Ketamine inhibits long-term, but not intermediate-term memory formation in Lymnaea stagnalis

Neuroscience 155 (2008) 613– 625

KETAMINE INHIBITS LONG-TERM, BUT NOT INTERMEDIATE-TERM MEMORY FORMATION IN LYMNAEA STAGNALIS K. BROWNING* AND K. LUKOWIAK*

ative memory is the storage of skills or procedures (Squire and Zola, 1996; Squire, 2004). To date, there have been a relatively small number of studies concerning ketamine’s effects on memory acquisition, consolidation and/or retrieval. While most have focused on ketamine’s effects on various forms of declarative memory, evidence from Morgan et al. (2004) suggests that at least in humans, ketamine also impairs non-declarative memory. To our knowledge this is the only study, in any species, that has focused directly on ketamine’s effects on non-declarative memory. Consequently, we set out to determine further the effects of ketamine on non-declarative intermediate-term (ITM) and long-term memory (LTM), in a well-established operant conditioning model, the fresh water pond snail, Lymnaea stagnalis, in which both, ITM and LTM have been previously demonstrated (Lukowiak et al., 2000). Lymnaea is a bimodal breather; that is, it is able to satisfy its respiratory requirements both cutaneously and aerially. Aerial respiration is accomplished at the water–air interface via the snail opening its respiratory orifice, termed the pneumostome, while at the same time contracting and relaxing its respiratory muscles (Lukowiak et al., 1996). Snails typically only resort to increased aerial respiration as their environment becomes hypoxic (Lukowiak et al., 1996). Aerial respiratory behavior, as a result, can therefore be operantly conditioned in a hypoxic environment. Conditioning results in fewer attempted openings in later memory tests and serves as our operational definition of memory. Since snails can still perform cutaneous respiration in hypoxia, snails trained not to perform aerial respiration are not harmed as a result of training (Lukowiak et al., 1996, 1998; Martens et al., 2007a). Recently, a onetrial training (1TT) procedure has been established in our laboratory in which the snail’s first aerial respiration attempt, when placed in a hypoxic environment, is followed by a single training event (Martens et al., 2007b). This single training event has been shown to be sufficient to produce LTM (Martens et al., 2007a,b). The advantage of the 1TT paradigm for the present ketamine studies is that training takes place over a short 35 s interval, requires only one training exposure and therefore, the time at which consolidation begins can be accurately estimated to occur directly after exposure to the single training event (Martens et al., 2007b). This allowed us to manipulate ketamine administration around the consolidation period, with confidence, in a precise and consistent manner. Depending on the training procedure used in Lymnaea, either ITM (persisting up to 3 h) or LTM (persisting at least 24 h) results following operant conditioning of aerial respiratory behavior (Lukowiak et al., 1998, 2000, 2003). We

Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 2104 Heath Sciences Centre, 3330 Hospital Drive, Calgary, Alberta, Canada T2N 4N1

Abstract—We investigated the effects of the drug ketamine on procedural intermediate- and long-term memory formation in a well-established operant learning and memory model system, Lymnaea stagnalis. Animals were administered ketamine at discrete time points, ranging from 2 h pre-one-trial training (1TT) to 23 h post-1TT. Our results demonstrated that ketamine causes impairment of procedural memory formation, and that ketamine acts differentially, inhibiting only long-term memory (LTM) formation while having no effect on intermediate-term memory (ITM) formation. Ketamine’s ability to inhibit LTM was found not to be due to state dependent learning implying that ketamine’s effects are therefore specific to the molecular process involved in procedural LTM formation. Given past data from our laboratory, this suggests that ketamine may be exerting its differential effects by altering the gene transcription processes necessary and specific for LTM formation. Additionally, ketamine was found to have no effect on retrieval when administered 1 h before testing. However, ketamine was able to disrupt LTM formation when administered immediately before 1TT and up to 2 h after 1TT. Our findings suggest a longer period of consolidation after 1TT than previously demonstrated in Lymnaea, during which the procedural long-term memory remains labile and is vulnerable to disruption via amnestic agents, such as ketamine. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: pharmacology, consolidation, gene transcription, invertebrate, fresh-water pond snail.

Ketamine, a derivative of phencyclidine (Domino et al., 1965), is known to possess dissociative, anesthetic and psychomimetic properties and is commonly used in emergency and pediatric medicine (Wolff and Winstock, 2006). However, despite its continued use in medical and ‘recreational’ domains, its effects on various higher-level cognitive processes, such as its ability to cause amnestic effects, are not well understood (Morgan and Curran, 2006). Memory can be parsed into two main categories: declarative and non-declarative forms. Declarative memory is that aspect of memory that stores facts; while non-declar*Corresponding author. Tel: ⫹1-403-220-4493; fax: ⫹1-403-283-2700. E-mail addresses: [email protected] (K. Browning), lukowiak@ ucalgary.ca (K. Lukowiak). Abbreviations: ITM, intermediate-term memory; K-S, KolmogorovSmirnov; LTM, long-term memory; NMDA, N-methyl-D-aspartate; PHP, post hoc power; Post-Obs, post-observational; Pre-Obs, preobservational; PW, pond water; RM-ANOVA, repeated measures analysis of variance; RPeD1, right pedal dorsal 1; TBT, total breathing time; 1TT, one-trial training.

0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.06.012

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have also found that while both ITM and LTM depend upon new protein synthesis there is an additional requirement of altered gene activity (i.e. transcription) for LTM formation (Sangha et al., 2003b,d). Importantly, we have also shown the necessary requirement for the soma of the neuron, which is responsible for initiating rhythmogenesis of the aerial respiratory central pattern generator (CPG), right pedal dorsal 1 (RPeD1) (i.e. the genes) to be present in order for LTM to form (Scheibenstock et al., 2002). In fact, RPeD1’s soma must be intact for other aspects of memory to be seen as well, including: reconsolidation, extinction and forgetting (Sangha et al., 2003a,c, 2004, 2005). However, ITM continues to form in snails in which RPeD1’s soma has been ablated (Scheibenstock et al., 2002; Parvez et al., 2005, 2006). Ketamine’s ability to alter gene transcription was the primary factor for choosing to investigate its effects on memory formation in our Lymnaea model system. Ketamine’s ability to act as a non-competitive antagonist at N-methyl-D-aspartate (NMDA) receptor sites is typically what is thought to endow it with its dissociative properties and its ability to disrupt declarative memory formation (Wolff and Winstock, 2006). However, more recently it has been demonstrated that ketamine may exert some of its effects via less studied mechanisms, such as by altering gene transcription (Sakai et al., 2000; Chen et al., 2005; Yu et al., 2007). As ketamine has the ability to alter gene transcription, we hypothesized that it could selectively block the formation of LTM, while not interfere with the formation of ITM (see below) in our Lymnaea model. In the present study, we extend the Martens et al. (2007b) findings to establish that, in addition to LTM, 1TT is sufficient to also produce ITM. We then go on to demonstrate that ketamine exerts differential effects on LTM and ITM. That is, ketamine was found to inhibit LTM formation while having no effect on ITM formation. Additionally, ketamine’s inhibitory effects on LTM formation were observed to occur in a time dependent manner. Further, we found that ketamine does not affect retrieval and its effects on LTM are not explained by state dependent learning. This finding substantiates the notion that ITM and LTM work via different molecular mechanisms in Lymnaea stagnalis despite the training regimen and that ketamine possibly exerts its effects by acting to prevent the gene transcription mechanisms required for LTM formation.

EXPERIMENTAL PROCEDURES Animals The stocks of Lymnaea stagnalis used originate from populations derived from those at Vrije Universeit (Amsterdam) in the late 1950s, collected from a polder near Eemnes in Utrecht. Snails are reared at the snail facility at the University of Calgary. They are kept at room temperature (18 –20 °C) in aquariums containing well-aerated and filtered eumoxic pond water (PW) (i.e. containing normal levels of O2 measured to approximately 6 ml O2/l) and are fed a diet of Lactuca sativa–romaine lettuce. Animal shell lengths used for all experiments ranged from 21 to 25 mm.

1TT 1TT will be described in brief; for a more complete review of the development of the technique, please see Martens et al. (2007b). PW (500 ml) was made hypoxic by bubbling N2 uniformly through out a beaker for at least 20 min (⬍0.1 ml O2/l). Individually marked animals were then removed from their eumoxic home aquarium and placed in the beaker containing 500 ml of room temperature hypoxic PW. Subsequently, animals were left for a 10 min acclimatization period and then total breathing time (TBT) for the animals was recorded over a half-hour period. This is known as the pre-observational (Pre-Obs) breathing session. The animals were then placed back in their eumoxic home aquarium for 24 h and re-subjected to the hypoxic challenge for a second time. However, in this second session instead of a 10 min acclimatization period followed by a half-hour breathing session animals were plucked out of the beaker upon initial pneumostome opening and placed in a Petri dish (37 mm) containing a noxious stimulus (4 ml of 25 mM potassium chloride; KCl) for 35 s, after which they were placed back in their eumoxic home aquarium and tested at one of two specific time points (2 h post-1TT for ITM or 24 h post-1TT for LTM). Testing (i.e. a probe session) requires that the animals undergo an additional hypoxic challenge first with a 10 min acclimatization period followed by a 30 min session, during which TBT was recorded; this is known as the post-observational (Post-Obs) breathing session.

Yoked controls Yoked control groups were treated identical to that of the corresponding experimental groups. The primary difference between yoked and experimental animals is that yoked animals do not receive the noxious KCl stimulus contingent upon their pneumostome opening, but instead they receive a noxious KCl stimulus at the time the snail from the experimental group to which they are yoked received its noxious KCl stimulus. Therefore, the animal would not have had its pneumostome open at this time; ergo, there is no contingency between pneumostome activity and KCl stimulation for yoked animals. Consequently, if operant learning and memory formation is occurring as a result of the aversive KCl stimulus, yoked animals should show no difference between Pre-Obs and Post-Obs breathing sessions, while the experimental snails should. Yoked animals received the Pre-Obs breathing session on the first day, yoked non-contingent noxious KCl (25 mM) stimuli 24 h later and a Post-Obs breathing session at either, 2 h or 24 h post-1TT, depending on the memory paradigm of interest (ITM or LTM, respectively).

Ketamine dosage and administration Ketamine concentration. Appropriate dosing of ketamine (Ketalean®; Bimeda-MTC Animal Health Inc.) was assessed by first constructing a breathing dose response curve specific to Lymnaea. Animals were given a Pre-Obs half-hour breathing session in hypoxic-PW during which their TBT was recorded. This was termed the pre-ketamine session, after which they were left for 1 h in their eumoxic home aquariums. They were then placed in a hypoxic ketamine-PW bath at one of several varying concentrations (0.08, 0.04, 0.004, 0.0004 or 0.00004 mg/ml, respectively), allowed to acclimatize for 10 min, after which their TBT over a half-hour period was recorded. This was termed the ketamine session. Animals were then placed in their eumoxic home aquarium for 1 h to recover from ketamine influence and a halfhour Post-Obs breathing session again in hypoxic-PW was performed. This was termed the recovery session. A curve was constructed as percent decrease in aerial respiration plotted as a function of ketamine concentration (Fig. 1). We used the curve to extrapolate a concentration to investigate ketamine’s effects on

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Fig. 1. Percent change in aerial respiration as a function of ketamine bath concentration. Points are plotted as percent mean⫾S.E.M. A bioassay was conducted in order to elucidate an appropriate concentration of ketamine with which to investigate its effects on memory in subsequent experiments. Ultimately, we wanted a single concentration in which there were notable effects on behavior during administration but that did not incapacitate the animals or have long lasting observable effects on aerial respiration after cessation of administration. Several concentrations were investigated via bath application: 0.00004, 0.00022, 0.0004, 0.004, 0.04 and 0.08 mg/ml. Two concentrations, 0.00004 and 0.00022 mg/ml, resulted in an increase in breathing from pre-ketamine values of: 27⫾19% and 9⫾15%, respectively. The remaining concentrations, 0.0004, 0.004, 0.04 and 0.08 mg/ml, however, resulted in decreases in aerial respiration from pre-ketamine values of: 14⫾7%, 39⫾11%, 94⫾3% and 100%, respectively. We decided that a concentration that moderately affected the animals’ aerial respiratory behavior during administration would be best to further investigate ketamine’s effects on memory. Therefore, we chose to use the concentration that produced approximately a 40% decrease in aerial respiration during bath administration— 0.004 mg/ml— but, which allowed for quick recovery of aerial respiratory behavior of approximately ⱕ1 h upon cessation of administration so as not to interfere with memory testing in subsequent experiments (Fig. 2A). At all the concentrations investigated aerial respiratory behavior returned to pre-ketamine values approximately 1 h after cessation of administration. This includes the concentration chosen to investigate ketamine’s effects on memory. Neither administration of ketamine in memory experiments nor the time it took animals to recover to pre-ketamine values of aerial respiratory behavior upon cessation of ketamine administration overlapped with memory testing sessions.

memory in subsequent experiments. We chose to use the concentration that caused a decrease in aerial respiration of 40% when administered in hypoxic ketamine-PW, 0.004 mg/ml. Righting reflex. The animals’ righting reflex (Fei et al., 2007; Orr et al., 2007) was investigated, both on control animals (no ketamine) and on animals administered ketamine previous to assessment. Righting for animals in both PW and ketamine-PW was assessed via the same procedure. Previous to righting measurement ketamine was administered via a eumoxic ketamine bath for 15 min with the concentration derived from the breathing dose response curve (0.004 mg/ml; see above). Animals were placed in a Petri dish (150 mm) and left to explore for 10 min. Subsequently, they were flipped onto their backs and timed for righting. Aerial respiratory behavior. In order to gauge if 0.004 mg/ml ketamine, when administered in eumoxic-PW for 15 min, had

long-term side effects on respiratory behavior that would confound our Post-Obs breathing sessions in memory experiments we conducted a series of breathing control experiments. Animals were given a Pre-Obs breathing session followed by a 15 min eumoxic ketamine bath 24 h later. Subsequently, a Post-Obs breathing session was conducted at the times we would be testing the animals in future memory experiments, 2 or 24 h later (Fig. 1C–D). Ketamine and 1TT. The procedures for 1TT were repeated as described above for experimental animals. However, animals were subjected to a eumoxic ketamine bath (0.004 mg/ml) for 15 min at varying times before or after the 1TT procedure. Snails were exposed to the 15 min of ketamine at times ranging from 2 h before the 1TT event to 23 h after the 1TT event. We determined whether the specified exposure to ketamine altered the snails’ ability to form either ITM and/or LTM. Applying ketamine directly before 1TT and then right before testing assessed state depen-

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Fig. 2. The effects of the chosen ketamine concentration on the behavior of Lymnaea stagnalis. Graphs are plotted as mean⫾S.E.M. (A) In order to measure pre-ketamine aerial respiratory behavior snails were first observed for total aerial respiratory time over a half-hour breathing session in hypoxic PW. They were then left for 1 h in their eumoxic home aquarium and re-observed for an additional half-hour breathing session in hypoxic ketamine-PW. Following the hypoxic ketamine-PW breathing session, the snails were once again returned to their home aquaria for 1 h, after which they were again observed in hypoxic-PW for a half-hour to assess recovery. An RM-ANOVA run between groups was found to be significant (F(2, 42)⫽12.95; ** P⬍0.01). Post hoc, the Tukey-Kramer multiple comparisons test showed a significant decrease in total aerial respiratory behavior over a half-hour for the ketamine condition (119⫾23) when compared with the pre-ketamine condition (212⫾23; ** P⬍0.01) and the recovery condition (224⫾18; ** P⬍0.01). No difference was observed between the pre-ketamine and the recovery conditions (P⬎0.05). (B) Ketamine’s effects on the righting reflex were assessed by comparing average righting times. Two separate cohorts of snails, a control group (209⫾24; n⫽36) in which animals had no administration of ketamine prior to testing and an experimental group (255⫾23; n⫽38) in which animals were administered ketamine directly before testing was compared. No difference in righting times was observed between the two groups (P⬎0.05). (C, D) Breathing controls were used to assess possible long-term side effects of residual ketamine on aerial respiratory behavior. Animals were observed for a half-hour in hypoxic-PW, they were then administered ketamine in eumoxic-PW via a 15 min bath application process 24 h later identical to that given in subsequent memory experiments. Animals were then tested either 2 (C) or 24 (D) h later in hypoxic-PW. No differences between Pre-Obs breathing and Post-Obs breathing were observed at either 2 (182⫾25 and 211⫾37, respectively; n⫽20; P⬎0.05) or 24 h (163⫾15 and 176⫾16, respectively; n⫽24; P⬎0.05) following the ketamine administration.

dent memory formation. Specifically, animals were trained under the influence of ketamine and then administered ketamine 1 h before testing.

Statistics Percent decrease in aerial respiratory behavior was calculated for each concentration by dividing the ketamine TBT value minus the pre-ketamine TBT by the pre-ketamine TBT, and then multiplying by 100 (Fig. 1): Percent decrease in aerial respiration⫽[(ketamine TBT⫺preketamine TBT)/pre-ketamine TBT)]⫻100. Repeated measures analysis of variance (RM-ANOVA) was used to assess differences between pre-ketamine, ketamine and recovery breathing sessions for the concentration chosen (0.004

mg/ml) to investigate ketamine’s effects on memory (InStat 3.0b; Fig. 2A). Isolated group comparisons were analyzed post hoc using a Tukey-Kramer multiple comparisons test (Fig. 2A). Righting response data were transformed in order to meet normality requirements using a log transformation, as data were positively skewed, and were subsequently analyzed using a Student t-test (Fig. 2B). Ketamine breathing control experimental data were analyzed using paired Student t-tests (Fig. 2C–D). For 1TT experiments two-tailed Student t-tests, each with a 95% confidence interval, were used to assess differences between Pre-Obs and Post-Obs breathing sessions (InStat 3.0b). For groups in which difference distributions did not meet normality as determined by the Kolmogorov-Smirnov test (K-S test) log transformations were conducted for both Pre-Obs and Post-obs

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Fig. 3. ITM and LTM following 1TT. Graphs are plotted as mean⫾S.E.M. (A, B) ITM formation was observed 2 h following 1TT (A). The Post-Obs TBT (185⫾13) was found to be significantly lower than the Pre-Obs TBT (272⫾17; n⫽22; ** P⬍0.01). (B) Yoked animals showed no decrease in breathing when assessed at 2 h. The Post-Obs TBT (287⫾57) was not significantly different from the Pre-Obs TBT (282⫾76; n⫽10; P⬎0.05). The observed effect sizes for experimental and yoked ITM controls were calculated to be 1.21 and ⫺0.026, respectively. (C, D) LTM was observed 24 h following 1TT (C). The Post-Obs TBT (186⫾11) was found to be significantly lower than the Pre-Obs TBT (305⫾36; n⫽28; ** P⬍0.01). (D) Yoked animals showed no decrease in breathing when assessed at 24 h. The Post-Obs TBT (185⫾18) was not significantly different from the Pre-Obs TBT (192⫾28; n⫽12; P⬎0.05). The observed effect sizes for experimental and yoked LTM controls were calculated to be 0.90 and 0.070, respectively.

data, normality was then reassessed using the K-S test. If the transformed difference distribution passed normality the transformed data were then assessed using paired Student t-tests; only the LTM control experiment (Fig. 3C) and the 3 h delay experiment required transformations (Fig. 6C). Raw data are presented graphically throughout. Post hoc power (PHP) analysis was conducted for both control ITM and LTM 1TT paradigms (Origin Laboratory 8.0; Fig. 3). We set the minimum power acceptable to detect a difference between Pre-Obs and Post-Obs to be 80% for both the ITM and LTM, 1TT paradigms; they were calculated to have a power of 98% and 90%, respectively using PHP analysis. It was concluded that since the power calculated for both the ITM and LTM 1TT paradigms was above 80% that were was sufficient sensitivity for detecting differences between Pre-Obs and Post-Obs breathing sessions in subsequent experiments investigating ketamine’s effects on memory using the 1TT procedure. Effect size is an index used to express the magnitude of an effect. Using the Cohen’s d method we calculated observed effect sizes for control ITM and LTM data (Fig. 3). Calculated values were then set as the minimum effect size needed to constitute a large effect in subsequent ketamine experiments. For the ITM 1TT paradigm a large observed effect size was considered to be ⱖ1.00, medium ⱖ0.70 and small ⱖ0.40. For the LTM 1TT paradigm an observed large effect size was considered ⱖ0.90, me-

dium ⱖ0.60 and small ⱖ0.30. Observed effect sizes for ketamine experiments were then calculated in the same manner and were referenced to that of the controls. However, we do not discuss practical significance in relation to the calculated observed effect sizes, but simply report them as an interpretive gauge for the interested reader.

RESULTS Effects of ketamine on the behavior of Lymnaea stagnalis In a series of pilot experiments, we investigated the effects of a range of ketamine concentrations (0.08 – 0.00004 mg/ ml) on aerial respiratory behavior in Lymnaea. The percent decrease in aerial respiration is plotted as a function of the tested concentration range in Fig. 1. We were seeking to use a concentration at which animals’ aerial respiratory behavior would be approximately 40% decreased during administration, as to ensure ketamine was being absorbed during bath administration and thus having an effect on the animals. We found that a concentration of 0.004 mg/ml ketamine when bath applied via hypoxic-PW resulted in

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approximately a 40% decrease in aerial respiratory behavior and that the animals also recovered to pre-ketamine breathing levels following a 1 h interval in their eumoxic home aquarium; these data are plotted in Fig. 2A. We chose this concentration to investigate ketamine’s effects on memory. Note that while aerial respiration was found to be significantly decreased by approximately 40% when animals were administered ketamine at the 0.004 mg/ml concentration, they are, in addition to aerial respiration, still performing cutaneous respiration in hypoxic conditions. Further, ketamine administration in subsequent experiments was administered in eumoxic-PW not hypoxic-PW and therefore changes in aerial respiration were not a factor, as animals are only driven to breathe aerially when there is a shortage of oxygen in their environment. Consequently, we do not feel that any decrease in aerial respiration as a result of ketamine administration would have had an impact on our subsequent experimental results, especially because the ability for ketamine to cause a decrease in aerial respiration is limited to the duration of administration in a hypoxic environment and because there is a short recovery period thereafter of ⱕ1 h. That is, during administration of ketamine in all other experiments, except those used to collect the dose response data, animals would be primarily breathing cutaneously. Further, the timing of administration in subsequent experiments was conducted in a way as not to influence Pre-Obs or Post-Obs breathing behavior observations (see below). In order to further assess the concentration of 0.004 mg/ml ketamine on behavior in Lymnaea we examined the righting reflex (Fei et al., 2007; Orr et al., 2007). The ability of the snail to right itself after being placed on its back was assessed in eumoxic-PW and eumoxic ketamine-PW, which was administered for 15 min prior to the righting experiment. No significant difference was observed in the time necessary for the snail to right itself in PW compared with ketamine-PW (Fig. 2B). We therefore concluded that because there was no effect on righting reflex that the animals were not anesthetized as a result of ketamine administration in eumoxic-PW using the 0.004 mg/ml concentration. We performed an additional series of controls to eliminate the possibility that administered to 0.004 mg/ml ketamine for 15 min would alter subsequent aerial respiratory behavior at 2 or 24 h after ketamine administration, the times we would—in subsequent experiments—test for ITM and LTM formation, respectively. These data are presented in Fig. 2C–D. There was no significant difference in TBT between the Pre-Obs and the Post-Obs breathing sessions with the interposed 15 min eumoxic ketamine exposure, at either the 2 h or 24 h time point. ITM and LTM following 1TT Initially, we set out to demonstrate that the 1TT procedure resulted in both ITM and LTM. In our previous work (Martens et al., 2007b) we only looked at LTM formation (i.e. memory at 24, 36 and 48 h after the 1TT event). We therefore performed a series of experiments that demonstrated our ability to use the 1TT procedure to produce

both ITM and LTM (Fig. 3). Snails underwent a Pre-Obs breathing session followed by ITT 24 h later and a subsequent Post-Obs breathing session either 2 or 24 h after training. Animals who underwent ITT with a subsequent 2 h Post-Obs breathing session demonstrated ITM formation. That is, the Post-Obs TBT was found to be significantly lower than the Pre-Obs TBT (Fig. 3A). Snails who underwent the yoked control procedure did not exhibit a decrease in breathing in the Post-Obs breathing session 2 h later (Fig. 3B). Likewise when we tested for LTM formation after 1TT we found that there was a significant decrease in the TBT in the 24 h Post-Obs session compared with the Pre-Obs session (Fig. 3C) but not in the yoked animals (Fig. 3D). Thus, our results are consistent with the Martens et al. (2007b) report and extend those findings, showing that ITM is also formed as a result of 1TT. The effects of pre-administered ketamine on ITM and LTM Having demonstrated that we were capable of inducing both ITM and LTM using the 1TT procedure (Fig. 3A, C) we then asked what effect, if any, a 15 min ketamine administration before 1TT had on memory formation (Fig. 4). We first determined the effect of ketamine exposure immediately before the 1TT event on the formation of ITM (Fig. 4A). A cohort of naïve snails’ breathing behavior in hypoxic-PW was observed (Pre-Obs session). On the following day, the snails were administered ketamine for 15 min; immediately after administration the animals received the 1TT procedure. We then tested for memory 2 h later. As can be seen the TBT in the Post-Obs session was significantly less than in the Pre-Obs session (Fig. 4A). Yoked control snails were given a similar procedure as described above except that their placement into KCl was not contingent upon opening of their pneumostome. We found that their TBT in the Post-Obs session was not significantly different from that in the Pre-Obs session (Fig. 4B). We therefore concluded that ketamine administration before the 1TT did not block ITM formation. We performed a similar series of experiments on another cohort of naïve snails, only this time we tested whether ketamine exposure altered LTM formation. We found that ketamine administration for 15 min immediately before the ITT event blocked LTM formation. That is, the TBT in the Post-Obs session was not significantly different from the TBT in the Pre-Obs session (Fig. 4C). Yoked control snails gave similar results: the TBT in the Post-Obs session was not significantly different from the TBT in the Pre-Obs session (data not shown). Ketamine administration thus blocked LTM formation when snails were exposed to it immediately before training. We next set out to determine if we administered snails ketamine 2 h before training if ketamine would still block LTM formation. When we performed this experiment, ketamine did not block LTM formation. That is, the TBT in the Post-Obs session was significantly less than in the Pre-Obs session (Fig. 4D). Thus, we concluded that ketamine administration immediately before the 1TT had the ability to block the formation

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Fig. 4. The effects of pre-administered ketamine on ITM and LTM formation. Graphs are plotted as mean⫾S.E.M., where the concentration of ketamine used in the 15 min eumoxic-PW bath administration was 0.004 mg/ml. (A, B) Animals administered ketamine immediately before 1TT, exhibited ITM formation when tested 2 h following 1TT (A). The Post-Obs TBT (117⫾11) was found to be significantly lower than the Pre-Obs TBT (206⫾19; n⫽27; ** P⬍0.01). (B) Yoked animals that were administered ketamine immediately before the yoking procedure and tested 2 h later, did not exhibit a significant decrease in breathing. The Post-Obs TBT (258⫾32) was not significantly different from the Pre-Obs TBT (276⫾40; n⫽12; P⬎0.05). The observed effect sizes for experimental ITM and yoked pre-treated ketamine groups were calculated to be 1.10 and 0.14, respectively. (C) Animals administered ketamine immediately before 1TT, did not exhibit LTM formation when tested 24 h following 1TT (n⫽26; P⬎0.05). The Post-Obs TBT (185⫾19) was not significantly different than the Pre-Obs TBT (178⫾15). Yoked animals that were administered ketamine immediately before the yoking procedure and tested 24 h later, did not exhibit a significant decrease in breathing (data not shown; n⫽11; P⬎0.05). The Post-Obs TBT (164⫾40) was not significantly different from the Pre-Obs TBT (169⫾24). The observed effect sizes for experimental LTM and yoked pre-treated ketamine groups were calculated to be ⫺0.071 and 0.042, respectively. (D) Animals administered ketamine 2 h before 1TT, did exhibit LTM formation when tested 24 h following 1TT (n⫽18; ** P⬍0.01). The Post-Obs TBT (91⫾13) was found to be significantly lower than the Pre-Obs TBT (197⫾24). The observed effect size for the 2 h pre-treated ketamine group was calculated to be 1.26.

of LTM, but that ketamine’s ability to block LTM diminished if the interval between pre-administered ketamine and training is increased to 2 h. The effects of post-administered ketamine on ITM and LTM A similar series of experiments (see above) was then performed only this time snails were administered ketamine immediately after the 1TT event (Fig. 5). We found, in another cohort of naïve snails, that ketamine administration immediately after 1TT did not block ITM formation. That is, the TBT in the Post-Obs was significantly less than in the Pre-Obs session (Fig. 5A). However, corresponding yoked control snails TBT in the Post-Obs session was not significantly different from the TBT in the Pre-Obs session

(Fig. 5B). Thus, ITM formation was not disrupted by ketamine administration immediately after the training event. However, similar to the results shown in Fig. 4, ketamine administration immediately after the 1TT event blocked LTM formation. These data are shown in Fig. 5C. When we tested a cohort of snails for memory 24 h after the 1TT and immediate ketamine administration for 15 min we found that the TBT in the Post-Obs session 24 h later was not significantly less than in the Pre-Obs session (Fig. 5C). Yoked control snails exhibited similar results; the TBT in the Post-Obs session was not significantly different from the TBT in the Pre-Obs session (Fig. 5D). Thus, LTM formation was also blocked by immediate administration of ketamine after the 1TT event, whereas ITM was not.

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Fig. 5. The effects of post-administered ketamine on ITM and LTM formation. Graphs are plotted as mean⫾S.E.M., where the concentration of ketamine used in the 15 min eumoxic-PW bath administration was 0.004 mg/ml. (A, B) Animals administered ketamine immediately after 1TT, exhibited ITM formation when tested 2 h following 1TT (A). The Post-Obs TBT (237⫾16) was found to be significantly lower than the Pre-Obs TBT (178⫾10; n⫽44; ** P⬍0.01). (B) Yoked animals that were administered ketamine immediately after the yoking procedure and tested 2 h later, did not exhibit a significant decrease in breathing. The Post-Obs TBT (129⫾22) was not significantly different from the Pre-Obs TBT (162⫾23; n⫽18; P⬎0.05). The observed effect sizes for experimental ITM and yoked post-treated ketamine groups were calculated to be 0.65 and 0.34, respectively. (C, D) Animals administered ketamine immediately after 1TT, did not exhibit LTM formation when tested 24 h following 1TT (C). The Post-Obs TBT (215⫾16) was not significantly different than the Pre-Obs TBT (218⫾20; n⫽29; P⬎0.05). (D) Yoked animals that were administered ketamine immediately after the yoking procedure and tested 24 h later, did not exhibit a significant decrease in breathing. The Post-Obs TBT (171⫾24) was not significantly different from the Pre-Obs TBT (200⫾34; n⫽12; P⬎0.05). The observed effect sizes for experimental LTM and yoked post-treated ketamine groups were calculated to be 0.032 and 0.29, respectively.

Time dependent effects of ketamine administration We next set out to determine the post-temporal boundaries in which ketamine administration was capable of blocking LTM formation (Fig. 6). That is, we asked how long we could delay ketamine administration after 1TT and still block LTM formation. We found that ketamine was still capable of blocking LTM formation if given both 1 and 2 h following the 1TT event (Fig. 6A–B). That is, the TBT in the Post-Obs session was not significantly less than in the Pre-Obs session if snails were administered ketamine for 15 min 1 or 2 h after training (Fig. 6A–B). However, if we delayed the administration of ketamine until 3 h after 1TT, then LTM was observed. That is, the TBT in the Post-Obs session was significantly less than in the Pre-Obs session (Fig. 6C). Thus, ketamine’s ability to block LTM formation extended at least 2 h after the 1TT event.

Ketamine’s effects on retrieval and state dependency learning We examined whether ketamine had any effects on retrieval of LTM facilitated by 1TT and whether ketamine’s ability to block LTM formation could be a result of state dependent learning. In order to investigate ketamine’s effects on retrieval we administered ketamine for 15 min 1 h prior to the Post-Obs breathing session. By administering ketamine 1 h prior to testing we were confident that any significant decrease in breathing would not be due to residual ketamine, as previously determined by the above breathing control experiment shown in Fig. 2A, but would instead be a result of LTM formation. We found that ketamine did not have any effect on the retrieval of LTM. That is, the TBT in the Post-Obs session was significantly less than in the Pre-Obs session when ketamine was adminis-

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Fig. 6. Time dependent effects of ketamine administration following 1TT. Graphs are plotted as mean⫾S.E.M., where the concentration of ketamine used in the 15 min eumoxic-PW bath administration was 0.004 mg/ml. (A, B) Ketamine administered for a 15 min duration 1 or 2 h after 1TT was able to inhibit LTM formation (P⬎0.05). That is, the Post-Obs TBT (220⫾21 and 160⫾30, respectively) was not significantly different than the Pre-Obs TBT (205⫾21 and 178⫾26, respectively). The observed effect sizes for the 1 and 2 h delay groups were calculated to be ⫺0.18 and 0.18, respectively. (C) Ketamine administered 3 h after 1TT did not block LTM formation (** P⬍0.01). The Post-Obs TBT (206⫾29) was found to be significantly lower than the Pre-Obs TBT (298⫾20). From these results we concluded that there is a post-administration window lasting up to at least 2 h after the 1TT event within which LTM formation can be inhibited by ketamine. The observed effect size was calculated to be 0.95.

tered for 15 min 1 h before testing (Fig. 7A). Lastly, we wanted to eliminate the possibility that ketamine’s ability to inhibit LTM but not ITM formation was not due to state dependent learning. That is, we reasoned that because when administered immediately before 1TT, ketamine could be present in residual forms in the snails at the time of testing in the ITM paradigm, it could set the stage for state dependent learning, possibly accounting for the differential effects of ketamine between ITM and LTM paradigms. Ergo, the snails would have been under the influence of ketamine both during training and testing in the ITM paradigm. Therefore this effect, although relevant to the LTM paradigm, would not have been tested as the ketamine in the snails may have completely cleared at the time of testing 24 h after 1TT. In order to test this hypothesis we first administered ketamine for 15 min immediately before training and 1 h before testing 24 h later. If a state dependent effect were taking place we would expect to see a significant decrease in Pos-Obs breathing when compared with Pre-Obs breathing. We observed no such effect (Fig. 7B).

Therefore we concluded that ketamine’s ability to inhibit LTM formation was not due to state dependent learning.

DISCUSSION In the present study, we have demonstrated that ketamine administration has the ability to inhibit non-declarative LTM formation, but not ITM formation, if administered immediately before and up to 2 h following the 1TT event. Additionally, ketamine when administered 1 h prior to testing has no effect on retrieval. Ketamine’s ability to block LTM formation is not due to state dependent learning or anesthetic effects, as the concentration of ketamine administered did not alter breathing behavior, the righting reflex or the ability to form ITM. Since ketamine administration does not interfere with ITM formation, but only LTM formation, and this effect is not due to state dependent learning we hypothesize that ketamine is acting at the required gene transcription processes in neurons (e.g. RPeD1) necessary for LTM formation.

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Fig. 7. Ketamine’s effects on retrieval and state dependent learning. Graphs are plotted as mean⫾S.E.M., where the concentration of ketamine used in the 15 min eumoxic-PW bath administration was 0.004 mg/ml. (A) In order to test ketamine’s effects on retrieval ketamine was administered for 15 min 1 h prior to testing for LTM. Ketamine was found to have no effect on retrieval (n⫽19; ** P⬍0.01). The Post-Obs TBT (169⫾24) was found to be significantly lower than the Pre-Obs TBT (302⫾34). The observed effect size was calculated to be 1.04. (B) In order to assess whether state dependent learning could have accounted for the differential effects ketamine exerted between ITM and LTM we administered ketamine immediately before 1TT and 1 h before testing. Ketamine’s ability to inhibit LTM was not found to be caused by state dependent learning, as no evidence of LTM was present when animals were trained and then tested in association with ketamine (n⫽19; P⬎0.05). That is, the Post-Obs TBT (275⫾23) was not significantly different than the Pre-Obs TBT (288⫾28). The observed effect size was calculated to be 0.13.

We have previously shown in our Lymnaea model system that we have the ability to differentially block the formation of LTM, while leaving intact ITM formation. We are able to do this in two ways. Firstly, if we injected the transcription blocker, actinomycin D prior to training LTM was blocked, while ITM formation was not. If, however, anisomycin, a translation blocker was injected prior to training, both ITM and LTM formation were blocked (Sangha et al., 2003b). Second, if we ablate the soma of RPeD1 (i.e. removing the nucleus where transcription occurs) prior to training, ITM formation still occurs, but LTM formation is blocked (Scheibenstock et al., 2002). Moreover, RPeD1 soma ablation also prevents snails from al-

tering their behavioral phenotype as a result of processes that require altered gene activity such as can be seen with extinction, reconsolidation, and forgetting (Sangha et al., 2003a,c, 2005). These data suggest that ITM which persists for 2–3 h requires new protein synthesis from preexisting mRNA can occur extra-somally (Scheibenstock et al., 2002; Van Minnen et al., 1997; Spencer et al., 2000); while, LTM is dependent on both new protein synthesis and altered gene activity. We reasoned that because RPeD1 also needs to be present in order for LTM formation to occur after 1TT (Martens et al., 2007b) that the 15 min ketamine administration used in the present study may interfere with the gene transcription process, thus blocking LTM formation. Drawing from the current data in conjunction with the above mentioned studies we concluded that ketamine may not block ITM formation because it does not interfere with the translation of pre-existing mRNA. A question that therefore must be addressed is whether there are data supporting the hypothesis that ketamine can interfere with the transcription process in cells. Chen et al. (2005) found that ketamine had the ability to suppress eNOS expression, as seen by a decrease in eNOS mRNA over a 24 h period. This effect could be seen as early as 1 h after ketamine administration to human umbilical vein endothelial cells and was still observable 24 h later. This effect was correlated to a time dependent decrease in eNOS protein production and nitrite, thus demonstrating ketamine’s ability to exert its ultimate effects via a pre-translational mechanism (Chen et al., 2005). Further evidence for ketamine’s direct role in transcription regulation comes from studies outlining its anti-inflammatory properties. Ketamine has been demonstrated to decrease endotoxin-induced nuclear factor-kappa B (NF-␬B) expression, a latent cytoplasm transcription factor, thus dampening gene expression of pro-inflammatory cytokines (Sakai et al., 2000; Yu et al., 2007). These studies substantiate ketamine’s role in regulating transcription events and therefore lend support to our hypothesis that ketamine’s conjectured role in blocking LTM formation is executed via regulation of transcription events. Moreover, these results are consistent with our previous findings regarding the necessary molecular processes underlying both ITM and LTM formation (see below). Since ketamine does not block ITM, which is dependent on only new protein synthesis, but does block LTM, which is dependent upon both new protein synthesis and altered gene activity, the conclusion we draw is that ketamine’s inhibitory effect on LTM formation is due to its ability to interfere with the gene transcription process. As mentioned above, our present data are consistent with previous work in our laboratory concerning the necessary steps in LTM formation. Based on a series of earlier studies (Smyth et al., 2002; Parvez et al., 2005, 2006), we hypothesized that LTM formation first requires the formation of ITM. That is, they occur in series rather than in parallel (Parvez et al., 2006). While this hypothesis implicates translation as the primary event underscoring ITM formation, it also suggests that these translation events are the first step to establishing LTM, with the second part of the modus operandi underlined by transcription events. As

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ketamine is able to block LTM formation and not ITM formation our data are consistent with this view of memory formation in Lymnaea. Ketamine appears to act solely on transcription events that are distinct for LTM formation, at least in regard to behavioral phenotype. While NMDA receptors have been reported in Aplysia (Glanzman, 2008), a close relative of Lymnaea, and NMDA-like receptors have been reported in Lymnaea (Ha et al., 2006) and therefore could be implemented in effects involving acquisition, we believe ketamine’s effects are exerted specifically at the consolidation phase of memory formation. We reasoned that if ketamine were disrupting acquisition as well as consolidation, ITM formation would have also been inhibited, as ITM formation requires acquisition the same as LTM formation. Therefore, our hypothesis leads to the idea that ketamine does not affect acquisition, but is acting specifically on consolidation. A somewhat surprising finding in regard to the post1TT administration of ketamine was the discovery that it was still possible to block LTM formation with a long interval between the training event and the administration of the snail to ketamine. That is, we found that it was possible to block LTM even if we delayed the administration of the snails to ketamine for 2 h. A major advantage to using the 1TT procedure is that the memory consolidation process (see below) must be initiated immediately after the single training event. In general, it has been widely accepted that learning does not induce instantaneous permanent memories, but that memory instead remains vulnerable to disruption for a period of time after learning. This is known as the consolidation period (Lechner et al., 1999). The time course of memory consolidation varies and is dependent on both the task and the training procedures (Lechner et al., 1999). How long the required molecular event(s) underlying memory formation persist is not certain, although earlier data in Lymnaea, both from our laboratory and others had suggested the consolidation period was relatively short. That is, interventions used to block memory formation had to occur within the first hour following training. Previous work by Fulton et al. (2005) (appetitive feeding) and Sugai et al. (2007) (taste aversion) in which a one-trial conditioning procedure was used to associatively condition a change in feeding behavior demonstrated that memory, under these conditions, forms rapidly. For example, in the Fulton et al. (2005) study they show that if they injected anisomycin into snails within 10 min of their ITT procedure, LTM was blocked. Similarly, if they injected actinomycin D within the first 10 min post-ITT, LTM was also blocked. They could not block LTM formation at injection times after that of the first 10 min post-ITT. They concluded that the consolidation processes involving protein synthesis is rapid following one trial appetitive conditioning, and is complete within 1 h after training (Fulton et al., 2005). In the Sugai et al. (2007) study, the authors also demonstrated that a cold block (immediate immersion into 4 °C PW) was only successful in blocking LTM formation if it occurred within 10 min of the 1TT event. Previously, we have shown that we could block the formation of LTM with 1TT if we placed snails into cold PW (4 °C PW) for 1 h

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immediately after training. If cooling was delayed for 1 h or more, the cold block did not prevent LTM formation (Martens et al., 2007b). Thus, it was assumed that the memory consolidation process in Lymnaea was a rather fast process vulnerable to disruption only briefly after 1TT. The results of the present study suggest that this may not in fact be the case, and that the consolidation phase and thus the ability to inhibit LTM may last up to at least 2 h after ITT, at least with regard to aerial respiratory behavior. In some model systems, the dynamics of the memory consolidation process appear to be interruptible for longer periods of time following learning. In fact, some authors have suggested that there are two distinct phases of protein synthesis during the consolidation process; that is, LTM formation may be blocked by protein synthesis blockers at multiple time points after training, (Grecksch and Matthies, 1980; Freeman et al., 1995; Epstein et al., 2003). Additionally, in the Martens et al. (2007b) study, cooling was used to block LTM formation. Cooling may not have worked as a sufficient block for memory when applied at later discrete time points in previous studies due to its hypothesized mechanism, which is to ‘slow things down.’ At the later time points investigated the molecular cascades needed for memory formation may have already been sufficiently activated and thus to simply ‘slow things down’ could not have prevented them from ‘starting back up’ once the cold block was abated (Sangha et al., 2003d). Ketamine however, may disrupt the molecular processes necessary for memory formation via a specific molecular mechanism and therefore be able to block memory formation at these later time points. Drugs such as actinomycin D or anisomycin were not used in the Martens et al. (2007b) study due in part to the fact that injection of these drugs close to the 1TT event causes excessive stress which itself inhibits LTM formation (Martens et al., 2007a) and to lingering adverse sideeffects of these drugs that are still present at the time it would have been necessary to test for LTM (Sangha et al., 2003b). In addition, while these drugs have often been used to validate the hypothesis that new protein synthesis is necessary for memory formation, they may have significant side-effects such as the ability to activate apoptosis (Rudy et al., 2006). Thus, it may be necessary to use a number of different procedures and/or agents before concluding that new protein synthesis is a necessary requirement of LTM formation. The data presented here suggest that ketamine might be a drug that should be added to the armamentarium used in studies attempting to test the hypothesis that new protein synthesis is a requirement of LTM formation. Finally, while in the past we have championed cooling as a quick, reversible agent for showing that protein synthesis is necessary for memory formation or forgetting (Sangha et al., 2003c), it must be remembered that cooling may not, in all organisms, block the formation of LTM if applied during the consolidation or reconsolidation process. Thus, for example, in Caenorhabditis elegans heating rather than cooling blocks the reconsolidation process (Rose and Rankin, 2006). Nevertheless, the use of pharmacological agents such as ketamine has and will con-

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tinue to aid in defining the molecular mechanisms important for different memory processes (Abel and Lattal, 2001).

CONCLUSION In conclusion, our data suggest that ketamine exerts differential effects on non-declarative memory formation. Ketamine inhibits LTM and not ITM formation. This implies, based on previous data in our laboratory, that ketamine may exert its distinct effects via gene transcription processes. Our investigations with ketamine helped further delineate the consolidation phase following the ITT paradigm with data suggesting that the consolidation phase previously thought to be completed within 1 h following training is in fact at least 2 h. It is possible that there are other steps of consolidation that occur at more distant time points than the ones tested here which may be susceptible to agents other than ketamine. Investigations more closely outlining ketamine’s unique effects on LTM formation are under further examination in our laboratory. Likewise, further efforts to delineate more precisely the length and dynamics of the consolidation phase following ITT in Lymnaea stagnalis are also under way. Acknowledgments—This work was supported by the Canadian Institute for Health Research (CIHR), Natural Sciences and Engineering Council of Canada (NSERC), the Alberta Heritage Foundation for Medical Research (AHFMR) and the University of Calgary.

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(Accepted 9 June 2008) (Available online 11 June 2008)