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The effects of the dopamine D1 receptor antagonist SCH23390 on memory reconsolidation following reminder-activated retrieval in day-old chicks
Neurobiology of Learning and Memory 83 (2005) 104–112 www.elsevier.com/locate/ynlme
The effects of the dopamine D1 receptor antagonist SCH23390 on memory reconsolidation following reminder-activated retrieval in day-old chicks Joanne M. Sherry, Matthew W. Hale, Simon F. Crowe* School of Psychological Science, La Trobe University, Bundoora 3086, Australia Received 19 November 2003; revised 3 May 2004; accepted 30 August 2004 Available online 8 December 2004
Abstract This series of experiments examined the involvement of the dopamine D1 receptor antagonist, SCH23390, on memory reconsolidation following reminder-activated retrieval. Day-old male New Hampshire · White Leghorn chicks were trained on a single trial passive avoidance task. A dose of 0.5 mg/kg of SCH23390 was administered subcutaneously 5 min before reminder trials, which were presented at 30, 60, and 90 min following training. Memory deficits were observed when reminder trials were presented at 30 and 60 min following training, but not when a reminder was presented at 90 min. No effect on memory retention was observed when reminder trials were not presented, suggesting that reconsolidation mechanisms were both contingent on the presentation of the reminder and independent of the consolidation process. Following a reminder presented at 60 min post-training, deficits in memory retention emerged between 45 and 60 min. The deficit was prolonged, lasting for up until 48 h after reminder presentation. The results indicate an important role for the D1 receptor in reconsolidation processes. 2004 Elsevier Inc. All rights reserved.
1. Introduction There is an increasing body of literature that implicates dopamine (DA) in learning and memory-related processes across a large number of species and learning paradigms (Floresco & Phillips, 2001; Ichihara, Nabeshima, & Kameyama, 1992; Schnabel & Braun, 1996). Research using the single-trial passive avoidance task and the young chick has revealed an important role for the D1 receptor in memory consolidation. Autoradiographic studies have demonstrated that the D1 receptor subtype is upregulated in the lobus parolfactorius (LPO) at about 30 min following avoidance training (Stewart et al., 1996). The LPO is known to be an important structure in the memory processing that follows avoidance training in the chick (Gigg, Patterson, & Rose, 1994; Hunter & Stewart, 1993; Lowndes & Stewart, 1994). *
Corresponding author.Fax: +61 3 9479 2471. E-mail address: [email protected] (S.F. Crowe).
1074-7427/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2004.08.004
Csillag (1999) proposed a model of peck suppression in the chick that describes the possible mechanism of the D1 receptor influence on behaviour observed following avoidance training. It was suggested that in the chick, a stable level of DA is maintained between the LPO-ventral tegmental area and the LPO-nigral circuits. The level of DA does not change in response to aversive training, however, the upregulation of D1 receptors make them supersensitive to existing levels of DA. This results in an enhanced signal to suppress pecking, which is subsequently observed as memory retention (i.e., avoidance) of the aversive stimulus. A recent study conducted in our laboratory (Hale & Crowe, 2003) employing a D1 agonist and antagonist revealed results consistent with Csillag (1999). Using the single-trial passive avoidance task, and day-old chicks, we observed that the D1 antagonist, SCH23390, disrupted memory for the task from 60 min post-training. Kabai, Stewart, Tarcali, and Csillag (2004) have also reported SCH23390-induced memory disruption
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in chicks trained with this task. Administration of the D1 agonist, SKF 38393, facilitated retention under weak training (20% methyl anthranilate (MeA) diluted in ethanol) conditions (Hale & Crowe, 2004). According to Csillag, antagonism of the D1 receptor would result in a weakened signal to suppress pecking, while stimulating the D1 receptor through application of an agonist may enhance the signal to suppress pecking. The results of Hale and Crowe and Kabai et al. are consistent with these predictions. The finding that blockade of the D1 receptor subtype disrupts memory at 60 min following training, may suggest a role for DA in the late modulation of memory (Rose, 2000). It is generally accepted that the consolidation of long-term memory (LTM) involves the transcription of genes and the formation of structural proteins (Davis & Squire, 1984; Dudai, 1989). An important transcription factor believed to be involved in the initial consolidation process is cAMP response element-binding protein (CREB) (Lamprecht, 1999). There is some evidence in the literature to suggest that stimulation of the D1 receptor may be involved in the pharmacological cascade that leads to the activation of CREB (Liu & Graybiel, 1996). Activation of CREB has also been observed following reminder activated retrieval in mice (Hall, Thomas, & Everitt, 2001; Kida et al., 2002), suggesting that, as with consolidation, the D1 receptor may play a role in the biochemical cascade activated following retrieval. The retention function observed following reactivation of a memory has been referred to as reconsolidation (Nader, 2003; Sara, 2000). Reconsolidation may be loosely defined as again rendering a memory labile and sensitive to disruption by amnesic agents after the process of initial consolidation has taken place (Nader, 2003; Sara, 2000). It has also been argued that a representation of the original trace is activated, rather than the initial trace itself (Nader, Schafe, & Le Doux, 2000b). This acts to preserve the integrity of the originally learned experience (Summers, Crowe, & Ng, 2003) and may be the reason why spontaneous recovery of retention is observed. Defined as such, reconsolidation can be separated from retrieval, which is the act of remembering something learned (Miller, Kasprow, & Schachtman, 1986). These two terms are, however, used interchangeably in the retrieval literature because the two phenomena cannot be separated experimentally. This has led to considerable confusion. In a series of experiments using reminder-activated retrieval and day-old chicks, it was proposed that two phases of retrieval are activated following a reminder (see Summers et al., 2003; for review). It may be contended, however, that the authors are reporting two phases of memory reconsolidation, as the chicks displayed a distressed reaction to the reminder stimulus indicating that the process of retrieval was functional
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in all experiments reported. The first phase of reconsolidation was activated when reminders were presented from 7.5 min after training, and can be inhibited by lanthanum chloride (Summers, Crowe, & Ng, 1996). Retention deficits were consistently shown 5 min after the reminder and subsequently resolved about 10–15 min later. The authors suggested that this phase functions as an immediate recall, allowing the organism to rapidly respond to a recognised stimulus. The second phase, which is more prolonged than the first, appears dependent on the time of memory reactivation following initial training, as the authors reported that the further from the original learned experience the memory is activated the shorter the duration of amnesia. Administration of monosodium glutamate (MSG) (Summers, Crowe, & Ng, 1995) and D ,L -2-amino-5-phosphonvaleric acid (AP5) (Summers, Crowe, & Ng, 1997) can disrupt this phase. The authors suggested that this phase might function to allow modification of the underlying trace to include new information gleaned at the time of retrieval. The deficits observed in both phases of reconsolidation were found to be transient. That is, retention levels returned to normal at a consistent point after the reminder stimulus. This observation supports the contention that with a reminder stimulus a representation of the original trace is activated and subsequently blocked, rather than the original consolidated trace (Nader et al., 2000b). In this way, the memory can be reinstated and retention recovers. Some authors have suggested that consolidation and reconsolidation processes may comprise similar pharmacological properties (Nader, 2003; Sara, 2000). The D1 receptor subtype has been implicated in the consolidation of a learned experience in day-old chicks (Csillag, 1999; Hale & Crowe, 2003). Consistent evidence also seems to indicate a particular role for DA in memory reconsolidation. The aim of the current series of experiments was to explore the effect of the D1 receptor antagonist, SCH23390, on memory reconsolidation following reminder-activated retrieval in the day-old chick.
2. Method 2.1. Animals and experimental housing Male day-old New Hampshire · White Leghorn chickens were obtained from a local hatchery on the morning of each experiment. One chick from each pair was marked on the head with a black mark to assist with identification during data recording. Chicks were housed in pairs to eliminate confounds such as stress from social isolation (Andrew, 1991). Wooden boxes (20 · 25 · 20 cm) were maintained at a temperature of between 26–29 C by a single 25 W white incandescent
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bulb. Chicken mash was made available ad libitum and water was provided when the chicks were kept for more than one day. In Experiment 1, 293 chicks were used. Experiment 2 used 288 chicks and Experiment 3 used 552 chicks. 2.2. Procedure Chicks were trained on a modified version of the single-trial passive avoidance task (Crowe & Hale, 2002). The task in the current study involved four components: pretraining, training, reminder trial, and retention test. 2.2.1. Pretraining Pretraining of the chicks occurred in two phases. A chrome bead (2 mm diameter) coated in water was presented to each pair of chicks for approximately 10 s to encourage the chickÕs natural tendency to peck. The procedure was repeated 20 min later to ensure optimal conditions for training. A water coated red bead (4 mm diameter) was then presented to the chicks, again for 10 s, with the number of pecks to this bead recorded on an electronic hand set connected to an on-line computer. The number of pecks at this bead acted as the chickÕs baseline level of pecking. 2.2.2. Training: experimental group Upon completion of the pretraining phase, the experimental chicks were trained to avoid a red bead visually identical to the one used in pretraining, which was coated in aversive methyl anthranilate (MeA). Chicks that pecked at the aversive bead showed a strong disgust reaction that included behaviours such as beak wiping, head shaking, and distress calls. 2.2.3. Control group Upon completion of the pretraining phase, the control chicks were trained on a water coated red bead visually identical to the stimulus used in pretraining to control for any non-specific effects of the drug not related to memory processes. For example, the DA D1 antagonist, SCH23390, has been shown to produce sedation in open field rat behaviour (Salmi & Ahlenius, 2000). Sedation due to administration of this drug has also been observed in day-old chicks (Hale & Crowe, 2003). Training on a water-coated bead allows examination drug effects on pecking rate independent of any effect the drug may have on memory. Chicks that failed to peck at the training bead within a 10-s period in either the MeA or the water condition were excluded from later analysis. 2.2.4. Reminder trial Memory retrieval for the learned stimulus was activated via a reminder trial. This involved presentation of a dry red bead, visually identical that used in training, for approximately 10 s. Possible lateralisation effects
were avoided by ensuring that the bead was seen with both eyes. Chicks were not permitted to peck at this bead, thus avoiding the possibility of a new trace (i.e., the association between the bead and the absence of its reinforcement properties) being initiated. With the presentation of the reminder stimulus, chicks reacted with distress behaviour, indicating that the presentation of the dry bead was a sufficient stimulus to reactivate the memory for the original learned experience. 2.2.5. Retention trial Retention for the task was measured by presenting the chicks with a dry red bead at various times relevant to the specific experiment. Pecks at this bead were recorded with an electronic handset connected to an on-line computer. An avoidance ratio (AR) was calculated as the number of pecks at the red pretraining bead divided by the number of pecks at the red test bead plus the number of pecks at the red pretraining bead (AR = peck pre/ peck + peck test). Typically, a low avoidance ratio is taken as indicative of a memory deficit. 2.3. Drug preparation and administration Drugs were administered subcutaneously into a ventral skin fold below the rib cage. All drug injections were blind and the codes were not broken until after the behavioural data had been collected. Injections were given freehand using a Terumo 1 ml syringe with a 27.5 gauge needle. As determined in our previous study (Hale & Crowe, 2003), a dose of 0.5 mg/kg of SCH23390 was prepared in saline to a total volume of 100 ll. Control chicks received 100 ll of saline injected in the same manner. SCH23390 was obtained from Sigma Chemicals (Sydney, Australia). 2.4. Statistical analysis Analysis in Experiments 1 and 2 were by independent samples t test, which compared the mean avoidance ratios of the drug and saline groups under each condition. Analysis in Experiment 3 was by orthogonal planned contrasts, which compared the drug and saline conditions at each of the seven retrieval-test intervals. All data were analysed using the statistical software package SPSS and type I error rate was set a priori at .05.
3. Results 3.1. Experiment 1: Effect of reminder trials presented at 30 min on SCH23390 induced disruption of memory at various times of injection The first experiment in the series investigated the effect of the DA D1 antagonist, SCH23390, on retention
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at 180 min after training when reminder trials were presented at 30 min after training. The reminder time of 30 min was chosen to sample the period where the DA D1 receptor has been shown to be upregulated following avoidance training (Stewart et al., 1996). The effects on retention were also compared when chicks received injections 5 min before the training trial and 5 min before the reminder trial. These four comparisons were done to determine the appropriate time of injection in order to target reconsolidation mechanisms, and to determine whether the DA D1 receptor has a role to play in memory reconsolidation. It was expected that memory retention would be disrupted when injections were administered 5 min before training in the absence of a reminder, consistent with previous research (Hale & Crowe, 2003). It was also expected that retention following injections administered before the reminder trial would be disrupted. Independent sample t tests revealed a non-significant effect between drug and saline groups when chicks were injected before training and presented with a reminder (t(24) = 1.27, p = .22). Consistent with previous research (Hale & Crowe, 2003), a significant retention deficit was revealed in chicks that received an injection before training and were not presented with a reminder (t(34) = 3.77, p = .001) (see Fig. 1). Analysis of differences in retention when chicks received injections 5 min before the reminder was presented at 30 min post-training, revealed a significant effect of the drug (t(34) = 2.18, p = .036). No difference in retention was detected between chicks that received saline, or SCH23390, at 180 min post-training when no reminder was presented (t(29) = 0.16, p = .88), suggesting that the memory deficit produced by injections of SCH23390 was contingent on the presence of the reminder (see Fig. 2). To control for any non-specific effects of the drug not due to memory processes, such as the sedative effect previously noted, chicks were trained on a water coated red bead and administered injections of SCH23390 or saline at identical times to the birds trained on the MeA bead.
Fig. 1. Retention function after a reminder trial at 30 min and injections of SCH23390 or saline 5 min before training on mean avoidance ratio (+SEM) measured at 180 min following training (*p < .05).
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Fig. 2. Retention function after a reminder trial at 30 min and injections of SCH23390 or saline 5 min before reminder time on mean avoidance ratio (+SEM) measured at 180 min following training (*p < .05).
Non-significant effects were found, when injections were administered 5 min before the reminder was presented (t(38) = 1.52, p = .14), or when no reminder was presented (t(38) = 0.50, p = .62). Non-significant effects were also demonstrated, when birds received injections 5 min before training in both the reminder condition (t(35) = 1.07, p = .29), and in the no reminder condition (t(35) = 0.10, p = .92). The results indicate that the deficits observed in the birds trained on the aversive MeA bead, were due to memory-related deficits rather than to other non-specific effects of the drug. 3.2. Experiment 2: Effect of reminder trials presented at 60 and 90 min following training on SCH23390 induced disruption of memory retention The second experiment in the series investigated the effects of presenting reminders at 60 and 90 min posttraining on retention tested at 180 min. These times were chosen to sample the period of D1 upregulation in the LPO (Stewart et al., 1996), and the period where blockade of the D1 receptor has been shown previously to produce memory deficits during consolidation (Hale & Crowe, 2003). Experiment 1 indicated that the DA D1 receptor antagonist, SCH23390, produced deficits in memory retention following administration of the drug 5 min before a reminder trial presented at 30 min. The deficits were not seen in the absence of a reminder. Therefore injections were administered 5 mins before the reminder in the remaining two experiments. It was expected that administration of SCH23390 would disrupt memory for the passive avoidance task. It was also expected that deficits would not be present in the absence of a reminder consistent with the retrieval literature (Summers et al., 2003) and the results demonstrated in Experiment 1. Chicks were administered SCH23390 5 min before the reminder. To control for the effect of presenting a reminder, half the chicks received the same drug treatments as the experimental groups, without the
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effect was revealed in water-trained birds in the absence of a reminder when the drug was administered at 85 (t(38) = 2.14, p = .039) min following training. 3.3. Experiment 3: Retention function for chicks given a reminder trial at 60 min post-training
Fig. 3. Retention function after reminder trials at 30, 60, and 90 min and injections of SCH23390 or saline 5 min before the reminder trial on mean avoidance ratio (+SEM) measured at 180 min after training (*p < .05).
presentation of the reminder. To control for any nonspecific effects of the drug not due to the training, water trained control chicks received the same drug treatments as the experimental chicks. Independent sample t tests were conducted to compare mean avoidance ratios of chicks that received SCH23390 or saline in the presence, or absence, of a reminder at the times outlined above. Analysis revealed significant differences in retention at 180 min when a reminder was presented at 60 (t(35) = 2.54, p = .016) min post-training. No effect was detected at 180 min when chicks were presented with a reminder at 90 min (t(28) = 0.03, p = .98) post-training. To aid in comparison, all three-reminder times (i.e., 30, 60, and 90 min) are presented in Fig. 3. Chicks that received the same drug treatments as experimental chicks without the presentation of a reminder, demonstrated non-significant effects at 180 min when injections were administered at 55 (t(29) = 0.70, p = .49), and 85 (t(32) = 1.76, p = .09) min following training (see Fig. 4). Under water-trained conditions, independent samples t tests revealed non-significant differences between drug and saline conditions when reminders were presented at 60 (t(38) = 1.17, p = .25) and 90 (t(37) = 0.99, p = .33) min post-training. Non-significant effects were also revealed in the condition where no reminder was presented and an injection was administered at 55 (t(33) = 0.98, p = .37) min following training. However, a significant
Fig. 4. Retention function after injections of SCH23390 or saline at 25, 55, and 85 min after training on mean avoidance ratio (+SEM) measured at 180 min after training.
Experiment 3 investigated the nature of memory retention following a reminder. Results from Experiments 1 and 2 demonstrated that deficits in retention were present following reminders at 30 and 60 min. The deficit following a reminder at 30 min appeared slightly larger than that following a 60-min reminder. However, the reminder time of 60 min was chosen for Experiment 3 because previous research has revealed effects in retention from blockade of the D1 receptor at about 60 min following training (Hale & Crowe, 2003). This suggests that processes affected by DA blockade are active at this time. Chicks were administered either saline, or SCH23390, 5 min before a reminder at 60 min. Retention was tested at 45, 60, 90, 120, 180, 1440, and 2880 min following the reminder. Experiment 2 demonstrated that the memory deficit was not present in chicks that did not receive a reminder at 60 min, therefore it was not necessary to control for the presence of a reminder in Experiment 3. Consistent with previous research examining reminderactivated retrieval (Summers et al., 2003), the deficit in retention was expected to be transient. It was also expected that the deficit would begin from around 60 min following the reminder consistent with previous research investigating the effects of this drug (Hale & Crowe, 2003). Planned orthogonal contrasts were used to analyse differences in retention following a reminder at 60 min posttraining between the drug and saline conditions at each of the seven retrieval-test intervals (see Fig. 5). Equal variance was not assumed. Significant effects were detected at 60 min (t(35.94) = 2.23, p = .032), 90 min (t(31.02) = 3.55, p = .001), 120 min (t(27.46) = 3.35, p = .002), 180 min (t(21.64) = 3.26, p = .004), 1440 min (t(27.72) = 2.10, p = .045), and 2880 min (t(23.88) = 2.31, p = .03). No effect was detected at 45 min following the reminder (t(36.73) = 0.21, p = .83). The results illustrated in Fig. 5 indicate that SCH23390 produced a deficit in memory retention, which began from between 45 and 60 min post-reminder. Levels of retention had not significantly recovered by 2880 min (48 h), post reminder. The deficit can therefore not be considered to be transient, however, retention appeared to be improving from 180 min following the reminder. Water-trained controls that received the same drug treatments as the experimental chicks in Experiment 3 were used to control for any non-specific effects of the drug that may have affected pecking rates. Planned orthogonal contrasts found no significant effects at the
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Fig. 5. Retention function after a reminder trial at 60 min at seven retrieval-test intervals on mean avoidance ratio ( ± SEM) (*p < .05).
seven retrieval-test intervals, indicating that the memory deficit observed in the birds trained on the aversive stimulus was a result of the drugÕs action on reconsolidation processes, rather than due to non-specific effects.
4. Discussion The results from the current series of experiments indicate an important role for the DA D1 receptor in reconsolidation processes. The D1 receptor has been implicated in consolidation of the one-trial passive avoidance task in young chicks (Csillag, 1999; Hale & Crowe, 2003; Kabai et al., 2004; Stewart et al., 1996), however, this is the first study which has addressed the issue of reconsolidation, with reference to the D1 receptor subtype using the passive avoidance paradigm. Experiment 1 revealed a significant difference in retention, when the drug was administered 5 min before training and no reminder was presented, consistent with previous research conducted in our laboratory (Hale & Crowe, 2003). However, no effect was detected at 180 min under the same injection protocol in the presence of a reminder at 30 min. This suggests that a reminder trial presented to chicks 30 min after training overcame the SCH23390-induced amnesia. Previous research has demonstrated that a trace consolidated under weak training conditions may be strengthened to levels seen with strong learning when the chicks are presented with reminder trials (Crowe, Ng, & Gibbs, 1989; Summers, Crowe, & Ng, 2000). The authors proposed that the strengthened trace demonstrated that memories could be modified after initial consolidation. The results of Experiment 1, when injections are administered before training, are consistent with this proposition. In the absence of a reminder, it is clear that D1 activation is necessary for consolidation, as a memory deficit was observed. Therefore, presenting a reminder may act to assist in recovery of the memory trace under these conditions.
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Significant effects were demonstrated when the drug was administered 5 min before the presentation of the reminder at 30 min. No effect was found under this protocol, when no reminder was presented. This suggests that the deficit observed was contingent on the reminder presentation, rather than the time of injection, as without the presentation of the reminder and the reactivation of the trace, memory retention at 180 min was unchanged. This result suggests that: (1) no deficit was observed when no reminder was present because the memory was not reactivated; therefore no reconsolidation phase could be initiated and (2) administration of the drug at this time has no effect on the formation of the initial trace. If it did, a deficit would be observed regardless of whether the memory was reactivated with a reminder because the original ÔcopyÕ of the memory would be compromised. The protocol of administering the drug 5 min before the reminder to target reconsolidation processes independent of consolidation mechanisms is therefore justified by this result. Experiment 2 revealed a deficit in memory retention at 180 min following training when a reminder trial was presented at 60 min. The deficit was not present in the absence of a reminder or when a reminder was presented at 90 min. The lack of effect at 90 min suggests two possibilities. One, that activation of the reconsolidation phase dependent on D1 activation is contingent upon the time of reminder presentation, that is, that after this time the memory mechanisms reliant on D1 activation are now fixed, or two, that it is more difficult at this time to return the underlying trace to a reactivated state, so that reconsolidation is not initiated. The second possibility may be explored by applying a stronger reactivation trial. For example, presenting the reminder stimulus for longer than 10 s may push the memory trace beyond threshold so that it is reactivated and reconsolidation can occur. Alternatively, the lack of an effect may indicate that D1 activation is no longer actively participating in reconsolidation processes. Autoradiographic studies have shown that the D1 receptor is upregulated at about 30 min following avoidance training (Stewart et al., 1996). The upregulation of this receptor subtype appears to be an important factor in consolidating memory following passive avoidance training. CsillagÕs (1999) model predicts that blockade of the D1 receptor may lead to a weakened signal to suppress pecking, and thus low avoidance of the previously aversive stimulus. It is possible that by 90 min post-training, the upregulation of the D1 receptor in the LPO has resolved and DA-dependent mechanisms no longer participate in the retrieval process. Blocking these receptors with antagonists would therefore have no effect on subsequent retention, consistent with the result demonstrated in the current study.
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Experiment 3 explored the effect on retention across seven retrieval-test intervals when a reminder was presented at 60 min. Significant retention deficits were found at 60 min. A transient deficit was expected consistent with previous research (Summers et al., 2003). This suggests that a representative trace is activated during reconsolidation (Sara, 2000) leaving the underlying trace established during consolidation preserved. Therefore, when reconsolidation ceases, retention can recover because the original underlying trace takes over the task of remembering. Observing this phenomenon strengthens the assertion that the process affected by drug administration is reconsolidation. A transient deficit was not found in the current study, however, retention levels appeared to be recovering by 180 min suggesting that they were resolving and retention was returning to baseline level. The current series of experiments were repeated with chicks trained on a water-coated red bead to control for non-specific effects of the drug. The DA D1 antagonist, SCH23390, has been shown previously to cause sedation both in the rat (Salmi & Ahlenius, 2000) and in the dayold chick (Hale & Crowe, 2003). The sedation could have decreased the rate of pecking and over inflated the avoidance ratios. Therefore, differences between chicks that received drug or saline may not have been solely attributable to memory processes. With the exception of one data point, no differences between drug and saline conditions were detected when trained on the water-coated bead, indicating that the rate of pecking was not affected by non-specific effects such as sedation. From this, the conclusion can be drawn that deficit observed in the experimental condition was due to the drugs action on D1 receptors and their subsequent effect on memory related processes. A significant effect under water-trained conditions was detected in the condition where no reminder was presented at 90 min. It is unclear why a difference was detected at this point as no other effects were detected with or without a 90-min reminder. The difference is unlikely to be a result of sedation however, as previous research has demonstrated that pecking rates return to normal by 45 min following drug injection (Hale & Crowe, 2003). This result was also found in the current study when no difference between drug and saline birds was detected at 45 min when trained on the watercoated bead in Experiment 3. Therefore, the significant difference detected was probably attributable to sampling error. The phase of reconsolidation dependent on D1 activation identified in the current study appears analogous to Summers et al.Õs (2003) proposed second phase of memory reconsolidation, which is suggested to function as a mechanism to allow modification of the underlying trace to occur. The delay between time of reminder activation and the observed deficit indicates that the D1-de-
pendent phase could not act as an immediate recall mechanism, similar to the proposed first phase of memory reconsolidation. Instead, similar to the observed disruption by MSG (Summers et al., 1995) and AP5 (Summers et al., 1997), the deficit produced by blockade of the D1 receptor was prolonged and appeared dependent on the time of reminder activation, as a reminder trial at 90 min did not reactivate the trace. Due to these similarities, it is feasible to suggest that the D1-dependent phase of memory retrieval may also function to allow modification of the underlying trace. If the D1 receptor is involved in the pharmacological cascade leading to modification of the underlying trace, it is possible that this cascade may lead to activation of the transcription factor CREB, and the subsequent formation of structural proteins responsible for LTM. This cascade has been implicated in the initial consolidation of LTM. Briefly, stimulation of the D1 receptor activates the enzyme adenylyl cyclase, which subsequently increases the levels of intracellular cyclic AMP (cAMP) between 30 and 60 min post-training (Brown, 1984; Palermo-Neto, 1997). As a consequence of this increase, cAMP-dependent protein kinase A (PKA) is activated. Blockade of PKA has also been demonstrated to disrupt memory around 60 min following training in young chicks (Serrano et al., 1994; Zhao et al., 1995). It is suggested that PKA may then act to phosphorylate both CREB (Bacskai et al., 1993) and dopamine and adenosine 3 0 ,5 0 -monophosphate-regulated phosphoprotein (DARPP-32) (Liu & Graybiel, 1996). When activated, DARPP-32 inhibits protein phosphotase 1 (PP-1) (Greengard, Allen, & Nairn, 1999). This compound dephosphorylates CREB (Liu & Graybiel, 1996); therefore inhibition of PP-1 by DARPP-32 acts to prolong the activity of CREB by preventing dephosphorylation. Increased levels of CREB following retrieval have been reported in mice (Guzowski, 2002; Kida et al., 2002). Additionally, the time of disruption in the current study corresponds to the reported increased level of intracellular cAMP at 60 min post-training (Brown, 1984), and the time at which PKA inhibitors have been shown to disrupt memory (Serrano et al., 1994; Zhao et al., 1995). If the phase of reconsolidation dependent on D1 activation acts to modify the underlying trace, analogous with the second phase of reconsolidation identified by Summers et al. (2003), it is feasible to suggest that this modification may involve additional gene transcription and structural protein changes that cement the new information into LTM. Through CREB activation and the subsequent morphological changes responsible for maintaining LTM, the newly acquired information could be retained within the underlying trace. Previous studies have demonstrated that protein synthesis inhibitors can inhibit reconsolidation after reminder presentations (Litvin & Anokhin, 2000).
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Although CREB activation has been demonstrated in mice, future research should examine the compounds involved in the cascade outlined above following reminder-activated retrieval in the young chick. Establishing that cAMP, PKA, and CREB are activated during reconsolidation in the chick, will add support to the proposition that modifying a trace with information gleaned at the time of retrieval leads to the transcription of genes and long-term morphological changes. That these changes occur during initial consolidation is generally accepted, however, this is a relatively new idea within the retrieval and reconsolidation literature.
Acknowledgments The authors acknowledge the Australian Research Council Discovery Grant (DP0343051) awarded to Professor Crowe, which supported this work.
References Andrew, R. (1991). The chick in experiment: techniques and tests. In R. J. Andrew (Ed.), Neural and behavioural plasticity: the use of the domestic chick as a model. New York: Oxford University Press. Bacskai, B., Hochner, B., Mahaut-Smith, M., Adams, S., Kaang, B., & Kandel, E. (1993). Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons. Science, 260, 222–226. Brown, D. (1984). Changes in brain cAMP accumulation at specific times after learning. In: The role of adenosine 3 0 ,5 0 -cyclic monophosphate (cAMP) in memory formation. A behavioural, pharmacological and biochemical study, PhD thesis, Department of Psychology, La Trobe University. Crowe, S., & Hale, M. (2002). Carryover effects associated with the single-trial passive avoidance learning task in the young chick. Neurobiology of Learning and Memory, 78, 321–331. Crowe, S., Ng, K., & Gibbs, M. (1989). Effect of retraining trials on memory consolidation in weakly reinforced learning. Pharmacology, Biochemistry and Behaviour, 33, 889–894. Csillag, A. (1999). Striato-telencephalic and striato-tegmental circuits: relevance to learning in domestic chicks. Behavioural Brain Research, 98, 227–236. Davis, H., & Squire, L. (1984). Protein synthesis and memory. A review. Psychological Bulletin, 96, 518–559. Dudai, Y. (1989). The neurobiology of memory concepts findings, trends. Oxford: Oxford University Press. Floresco, S., & Phillips, A. (2001). Delay-dependent modulation of memory retrieval by infusion of a dopamine D1 agonist into the rat medial prefrontal cortex. Behavioural Neuroscience, 115(4), 934–939. Gigg, J., Patterson, T., & Rose, S. (1994). Increases in neuronal neural burst recorded from the chick lobus parolfactorius after training are both time dependent and memory specific. European Journal of Neuroscience, 6, 313–319. Greengard, P., Allen, P., & Nairn, A. (1999). Beyond the dopamine receptor: the DARPP-32/ protein phosphotase-1 cascade. Neuron, 23, 435–447. Guzowski, J. (2002). Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches. Hippocampus, 12, 86–104.
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The effects of the dopamine D1 receptor antagonist SCH23390 on memory reconsolidation following reminder-activated retrieval in day-old chicks Joanne M. Sherry, Matthew W. Hale, Simon F. Crowe* School of Psychological Science, La Trobe University, Bundoora 3086, Australia Received 19 November 2003; revised 3 May 2004; accepted 30 August 2004 Available online 8 December 2004
Abstract This series of experiments examined the involvement of the dopamine D1 receptor antagonist, SCH23390, on memory reconsolidation following reminder-activated retrieval. Day-old male New Hampshire · White Leghorn chicks were trained on a single trial passive avoidance task. A dose of 0.5 mg/kg of SCH23390 was administered subcutaneously 5 min before reminder trials, which were presented at 30, 60, and 90 min following training. Memory deficits were observed when reminder trials were presented at 30 and 60 min following training, but not when a reminder was presented at 90 min. No effect on memory retention was observed when reminder trials were not presented, suggesting that reconsolidation mechanisms were both contingent on the presentation of the reminder and independent of the consolidation process. Following a reminder presented at 60 min post-training, deficits in memory retention emerged between 45 and 60 min. The deficit was prolonged, lasting for up until 48 h after reminder presentation. The results indicate an important role for the D1 receptor in reconsolidation processes. 2004 Elsevier Inc. All rights reserved.
1. Introduction There is an increasing body of literature that implicates dopamine (DA) in learning and memory-related processes across a large number of species and learning paradigms (Floresco & Phillips, 2001; Ichihara, Nabeshima, & Kameyama, 1992; Schnabel & Braun, 1996). Research using the single-trial passive avoidance task and the young chick has revealed an important role for the D1 receptor in memory consolidation. Autoradiographic studies have demonstrated that the D1 receptor subtype is upregulated in the lobus parolfactorius (LPO) at about 30 min following avoidance training (Stewart et al., 1996). The LPO is known to be an important structure in the memory processing that follows avoidance training in the chick (Gigg, Patterson, & Rose, 1994; Hunter & Stewart, 1993; Lowndes & Stewart, 1994). *
Corresponding author.Fax: +61 3 9479 2471. E-mail address: [email protected] (S.F. Crowe).
1074-7427/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2004.08.004
Csillag (1999) proposed a model of peck suppression in the chick that describes the possible mechanism of the D1 receptor influence on behaviour observed following avoidance training. It was suggested that in the chick, a stable level of DA is maintained between the LPO-ventral tegmental area and the LPO-nigral circuits. The level of DA does not change in response to aversive training, however, the upregulation of D1 receptors make them supersensitive to existing levels of DA. This results in an enhanced signal to suppress pecking, which is subsequently observed as memory retention (i.e., avoidance) of the aversive stimulus. A recent study conducted in our laboratory (Hale & Crowe, 2003) employing a D1 agonist and antagonist revealed results consistent with Csillag (1999). Using the single-trial passive avoidance task, and day-old chicks, we observed that the D1 antagonist, SCH23390, disrupted memory for the task from 60 min post-training. Kabai, Stewart, Tarcali, and Csillag (2004) have also reported SCH23390-induced memory disruption
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in chicks trained with this task. Administration of the D1 agonist, SKF 38393, facilitated retention under weak training (20% methyl anthranilate (MeA) diluted in ethanol) conditions (Hale & Crowe, 2004). According to Csillag, antagonism of the D1 receptor would result in a weakened signal to suppress pecking, while stimulating the D1 receptor through application of an agonist may enhance the signal to suppress pecking. The results of Hale and Crowe and Kabai et al. are consistent with these predictions. The finding that blockade of the D1 receptor subtype disrupts memory at 60 min following training, may suggest a role for DA in the late modulation of memory (Rose, 2000). It is generally accepted that the consolidation of long-term memory (LTM) involves the transcription of genes and the formation of structural proteins (Davis & Squire, 1984; Dudai, 1989). An important transcription factor believed to be involved in the initial consolidation process is cAMP response element-binding protein (CREB) (Lamprecht, 1999). There is some evidence in the literature to suggest that stimulation of the D1 receptor may be involved in the pharmacological cascade that leads to the activation of CREB (Liu & Graybiel, 1996). Activation of CREB has also been observed following reminder activated retrieval in mice (Hall, Thomas, & Everitt, 2001; Kida et al., 2002), suggesting that, as with consolidation, the D1 receptor may play a role in the biochemical cascade activated following retrieval. The retention function observed following reactivation of a memory has been referred to as reconsolidation (Nader, 2003; Sara, 2000). Reconsolidation may be loosely defined as again rendering a memory labile and sensitive to disruption by amnesic agents after the process of initial consolidation has taken place (Nader, 2003; Sara, 2000). It has also been argued that a representation of the original trace is activated, rather than the initial trace itself (Nader, Schafe, & Le Doux, 2000b). This acts to preserve the integrity of the originally learned experience (Summers, Crowe, & Ng, 2003) and may be the reason why spontaneous recovery of retention is observed. Defined as such, reconsolidation can be separated from retrieval, which is the act of remembering something learned (Miller, Kasprow, & Schachtman, 1986). These two terms are, however, used interchangeably in the retrieval literature because the two phenomena cannot be separated experimentally. This has led to considerable confusion. In a series of experiments using reminder-activated retrieval and day-old chicks, it was proposed that two phases of retrieval are activated following a reminder (see Summers et al., 2003; for review). It may be contended, however, that the authors are reporting two phases of memory reconsolidation, as the chicks displayed a distressed reaction to the reminder stimulus indicating that the process of retrieval was functional
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in all experiments reported. The first phase of reconsolidation was activated when reminders were presented from 7.5 min after training, and can be inhibited by lanthanum chloride (Summers, Crowe, & Ng, 1996). Retention deficits were consistently shown 5 min after the reminder and subsequently resolved about 10–15 min later. The authors suggested that this phase functions as an immediate recall, allowing the organism to rapidly respond to a recognised stimulus. The second phase, which is more prolonged than the first, appears dependent on the time of memory reactivation following initial training, as the authors reported that the further from the original learned experience the memory is activated the shorter the duration of amnesia. Administration of monosodium glutamate (MSG) (Summers, Crowe, & Ng, 1995) and D ,L -2-amino-5-phosphonvaleric acid (AP5) (Summers, Crowe, & Ng, 1997) can disrupt this phase. The authors suggested that this phase might function to allow modification of the underlying trace to include new information gleaned at the time of retrieval. The deficits observed in both phases of reconsolidation were found to be transient. That is, retention levels returned to normal at a consistent point after the reminder stimulus. This observation supports the contention that with a reminder stimulus a representation of the original trace is activated and subsequently blocked, rather than the original consolidated trace (Nader et al., 2000b). In this way, the memory can be reinstated and retention recovers. Some authors have suggested that consolidation and reconsolidation processes may comprise similar pharmacological properties (Nader, 2003; Sara, 2000). The D1 receptor subtype has been implicated in the consolidation of a learned experience in day-old chicks (Csillag, 1999; Hale & Crowe, 2003). Consistent evidence also seems to indicate a particular role for DA in memory reconsolidation. The aim of the current series of experiments was to explore the effect of the D1 receptor antagonist, SCH23390, on memory reconsolidation following reminder-activated retrieval in the day-old chick.
2. Method 2.1. Animals and experimental housing Male day-old New Hampshire · White Leghorn chickens were obtained from a local hatchery on the morning of each experiment. One chick from each pair was marked on the head with a black mark to assist with identification during data recording. Chicks were housed in pairs to eliminate confounds such as stress from social isolation (Andrew, 1991). Wooden boxes (20 · 25 · 20 cm) were maintained at a temperature of between 26–29 C by a single 25 W white incandescent
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bulb. Chicken mash was made available ad libitum and water was provided when the chicks were kept for more than one day. In Experiment 1, 293 chicks were used. Experiment 2 used 288 chicks and Experiment 3 used 552 chicks. 2.2. Procedure Chicks were trained on a modified version of the single-trial passive avoidance task (Crowe & Hale, 2002). The task in the current study involved four components: pretraining, training, reminder trial, and retention test. 2.2.1. Pretraining Pretraining of the chicks occurred in two phases. A chrome bead (2 mm diameter) coated in water was presented to each pair of chicks for approximately 10 s to encourage the chickÕs natural tendency to peck. The procedure was repeated 20 min later to ensure optimal conditions for training. A water coated red bead (4 mm diameter) was then presented to the chicks, again for 10 s, with the number of pecks to this bead recorded on an electronic hand set connected to an on-line computer. The number of pecks at this bead acted as the chickÕs baseline level of pecking. 2.2.2. Training: experimental group Upon completion of the pretraining phase, the experimental chicks were trained to avoid a red bead visually identical to the one used in pretraining, which was coated in aversive methyl anthranilate (MeA). Chicks that pecked at the aversive bead showed a strong disgust reaction that included behaviours such as beak wiping, head shaking, and distress calls. 2.2.3. Control group Upon completion of the pretraining phase, the control chicks were trained on a water coated red bead visually identical to the stimulus used in pretraining to control for any non-specific effects of the drug not related to memory processes. For example, the DA D1 antagonist, SCH23390, has been shown to produce sedation in open field rat behaviour (Salmi & Ahlenius, 2000). Sedation due to administration of this drug has also been observed in day-old chicks (Hale & Crowe, 2003). Training on a water-coated bead allows examination drug effects on pecking rate independent of any effect the drug may have on memory. Chicks that failed to peck at the training bead within a 10-s period in either the MeA or the water condition were excluded from later analysis. 2.2.4. Reminder trial Memory retrieval for the learned stimulus was activated via a reminder trial. This involved presentation of a dry red bead, visually identical that used in training, for approximately 10 s. Possible lateralisation effects
were avoided by ensuring that the bead was seen with both eyes. Chicks were not permitted to peck at this bead, thus avoiding the possibility of a new trace (i.e., the association between the bead and the absence of its reinforcement properties) being initiated. With the presentation of the reminder stimulus, chicks reacted with distress behaviour, indicating that the presentation of the dry bead was a sufficient stimulus to reactivate the memory for the original learned experience. 2.2.5. Retention trial Retention for the task was measured by presenting the chicks with a dry red bead at various times relevant to the specific experiment. Pecks at this bead were recorded with an electronic handset connected to an on-line computer. An avoidance ratio (AR) was calculated as the number of pecks at the red pretraining bead divided by the number of pecks at the red test bead plus the number of pecks at the red pretraining bead (AR = peck pre/ peck + peck test). Typically, a low avoidance ratio is taken as indicative of a memory deficit. 2.3. Drug preparation and administration Drugs were administered subcutaneously into a ventral skin fold below the rib cage. All drug injections were blind and the codes were not broken until after the behavioural data had been collected. Injections were given freehand using a Terumo 1 ml syringe with a 27.5 gauge needle. As determined in our previous study (Hale & Crowe, 2003), a dose of 0.5 mg/kg of SCH23390 was prepared in saline to a total volume of 100 ll. Control chicks received 100 ll of saline injected in the same manner. SCH23390 was obtained from Sigma Chemicals (Sydney, Australia). 2.4. Statistical analysis Analysis in Experiments 1 and 2 were by independent samples t test, which compared the mean avoidance ratios of the drug and saline groups under each condition. Analysis in Experiment 3 was by orthogonal planned contrasts, which compared the drug and saline conditions at each of the seven retrieval-test intervals. All data were analysed using the statistical software package SPSS and type I error rate was set a priori at .05.
3. Results 3.1. Experiment 1: Effect of reminder trials presented at 30 min on SCH23390 induced disruption of memory at various times of injection The first experiment in the series investigated the effect of the DA D1 antagonist, SCH23390, on retention
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at 180 min after training when reminder trials were presented at 30 min after training. The reminder time of 30 min was chosen to sample the period where the DA D1 receptor has been shown to be upregulated following avoidance training (Stewart et al., 1996). The effects on retention were also compared when chicks received injections 5 min before the training trial and 5 min before the reminder trial. These four comparisons were done to determine the appropriate time of injection in order to target reconsolidation mechanisms, and to determine whether the DA D1 receptor has a role to play in memory reconsolidation. It was expected that memory retention would be disrupted when injections were administered 5 min before training in the absence of a reminder, consistent with previous research (Hale & Crowe, 2003). It was also expected that retention following injections administered before the reminder trial would be disrupted. Independent sample t tests revealed a non-significant effect between drug and saline groups when chicks were injected before training and presented with a reminder (t(24) = 1.27, p = .22). Consistent with previous research (Hale & Crowe, 2003), a significant retention deficit was revealed in chicks that received an injection before training and were not presented with a reminder (t(34) = 3.77, p = .001) (see Fig. 1). Analysis of differences in retention when chicks received injections 5 min before the reminder was presented at 30 min post-training, revealed a significant effect of the drug (t(34) = 2.18, p = .036). No difference in retention was detected between chicks that received saline, or SCH23390, at 180 min post-training when no reminder was presented (t(29) = 0.16, p = .88), suggesting that the memory deficit produced by injections of SCH23390 was contingent on the presence of the reminder (see Fig. 2). To control for any non-specific effects of the drug not due to memory processes, such as the sedative effect previously noted, chicks were trained on a water coated red bead and administered injections of SCH23390 or saline at identical times to the birds trained on the MeA bead.
Fig. 1. Retention function after a reminder trial at 30 min and injections of SCH23390 or saline 5 min before training on mean avoidance ratio (+SEM) measured at 180 min following training (*p < .05).
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Fig. 2. Retention function after a reminder trial at 30 min and injections of SCH23390 or saline 5 min before reminder time on mean avoidance ratio (+SEM) measured at 180 min following training (*p < .05).
Non-significant effects were found, when injections were administered 5 min before the reminder was presented (t(38) = 1.52, p = .14), or when no reminder was presented (t(38) = 0.50, p = .62). Non-significant effects were also demonstrated, when birds received injections 5 min before training in both the reminder condition (t(35) = 1.07, p = .29), and in the no reminder condition (t(35) = 0.10, p = .92). The results indicate that the deficits observed in the birds trained on the aversive MeA bead, were due to memory-related deficits rather than to other non-specific effects of the drug. 3.2. Experiment 2: Effect of reminder trials presented at 60 and 90 min following training on SCH23390 induced disruption of memory retention The second experiment in the series investigated the effects of presenting reminders at 60 and 90 min posttraining on retention tested at 180 min. These times were chosen to sample the period of D1 upregulation in the LPO (Stewart et al., 1996), and the period where blockade of the D1 receptor has been shown previously to produce memory deficits during consolidation (Hale & Crowe, 2003). Experiment 1 indicated that the DA D1 receptor antagonist, SCH23390, produced deficits in memory retention following administration of the drug 5 min before a reminder trial presented at 30 min. The deficits were not seen in the absence of a reminder. Therefore injections were administered 5 mins before the reminder in the remaining two experiments. It was expected that administration of SCH23390 would disrupt memory for the passive avoidance task. It was also expected that deficits would not be present in the absence of a reminder consistent with the retrieval literature (Summers et al., 2003) and the results demonstrated in Experiment 1. Chicks were administered SCH23390 5 min before the reminder. To control for the effect of presenting a reminder, half the chicks received the same drug treatments as the experimental groups, without the
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effect was revealed in water-trained birds in the absence of a reminder when the drug was administered at 85 (t(38) = 2.14, p = .039) min following training. 3.3. Experiment 3: Retention function for chicks given a reminder trial at 60 min post-training
Fig. 3. Retention function after reminder trials at 30, 60, and 90 min and injections of SCH23390 or saline 5 min before the reminder trial on mean avoidance ratio (+SEM) measured at 180 min after training (*p < .05).
presentation of the reminder. To control for any nonspecific effects of the drug not due to the training, water trained control chicks received the same drug treatments as the experimental chicks. Independent sample t tests were conducted to compare mean avoidance ratios of chicks that received SCH23390 or saline in the presence, or absence, of a reminder at the times outlined above. Analysis revealed significant differences in retention at 180 min when a reminder was presented at 60 (t(35) = 2.54, p = .016) min post-training. No effect was detected at 180 min when chicks were presented with a reminder at 90 min (t(28) = 0.03, p = .98) post-training. To aid in comparison, all three-reminder times (i.e., 30, 60, and 90 min) are presented in Fig. 3. Chicks that received the same drug treatments as experimental chicks without the presentation of a reminder, demonstrated non-significant effects at 180 min when injections were administered at 55 (t(29) = 0.70, p = .49), and 85 (t(32) = 1.76, p = .09) min following training (see Fig. 4). Under water-trained conditions, independent samples t tests revealed non-significant differences between drug and saline conditions when reminders were presented at 60 (t(38) = 1.17, p = .25) and 90 (t(37) = 0.99, p = .33) min post-training. Non-significant effects were also revealed in the condition where no reminder was presented and an injection was administered at 55 (t(33) = 0.98, p = .37) min following training. However, a significant
Fig. 4. Retention function after injections of SCH23390 or saline at 25, 55, and 85 min after training on mean avoidance ratio (+SEM) measured at 180 min after training.
Experiment 3 investigated the nature of memory retention following a reminder. Results from Experiments 1 and 2 demonstrated that deficits in retention were present following reminders at 30 and 60 min. The deficit following a reminder at 30 min appeared slightly larger than that following a 60-min reminder. However, the reminder time of 60 min was chosen for Experiment 3 because previous research has revealed effects in retention from blockade of the D1 receptor at about 60 min following training (Hale & Crowe, 2003). This suggests that processes affected by DA blockade are active at this time. Chicks were administered either saline, or SCH23390, 5 min before a reminder at 60 min. Retention was tested at 45, 60, 90, 120, 180, 1440, and 2880 min following the reminder. Experiment 2 demonstrated that the memory deficit was not present in chicks that did not receive a reminder at 60 min, therefore it was not necessary to control for the presence of a reminder in Experiment 3. Consistent with previous research examining reminderactivated retrieval (Summers et al., 2003), the deficit in retention was expected to be transient. It was also expected that the deficit would begin from around 60 min following the reminder consistent with previous research investigating the effects of this drug (Hale & Crowe, 2003). Planned orthogonal contrasts were used to analyse differences in retention following a reminder at 60 min posttraining between the drug and saline conditions at each of the seven retrieval-test intervals (see Fig. 5). Equal variance was not assumed. Significant effects were detected at 60 min (t(35.94) = 2.23, p = .032), 90 min (t(31.02) = 3.55, p = .001), 120 min (t(27.46) = 3.35, p = .002), 180 min (t(21.64) = 3.26, p = .004), 1440 min (t(27.72) = 2.10, p = .045), and 2880 min (t(23.88) = 2.31, p = .03). No effect was detected at 45 min following the reminder (t(36.73) = 0.21, p = .83). The results illustrated in Fig. 5 indicate that SCH23390 produced a deficit in memory retention, which began from between 45 and 60 min post-reminder. Levels of retention had not significantly recovered by 2880 min (48 h), post reminder. The deficit can therefore not be considered to be transient, however, retention appeared to be improving from 180 min following the reminder. Water-trained controls that received the same drug treatments as the experimental chicks in Experiment 3 were used to control for any non-specific effects of the drug that may have affected pecking rates. Planned orthogonal contrasts found no significant effects at the
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Fig. 5. Retention function after a reminder trial at 60 min at seven retrieval-test intervals on mean avoidance ratio ( ± SEM) (*p < .05).
seven retrieval-test intervals, indicating that the memory deficit observed in the birds trained on the aversive stimulus was a result of the drugÕs action on reconsolidation processes, rather than due to non-specific effects.
4. Discussion The results from the current series of experiments indicate an important role for the DA D1 receptor in reconsolidation processes. The D1 receptor has been implicated in consolidation of the one-trial passive avoidance task in young chicks (Csillag, 1999; Hale & Crowe, 2003; Kabai et al., 2004; Stewart et al., 1996), however, this is the first study which has addressed the issue of reconsolidation, with reference to the D1 receptor subtype using the passive avoidance paradigm. Experiment 1 revealed a significant difference in retention, when the drug was administered 5 min before training and no reminder was presented, consistent with previous research conducted in our laboratory (Hale & Crowe, 2003). However, no effect was detected at 180 min under the same injection protocol in the presence of a reminder at 30 min. This suggests that a reminder trial presented to chicks 30 min after training overcame the SCH23390-induced amnesia. Previous research has demonstrated that a trace consolidated under weak training conditions may be strengthened to levels seen with strong learning when the chicks are presented with reminder trials (Crowe, Ng, & Gibbs, 1989; Summers, Crowe, & Ng, 2000). The authors proposed that the strengthened trace demonstrated that memories could be modified after initial consolidation. The results of Experiment 1, when injections are administered before training, are consistent with this proposition. In the absence of a reminder, it is clear that D1 activation is necessary for consolidation, as a memory deficit was observed. Therefore, presenting a reminder may act to assist in recovery of the memory trace under these conditions.
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Significant effects were demonstrated when the drug was administered 5 min before the presentation of the reminder at 30 min. No effect was found under this protocol, when no reminder was presented. This suggests that the deficit observed was contingent on the reminder presentation, rather than the time of injection, as without the presentation of the reminder and the reactivation of the trace, memory retention at 180 min was unchanged. This result suggests that: (1) no deficit was observed when no reminder was present because the memory was not reactivated; therefore no reconsolidation phase could be initiated and (2) administration of the drug at this time has no effect on the formation of the initial trace. If it did, a deficit would be observed regardless of whether the memory was reactivated with a reminder because the original ÔcopyÕ of the memory would be compromised. The protocol of administering the drug 5 min before the reminder to target reconsolidation processes independent of consolidation mechanisms is therefore justified by this result. Experiment 2 revealed a deficit in memory retention at 180 min following training when a reminder trial was presented at 60 min. The deficit was not present in the absence of a reminder or when a reminder was presented at 90 min. The lack of effect at 90 min suggests two possibilities. One, that activation of the reconsolidation phase dependent on D1 activation is contingent upon the time of reminder presentation, that is, that after this time the memory mechanisms reliant on D1 activation are now fixed, or two, that it is more difficult at this time to return the underlying trace to a reactivated state, so that reconsolidation is not initiated. The second possibility may be explored by applying a stronger reactivation trial. For example, presenting the reminder stimulus for longer than 10 s may push the memory trace beyond threshold so that it is reactivated and reconsolidation can occur. Alternatively, the lack of an effect may indicate that D1 activation is no longer actively participating in reconsolidation processes. Autoradiographic studies have shown that the D1 receptor is upregulated at about 30 min following avoidance training (Stewart et al., 1996). The upregulation of this receptor subtype appears to be an important factor in consolidating memory following passive avoidance training. CsillagÕs (1999) model predicts that blockade of the D1 receptor may lead to a weakened signal to suppress pecking, and thus low avoidance of the previously aversive stimulus. It is possible that by 90 min post-training, the upregulation of the D1 receptor in the LPO has resolved and DA-dependent mechanisms no longer participate in the retrieval process. Blocking these receptors with antagonists would therefore have no effect on subsequent retention, consistent with the result demonstrated in the current study.
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Experiment 3 explored the effect on retention across seven retrieval-test intervals when a reminder was presented at 60 min. Significant retention deficits were found at 60 min. A transient deficit was expected consistent with previous research (Summers et al., 2003). This suggests that a representative trace is activated during reconsolidation (Sara, 2000) leaving the underlying trace established during consolidation preserved. Therefore, when reconsolidation ceases, retention can recover because the original underlying trace takes over the task of remembering. Observing this phenomenon strengthens the assertion that the process affected by drug administration is reconsolidation. A transient deficit was not found in the current study, however, retention levels appeared to be recovering by 180 min suggesting that they were resolving and retention was returning to baseline level. The current series of experiments were repeated with chicks trained on a water-coated red bead to control for non-specific effects of the drug. The DA D1 antagonist, SCH23390, has been shown previously to cause sedation both in the rat (Salmi & Ahlenius, 2000) and in the dayold chick (Hale & Crowe, 2003). The sedation could have decreased the rate of pecking and over inflated the avoidance ratios. Therefore, differences between chicks that received drug or saline may not have been solely attributable to memory processes. With the exception of one data point, no differences between drug and saline conditions were detected when trained on the water-coated bead, indicating that the rate of pecking was not affected by non-specific effects such as sedation. From this, the conclusion can be drawn that deficit observed in the experimental condition was due to the drugs action on D1 receptors and their subsequent effect on memory related processes. A significant effect under water-trained conditions was detected in the condition where no reminder was presented at 90 min. It is unclear why a difference was detected at this point as no other effects were detected with or without a 90-min reminder. The difference is unlikely to be a result of sedation however, as previous research has demonstrated that pecking rates return to normal by 45 min following drug injection (Hale & Crowe, 2003). This result was also found in the current study when no difference between drug and saline birds was detected at 45 min when trained on the watercoated bead in Experiment 3. Therefore, the significant difference detected was probably attributable to sampling error. The phase of reconsolidation dependent on D1 activation identified in the current study appears analogous to Summers et al.Õs (2003) proposed second phase of memory reconsolidation, which is suggested to function as a mechanism to allow modification of the underlying trace to occur. The delay between time of reminder activation and the observed deficit indicates that the D1-de-
pendent phase could not act as an immediate recall mechanism, similar to the proposed first phase of memory reconsolidation. Instead, similar to the observed disruption by MSG (Summers et al., 1995) and AP5 (Summers et al., 1997), the deficit produced by blockade of the D1 receptor was prolonged and appeared dependent on the time of reminder activation, as a reminder trial at 90 min did not reactivate the trace. Due to these similarities, it is feasible to suggest that the D1-dependent phase of memory retrieval may also function to allow modification of the underlying trace. If the D1 receptor is involved in the pharmacological cascade leading to modification of the underlying trace, it is possible that this cascade may lead to activation of the transcription factor CREB, and the subsequent formation of structural proteins responsible for LTM. This cascade has been implicated in the initial consolidation of LTM. Briefly, stimulation of the D1 receptor activates the enzyme adenylyl cyclase, which subsequently increases the levels of intracellular cyclic AMP (cAMP) between 30 and 60 min post-training (Brown, 1984; Palermo-Neto, 1997). As a consequence of this increase, cAMP-dependent protein kinase A (PKA) is activated. Blockade of PKA has also been demonstrated to disrupt memory around 60 min following training in young chicks (Serrano et al., 1994; Zhao et al., 1995). It is suggested that PKA may then act to phosphorylate both CREB (Bacskai et al., 1993) and dopamine and adenosine 3 0 ,5 0 -monophosphate-regulated phosphoprotein (DARPP-32) (Liu & Graybiel, 1996). When activated, DARPP-32 inhibits protein phosphotase 1 (PP-1) (Greengard, Allen, & Nairn, 1999). This compound dephosphorylates CREB (Liu & Graybiel, 1996); therefore inhibition of PP-1 by DARPP-32 acts to prolong the activity of CREB by preventing dephosphorylation. Increased levels of CREB following retrieval have been reported in mice (Guzowski, 2002; Kida et al., 2002). Additionally, the time of disruption in the current study corresponds to the reported increased level of intracellular cAMP at 60 min post-training (Brown, 1984), and the time at which PKA inhibitors have been shown to disrupt memory (Serrano et al., 1994; Zhao et al., 1995). If the phase of reconsolidation dependent on D1 activation acts to modify the underlying trace, analogous with the second phase of reconsolidation identified by Summers et al. (2003), it is feasible to suggest that this modification may involve additional gene transcription and structural protein changes that cement the new information into LTM. Through CREB activation and the subsequent morphological changes responsible for maintaining LTM, the newly acquired information could be retained within the underlying trace. Previous studies have demonstrated that protein synthesis inhibitors can inhibit reconsolidation after reminder presentations (Litvin & Anokhin, 2000).
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Although CREB activation has been demonstrated in mice, future research should examine the compounds involved in the cascade outlined above following reminder-activated retrieval in the young chick. Establishing that cAMP, PKA, and CREB are activated during reconsolidation in the chick, will add support to the proposition that modifying a trace with information gleaned at the time of retrieval leads to the transcription of genes and long-term morphological changes. That these changes occur during initial consolidation is generally accepted, however, this is a relatively new idea within the retrieval and reconsolidation literature.
Acknowledgments The authors acknowledge the Australian Research Council Discovery Grant (DP0343051) awarded to Professor Crowe, which supported this work.
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