) examined the
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FIG. 5. Representative small PRER lesion appears on the left, while the corresponding normal brain slice appears on the right. This brain had damage to the perirhinal and entorhinal cortices, along with damage to the caudate and putamen and the ventral hippocampus. Taken from The Rat Brain in Stereotaxic Coordinates (Figs. 26, 35, 43, and 46) by Paxinos and Watson (1986).
effectiveness of a reminder treatment on rats with posttraining hippocampal lesions and found that hippocampal-lesioned rats that received a reminder treatment prior to testing performed significantly better than lesioned rats not receiving a reminder treatment. The reminder treatment phenomenon can be used to explain the decreased latencies and improved choice behavior found in the present experiment in all groups on probe
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FIG. 6. Representative large PRER lesion appears on the left, while the corresponding normal brain slice appears on the right. This brain had damage to the perirhinal and entorhinal cortices, along with damage to the ventral hippocampus. Taken from The Rat Brain in Stereotaxic Coordinates (Figs. 26, 35, 43, and 46) by Paxinos and Watson (1986).
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trials from Discrimination 2 to Discrimination 3. Presumably, the presentation of the probe trial for Discrimination 2 served as a reminder treatment and reactivated the memory for Discrimination 3, which led to better performance when rats were tested on Discrimination 3. The Discrimination 2 probe trial did not allow for new learning to take place because no reinforcement was provided (i.e., no FL were buried in the sand). In fact, because the Discrimination 2 probe trial was not reinforced, it served essentially as an extinction trial. So if rats learned anything from the Discrimination 2 probe trial it should have been that food was no longer available and that they should stop digging. In addition, previous pilot data from our lab suggests that Discrimination 3 (i.e., cumin versus garlic) is not easier to learn or retain than is Discrimination 2 (i.e., cocoa versus cinnamon). This reminder treatment improvement was seen in all groups and lends support to GisquetVerrier and Schenk (1994) and Land et al. (2000). In addition, the present finding of improved test performance in PRER rats extends the phenomenon of reminder treatmentinduced improvements in lesioned rats. GENERAL DISCUSSION The findings of the present experiment lend support to the Eichenbaum et al. theory of hippocampal system function. As expected, rats with lesions to the hippocampal formation performed similarly to controls on a nonspatial, odor-guided digging task. However, rats with lesions to the PRER were severely impaired on this simple discrimination task. The data from the pretraining manipulation were less clear. Based on the pretraining research described above, it would be expected that the pretraining manipulation would have resulted in improved performance for rats in those conditions. However, that appears not to be the case. It remains possible that alterations in training parameters (e.g., longer or shorter delay intervals between pretraining and surgery) or different lesion techniques (e.g., neurotoxic lesions rather than electrolytic or aspiration lesions) would enhance performance. The reminder treatment findings are interesting in that they show an improvement in performance for animals that are not impaired (i.e., fornix and control rats) as well as those exhibiting severe memory impairments (i.e., PRER-lesioned rats). REFERENCES Eichenbaum, H., Otto, T., & Cohen, N. J. (1994). Two functional components of the hippocampal memory system. Behavioral Brain Sciences, 17, 449–518. Eichenbaum, H., Otto, T., & Cohen, N. J. (1992). The hippocampus: What does it do? Behavioral and Neural Biology, 57, 2–36. Gisquet-Verrier, P., & Schenk, F. (1994). Selective hippocampal lesions in rats do not affect retrieval processes promoted by prior cuing with the conditioned stimulus or the context. Psychobiology, 22(4), 298–303. Gold, P. E., Bueno, O. F., & McGaugh, J. L. (1973). Training and task-related differences in retrograde amnesia thresholds determined by direct electrical stimulation of the cortex in rats. Physiology and Behavior, 11, 57–63. Gordon, W. C., & Mowrer, R. R. (1980). The use of an extinction trial as a reminder treatment following CS. Animal Learning and Behavior, 8, 363–367. Jensen, R. A., & Riccio, D. C. (1970). Effects of prior experience upon retrograde amnesia produced by hypothermia. Physiology and Behavior, 5, 1291–1294.
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