Effect of an NCAM mimetic peptide FGL on impairment in spatial learning and memory after neonatal phencyclidine treatment in rats

Behavioural Brain Research 199 (2009) 288–297

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Effect of an NCAM mimetic peptide FGL on impairment in spatial learning and memory after neonatal phencyclidine treatment in rats Thomas Secher a,∗ , Vladimir Berezin a , Elisabeth Bock a , Birte Glenthøj b a b

Protein Laboratory, Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Center for Neuropsychiatric Schizophrenia Research, University of Copenhagen, Psychiatric Centre Glostrup, Glostrup, Denmark

a r t i c l e

i n f o

Article history: Received 12 September 2008 Received in revised form 2 December 2008 Accepted 7 December 2008 Available online 14 December 2008 Keywords: Water maze Schizophrenia Animal model Reference memory Reversal learning Working memory

a b s t r a c t The FGL peptide is a neural cell adhesion molecule-derived fibroblast growth factor receptor agonist. FGL has both neurotrophic and memory enhancing properties. Neonatal phencyclidine (PCP) treatment on postnatal days 7, 9, and 11 has been shown to result in long-lasting behavioral abnormalities, including cognitive impairment relevant to schizophrenia. The present study investigated the effect of FGL on spatial learning and memory deficits induced by neonatal PCP treatment. Rat pups were treated with 30 mg/kg PCP on postnatal days 7, 9, and 11. Additionally, the rats were subjected to a chronic FGL treatment regimen where FGL was administered throughout development. Rats were tested as adults for spatial reference memory, reversal learning, and working memory in the Morris water maze. The PCP-treated rats demonstrated a robust impairment in working memory and reversal learning. However, the long-term memory component of the reference memory task was not affected by PCP. Chronic FGL treatment had no effect on the reversal learning impairment but ameliorated the working memory deficits almost to the levels of the control groups. In conclusion, the results suggest that the neonatal PCP treatment produced deficits in cognition relevant to schizophrenia. Moreover, working memory function was selectively protected by the neurotrophic peptide, FGL. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The neural cell adhesion molecule (NCAM) plays a pivotal role in development and plasticity of the nervous system [3,9]. NCAM signals through a direct interaction with the fibroblast growth factor (FGF) receptor [19,36,44], and several studies have shown that NCAM and the FGF receptor are involved in learning and memory [7,8,20,37,38,43]. The binding site of FGF receptor 1 has been mapped to the F and G ␤-strands and the interconnecting loop region of the second fibronectin type III module of NCAM’s extracellular domain. The corresponding 15-amino-acid peptide, termed FGL (from FG Loop), was subsequently synthesized and found to activate FGF receptor 1 [24]. Several important effects of the FGL peptide have been demonstrated. In vitro, the peptide stimulates neurite outgrowth, improves presynaptic function, and facilitates synapse formation [6,24,33].

Abbreviations: FGF, fibroblast growth factor; NCAM, neural cell adhesion molecule; NMDA, N-methyl-d-aspartate; PCP, phencyclidine; PND, postnatal day; T, trial; VEH-I/-II, vehicle-I/-II. ∗ Corresponding author at: Protein Laboratory, Panum Institute, Building 24.2, Blegdamsvej 3C, Copenhagen DK-2200, Denmark. Tel.: +45 3532 7331; fax: +45 3536 0116. E-mail address: [email protected] (T. Secher). 0166-4328/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2008.12.012

In vivo, FGL acts as a memory enhancer [6,39] and protects the nervous system against global ischemia [40]. Also, FGL has been shown to reduce neuropathology and cognitive impairment in an animal model of Alzheimer’s disease [25]. Furthermore, FGL crosses the blood–brain barrier after peripheral administration [39]. These properties make FGL an interesting pharmacological compound for therapeutic investigations. Cognitive impairment in schizophrenia is hypothesized to represent a core feature of the disease [49] that is strongly related to the functional outcome of affected individuals [16]. However, these impairments are poorly treated by current antipsychotic drugs [13,18,29,30], therefore development of new treatment strategies that improves cognition is important. Preclinical models are valuable in this context, because they can help to assess new pharmacological compounds. Neonatal treatment with the N-methyl-d-aspartate (NMDA) receptor antagonist phencyclidine (PCP) on postnatal days (PND) 7, 9, and 11 in rats has been proposed as a neurodevelopmental model of schizophrenia [45]. This treatment regimen is known to cause neuronal degeneration in brain regions relevant to the cognitive deficits of the human disease such as the frontal cortex and hippocampus [45–47]. So in effect, a neonatal pharmacological lesion is created, mimicking an early insult that disturbs normal brain development. Several behavioral deficits and molecular changes with relevance to schizophrenia have been demonstrated in adult animals treated with PCP in the

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neonatal period [1,5,11,12,17,45,48], including a number of cognitive deficits in working memory, reversal learning, selective attention, and attentional set-shifting. In the present study, a FGL treatment regimen was used where FGL was given before and in between the neonatal PCP treatment on PND7, 9, and 11 and subsequently throughout development. The effect of chronic FGL treatment on spatial learning and memory deficits induced by neonatal PCP treatment was investigated in adult animals. By using such a treatment regimen, we theorized that FGL with its growth factor-like properties would be able to reduce the deleterious effects of the lesion and to further provide trophic support during brain development. Cognitive disturbances in this animal model has been demonstrated for doses of 10–23 mg/kg PCP (PCP, HCl salt, see note in Section 2.1) [1,5,17,45]. However, higher PCP doses appear to result in larger deficits: performance in an attentional set-shifting paradigm was significantly lower after neonatal treatment with 23 mg/kg PCP compared to 11.5 mg/kg PCP in adult female rats [5]. Similar results are evident from our own preliminary studies using the Morris water maze (T. Secher, V. Berezin, E. Bock, B. Glenthøj, unpublished observations). It should be emphasized that the most pronounced acute effect of neonatal PCP treatment is neuronal death. The extent of the damage has been shown to be dose dependent, i.e. higher doses giving rise to increased number of degenerating neurons [21,46]. Therefore, 30 mg/kg PCP was used in the current study to provide a robust cognitive impairment. 2. Materials and methods 2.1. Drugs PCP was a generous gift from H. Lundbeck A/S (Copenhagen, Denmark). PCP, HCl was dissolved in isotonic saline (0.9% (w/v) NaCl, vehicle-I (VEH-I)). Please note that in some studies PCP dose is reported as mg/kg free base (243.4 g/mol) [1,5] whereas dose is given as mg/kg salt (279.9 g/mol) in the current study. For instance, the 8.7 mg/kg free base used by Andersen and Pouzet [1] thus equals 8.7 × 279.9/243.4 = 10.0 mg/kg salt. The pentadecapeptide FGL (EVYVVAENQQGKSKA), corresponding to residues E681–A695 of the second rat NCAM F3 module, was synthesized as a dimer (FGL2 , Polypeptide Laboratories, Hillerød, Denmark). The dimer was composed of two monomers linked by iminodiacetic acid (N[carboxymethyl]glycine) through their N-terminals. FGL2 was at least 95% pure as estimated by high performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (VG TOF Spec E, Fisons Instruments, Beverly, MA, USA). FGL2 was dissolved in isotonic glucose (55 g/L, vehicle-II (VEH-II)). 2.2. Animals The following animal experiment was performed in accordance with the European Communities Council Directive of 24 November 1986 and Danish legislation. The experiments were approved by the Danish Animal Experiments Inspectorate. All efforts were made to minimize the number of animals used and any possible suffering. Timed pregnant Sprague–Dawley rats (crl:CD(SD)BR) were obtained from Charles River (Sulzfeld, Germany). The dams arrived on gestational Day 15 and were housed separately until delivery. All dams gave birth on gestational Day 22 between 9:00 AM and 5:00 PM. The day of parturition was defined as PND0. Animals were kept in transparent Plexiglas cages with wood chip bedding in temperature- and humidity-controlled animal rooms on a 12 h light/dark cycle (lights on at 5:00 AM). Regular rat pellet food and water were available ad libitum. Pups from 12 litters were separated into males and females on PND4. The males were mixed and randomly assigned to four treatment groups. The pups were earmarked and randomly divided into six litters of 11 pups each, ensuring that the treatment groups were evenly distributed between litters. The female pups and excess dams were euthanized. The animals were weaned on PND23 and randomly divided into cages of three to four animals of the same treatment group. On PND39, the animals were divided again into two-animal cages of the same treatment group. There were uneven numbers of animals in some of the groups; thus one cage housed two rats from different treatment groups (PCP/FGL and VEH-I/VEH-II). Behavioral testing in the water maze (see below) was performed between PND53 and PND78. 2.3. Experimental design PCP and VEH-I were injected in combination with FGL and VEH-II providing four experimental groups: PCP/VEH-II (n = 12), PCP/FGL (n = 13), VEH-I/VEH-II (n = 15),

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and VEH-I/FGL (n = 14). PCP (30 mg/kg) or VEH-I was subcutaneously injected in a volume of 10 ml/kg on PND7, 9, and 11. The pups were subcutaneously injected with FGL (10 mg/kg) or an equal volume of VEH-II on PND4, 6, 8, and 10. The FGL treatment was continued, and the compound was injected on PND12 and 14 and, subsequently, twice a week until PND49, 4 days before behavioral testing began. This dose was chosen, because FGL2 was shown to cross into the brain and to improve social memory retention after subcutaneous injection at an 8.0 mg/kg dose [39]. At 10 mg/kg we attempted to increase the amount of FGL in the brain and still avoid solubility problems that arise at higher doses of FGL. The FGL injection volume started at 10 ml/kg on PND4–14. Subsequently, the volume was gradually decreased throughout development to 1 ml/kg on PND46–49. This was done to keep the injected volume within appropriate limits as the rats grew in size. The FGL injections were performed by a researcher who was blind to the FGL treatment status of the animals. Animals were weighed before each PCP or FGL injection to ensure that the correct dose was given. A total of 66 male rats were used in the experiment. Six animals were excluded due to various technical injection errors. One rat from the VEH-I/FGL was excluded because it would otherwise have been single-housed due to the uneven number of animals in the groups. Five pups died on PND7–8 after the first PCP treatment. Two of these pups were from the PCP/VEH-II-treated group, and three were from the PCP/FGL group. 2.4. Water maze testing Animals were tested in the reference memory, reversal learning, working memory, and visible-platform tasks of the Morris water maze [31]. 2.4.1. Apparatus The Morris water maze was a black circular tank, 160 cm in diameter and 60 cm in height. The water maze was placed on the floor in a room enriched with visual cues on the nearby walls (e.g. posters, electrical fittings, etc.). The room was dimly lit by indirect white light. The tank was filled with fresh water up to 20 cm from the top of the tank before each testing day. The water temperature was 21 ± 1 ◦ C. A black circular platform with a diameter of 10 cm was submerged 2.0 cm below the surface of the water where it was invisible to the swimming rats. A black and white video camera (Panasonic, Japan) was positioned above the water maze. The camera was connected to the MPEG encoder card (Canopus, Kobe, Japan) of a personal computer (Dimension 8400, Dell, USA) running Ethovision 3.1 (Noldus, Wageningen, The Netherlands). The swim paths of the rats were tracked, digitized, and stored for later behavioral analysis using Ethovision. The water maze was divided into four logical quadrants: north, south, east, and west, which also served as starting positions for the rats. Testing was performed daily between 8:30 AM and 5:30 PM. 2.4.2. Reference memory task The reference memory task consisted of 4 days of acquisition with the hidden platform followed by a probe test on the fifth day without the platform. The platform was fixed in a position in the middle of the north quadrant 40 cm from the maze wall during the first 4 days. Four swim trials were given per day, and each trial was started by placing the rats in the water close to the wall of the tank with their heads facing the wall. Trials were started in the middle of one of the three quadrants not occupied by the platform. The starting points were rotated through these three positions during the four trials each day. The rotation started in a new position every day. For each trial, the rats were allowed to swim for 60 s or until they located the platform which ended the trial. A rat that did not find the platform within 60 s was gently moved onto the platform by the researcher. After each trial, the rats were allowed to remain on the platform for 20 s at which time they were removed from the water maze for 10–15 s, and new trial was started. At the end of each daily session, the rats were dried with paper towels and returned to their home cages. The probe test was performed on the fifth day and consisted of one trial without the hidden platform. The rats were allowed to swim for 60 s, after which the trial was stopped. 2.4.3. Reversal learning task The reversal learning task was performed 2 days after the reference memory task. The platform was located in a novel position in the middle of the south quadrant 40 cm from the wall opposite to the location used for the reference memory task. The rats were tested for 4 days followed by a probe trial on the fifth day. Testing was performed as described above for the reference memory task. 2.4.4. Working memory task The working memory task was performed 2 days after the reversal learning task. The platform was placed in a novel position every day. The rats were tested for 5 days. No probe test was performed. The platform positions were chosen such that the whole maze was covered during the 5 days of testing. The platform was placed at least 35 cm from the wall to prevent the animals from jumping out of the pool. Platform positions on subsequent days were not in the same quadrant. Between trials, the rats were removed from the water maze area and carried behind a screen where they were unable to see the water maze. A new trial was started after a delay of 10–15 s. In all other respects, the testing was performed similarly to reference memory task.

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2.4.5. Visible-platform task The visible-platform task was performed 5 days after the working memory task. The rats were tested for 2 days. To make the platform visible, the tank was filled with water 1 cm below the surface of the platform. The platform was positioned 40 cm from the wall and placed in the north quadrant the first day and in the south quadrant the second day. Testing was performed similarly to the reference memory task.

When the results were averaged over days (working memory task), and thus there is no repeated measure, the data were analyzed with a two-way univariate ANOVA with PCP and FGL as fixed factors. Following the two-way ANOVA, post-tests were performed using the Bonferroni method detailed in pages 741–744, 771 in Neter et al. [34]. The post-tests were calculated using the online Quick Calcs calculator (www.graphpad.com/quickcalcs/posttest1.cfm). Growth rates of the treatment groups were estimated using simple linear regression on body weight and PND, i.e. the slope of the regression lines.

2.5. Statistics

3. Results All data are reported as mean ± S.E.M., and statistical significance was accepted at P = 0.05. All statistical analyses were performed using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Water maze performance is expressed as swim distance to locate the platform. Probe test results are expressed as percent distance moved within the four quadrants. Furthermore, escape latency data for the main water maze results are available as supplementary data (see Appendix A). Data were analyzed using a three-way mixed-model analysis with repeated measures and a first-order autoregressive covariance structure. PCP and FGL were included as fixed factors, and Day or Trial as repeated measures. In the water maze experiment, the levels of Day or Trial were clearly related, reflecting a learning process over time. This is better modeled with a first-order autoregressive covariance structure than the standard compound symmetry [35]. The compound symmetry covariance structure was used when the levels of the repeated factor were expected to be unrelated, i.e. the trial 1 data from the working memory task and swimming speed results.

Memory performance was evaluated in the Morris water maze. Testing was started at the emergence of puberty on PND53 and ended on PND78. The rats were tested in three paradigms: reference memory, reversal learning, and working memory. 3.1. Reference memory The reference memory task is known to be hippocampaldependent [32]. The average swim distances during the 4 days of acquisition are shown in Fig. 1A. A linear mixed-model analysis with repeated measures (factors: Day, PCP, and FGL) showed a significant effect of Day (F3,148.6 = 70.4, P < 0.001) and PCP (F1,71.5 = 11.1, P = 0.01).

Fig. 1. Reference memory task in the Morris water maze. The hidden platform was in a fixed position in the north quadrant throughout the 4 days of acquisition. (A) Mean swim distances during the 4 days of training. An average was computed across the four daily trials for each rat. There was an overall significant effect of PCP (P = 0.01). (B) Mean swim distances over the four trials. An average was computed across all four training days for each rat. Trial 1 from Day 1 was excluded as discussed in the text. There was no overall significant effect of PCP in trial 1 (P = 0.859); however, an effect was present in the subsequent trials 2–4 (trial 2: P < 0.001, trial 3: P = 0.001, trial 4: P = 0.002). (C) Single trial probe test without the platform. Mean distance moved (%) in the four quadrants for the first 40 s of the test. All treatment groups demonstrated the highest spatial preference for the target quadrant. FGL significantly improved the percent distance moved in the target quadrant (P = 0.020) whereas PCP had no effect. Swim distance is presented in cm and distance moved in the four quadrants is shown in percent of total distance moved. All data are expressed as mean ± S.E.M. There were between 12 and 15 animals per treatment group.

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There was a trend toward an FGL effect (F1,71.5 = 2.6, P = 0.115). None of the interactions were significant. Escape latency also was evaluated; these results were very similar to the swim distance data and are shown in Supplementary Fig. 1A. Performance in trial 1 was related to long-term reference memory because in this trial the rats only had knowledge of the platform’s position from previous training days. However, in trials 2–4, the rats also had the benefit of short-term memory of the platform’s position from within the daily session, and the performance in these trials had an additional short-term memory component [2]. The performance in the individual trials, averaged over days, is shown in Fig. 1B. Trial 1 from Day 1 was excluded because the animals were experimentally naïve in this trial, and therefore no long-term memory component existed. The graph suggests that there was little effect of PCP in trial 1, but an effect in the following trials 2–4. A linear mixed-models analysis with repeated measures (factors: Day, PCP, and FGL; with trial 1 from Day 1 excluded) performed for each trial separately showed that there was no PCP effect in trial 1 (F1,59.2 < 1, P = 0.859), but that PCP had an effect in subsequent trials (trial 2: F1,83.6 = 13.2, P < 0.001; trial 3: F1,77.6 = 11.0, P = 0.001; trial 4: F1,72.2 = 10.4, P = 0.002). None of the interactions were significant in any of the four trials. The four acquisition days were followed by a probe test on Day 5 to estimate the reference memory status of the rats. Only the first 40 s of

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the trial were analyzed because the rats had a tendency to “give up” and stop searching in the platform zone and start looking elsewhere (subjective observation). The results of the probe test are depicted in Fig. 1C. It is evident from the figure that all treatment groups demonstrated the highest spatial preference for the target quadrant. It should be noted that the rats started the probe test in the West quadrant which probably was the reason for the somewhat higher spatial preference for this quadrant compared to the two other non-target quadrants (east and south). A two-way ANOVA analysis on the results from the target quadrant (factors: PCP and FGL) showed a significant effect of FGL (F1,50 = 5.7, P = 0.020), but no effect of PCP (F1,50 < 1, P = 0.454). The interaction was not significant. Escape latency data for the probe test are shown in Supplementary Fig. 1B. The probe test on Day 5 assessed the long-term consolidated reference memory of the platform position. This is comparable to the trial 1 situation mentioned above where the rats only have knowledge of the platform position from the previous training days, but not from previous trials on the same day. Taken together, the results from the reference memory training and the probe test indicate that the PCP treatment did not affect long-term reference memory acquisition and performance in this task. However, there was a clear PCP effect in trials 2–4 over the four training days, suggesting that neonatal PCP treatment produces impairment in the

Fig. 2. Reversal learning task in the Morris water maze. The hidden platform was moved to a novel position in the south quadrant. The platform was fixed in this position during the 4 days of acquisition. (A) Mean swim distances during the 4 days of training. An average was computed across the four daily trials for each rat. There was an overall significant effect of PCP (P = 0.001). (B) Mean distance moved (%) in the four quadrants for first 40 s of the probe test. All treatment groups showed the highest spatial preference for the target quadrant. There was no effect of either PCP or FGL (C) Mean swim distances for all 16 trials performed during the 4 days of training. The animals were quick to relocate the platform. Swim distance are presented in cm and distance moved in the four quadrants is shown in percent of total distance moved. All data are expressed as mean ± S.E.M. There were between 12 and 15 animals per treatment group. T1–4: trials 1–4.

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short-term memory component of this task rather than the longterm memory component. Conversely, FGL improved performance in the probe test indicating that the peptide was able enhance the long-term reference memory of the animals. 3.2. Reversal learning Reversal learning is associated with response inhibition and behavioral flexibility, which is known to be sensitive to lesions of the prefrontal cortex [10,14,26–28]. The average swim distances during the 4 days of reversal learning acquisition are shown in Fig. 2A. A linear mixed-model analysis with repeated measures (factors: Day, PCP, and FGL) showed a significant effect of Day (F3,137.0 = 63.0, P < 0.001) and PCP (F1,62.0 = 11.8, P = 0.001). There was no effect of FGL, and no interactions were significant. Escape latency results were very similar to the swim distance data and are shown in Supplementary Fig. 2A. Performance in all 16 trials over the 4 days is shown in Fig. 2C. The figure illustrates that the rats were quick to relocate the platform. Long swim distances were seen on the very first reversal trial, which were in the vicinity of the scores obtained in the beginning of reference memory acquisition, presumably because the platform position was unknown. The two control groups (VEH-I/VEH-II and VEH-I/FGL) reached their maximum performance by the end of acquisition Day 2, whereas the two PCP-treated groups (PCP/VEH-II and PCP/FGL) still showed longer

swim distances at the beginning of Day 3. However, on acquisition Day 4, the scores from all groups were almost identical. The linear mixed-model analysis with repeated measures (factors: Day, PCP, and FGL) was run again, but without the first trial on Day 1 and without all of Day 4 to isolate the reversal effect. The analysis showed an effect of Day (F2,103.5 = 17.4, P < 0.001) and PCP (F1,60.4 = 14.9, P < 0.001) but no effect of FGL and no significant interactions. The four acquisition days were followed by a probe test on Day 5 to estimate the memory status of the rats. The result of the first 40 s of the probe test is shown in Fig. 2B. All treatment groups showed the highest spatial preference for the target quadrant. As with the reference memory probe test, the rats started the test in the west quadrant which probably was the reason for the somewhat higher preference for this quadrant compared to the two other nontarget quadrants (east and north). A two-way ANOVA analysis on the results from the target quadrant (factors: PCP and FGL) revealed no effect of PCP or FGL, and the interaction was not significant. The probe test reflects the memory status after Day 4 when all groups had reached their maximum performance, so it is not surprising that no differences were seen between groups. Escape latency data for the probe test are shown in Supplementary Fig. 2B. Taken together, the results from the reversal learning task show that rats treated neonatally with PCP were impaired in this task requiring behavioral flexibility. FGL treatment was not able to reverse this impairment.

Fig. 3. Working memory task in the Morris water maze. The hidden platform was moved to a new position every day, and the rats were tested for 5 days. No probe test was performed. (A) Mean swim distances over the four trials. An average was computed across all five testing days for each rat. There was a significant effect of PCP across all trials (P = 0.010) and in trial 2 alone (P = 0.006). (B) Mean trial 1 performance during the 5 days of testing. There was a significant effect of Day (P < 0.001), showing a considerable day-to-day fluctuation in performance. (C) Mean savings in swim distance between trials 1 and 2 (T1–T2). FGL significantly improved savings in the PCP-treated groups (PCP/VEH-II vs. PCP/FGL, P < 0.05). Swim distance and savings are expressed in cm. All data are expressed as mean ± S.E.M. There were between 12 and 15 animals per treatment group.

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3.3. Working memory The working memory task often is referred to as a delayedmatching-to-place task. The task tests fast one-trial learning as opposed to slower incremental learning over many trials and days which is characteristic of the reference memory task. Because the platform is moved to a new position every day, the rats cannot benefit from the memory formed during the previous days. Thus, performance does not increase over days of testing. Rats behavior in this task is characterized by long swim distances in trial 1 followed by substantially lower swim distances in the subsequent trials. The largest saving in swim distance occurs between trials 1 and 2, and therefore the performance in trial 2 is the main focus of this test, which reflects how well the rats “remember” the new position of the platform formed in trial 1 [41]. The rats improve their scores a little further in trials 3 and 4, approaching maximum performance. This ensures that the rats will enter the overnight delay at a similar performance level. Classically, the delayed-matching-to-place (or sample) tasks are known to be dependent on the prefrontal cortex. However, these types of tasks also are sensitive to lesions of the hippocampus [15,41,50]. The results of the working memory task averaged over days are shown in Fig. 3A. A mixed-model analysis with repeated measures (factors: Trial, PCP, and FGL) revealed a significant effect of Trial (F3,136.8 = 125.0, P < 0.001) and PCP (F1,61.6 = 7.1, P = 0.010) but no effect of FGL. None of the interactions were significant. Escape latency data for this test is shown in Supplementary Fig. 3A. Analysis of trial 2 performance with a two-way ANOVA (factors: PCP and FGL) showed a significant effect of PCP (F1,50 = 8.2, P = 0.006) but no effect of FGL and no PCP × FGL interaction. It is possible that the absolute performance in trial 2 was influenced by the performance in trial 1 [41]. A closer examination of trial 1 data revealed day-to-day fluctuations (Fig. 3B), especially on Day 4 when the platform was located close to the center of the pool and shorter swim distances were seen. A linear mixed-model analysis with repeated measures (factors: Day, PCP and FGL) revealed a significant effect of Day (F4,200 = 5.3, P < 0.001) but no effect of PCP or FGL (PCP: F1,50 < 0.1, P = 0.842; FGL: F1,50 = 0.4, P = 0.506) and no interactions. This means that other factors like platform position could have an impact on the swim distances in trial 1, even though the location of the platform was unknown. For instance, search strategies where the rats frequently cross the center of the maze should, by change, encounter platform positions near the center more often than platform positions around the periphery. It follows that a factor like platform position also could have an impact on trial 2 performance. Therefore, an analysis that considers the differences in swim distances between trials 1 and 2 should provide a more meaningful measure of memory performance. For this reason, savings were measured and defined as the difference in swim distances between trials 1 and 2 (T1–T2). This was done by subtracting the individual trial 2 scores from the trial 1 group mean scores from the same day with the expectation that larger savings reflect better performance. The savings in swim distances averaged over days are shown in Fig. 3C. A two-way ANOVA (factors: PCP and FGL) showed a significant effect of PCP (F1,50 = 9.8, P = 0.003), no effect of FGL (F1,50 = 1.9, P = 0.177), and a significant PCP × FGL interaction (F1,50 = 4.7, P = 0.034). A significant interaction made the main effects of PCP and FGL difficult to interpret. It indicated that the effect of PCP depended on whether the FGL treatment was given in combination. Subsequent Bonferroni post-tests showed that the PCP/VEH-II group had a significantly lower saving score than both the PCP/FGL (P < 0.05) and VEH-I/VEH-II groups, suggesting that the combination treatment with PCP and FGL significantly improved savings over the PCP treatment alone. Similar results were obtained using escape latency data (Supplementary Fig. 3B). Taken together, these results show that neonatal PCP treatment produces clear impairments in the working memory version of

Fig. 4. Visible-platform task in the Morris water maze. The tank was filled with water 1 cm below the surface of the platform to make it visible. The rats were tested for 2 days with the platform in a new position every day. Mean swim distances over the four trials are presented. An average was computed across the two testing days for each rat. There was no effect of either PCP (P = 0.659) or FGL (P = 0.837). Swim distance is presented in cm and expressed as mean ± S.E.M. There were between 12 and 15 animals per treatment group.

the Morris water maze. The PCP/FGL group showed better savings between trials 1 and 2 than the PCP/VEH-II group, indicating that FGL treatment improved this impairment. 3.4. Visible platform The visible-platform task is a non-spatial control test to ascertain whether the neonatal PCP treatment produces damage to the rat’s visual system, thereby impairing their ability to navigate by eyesight or in some way affecting their motivation to escape from the water. The swim distances from the four trials averaged over the 2 days of testing with the visible-platform task are shown in Fig. 4. A mixed-model analysis with repeated measures (factors: PCP, FGL, and Trial) showed a significant effect of Trial (F3,131.9 = 46.9, P < 0.001), but no effect of either PCP (F1,58.0 = 0.2, P = 0.659) or FGL (F1,58.0 < 0.1, P = 0.837) and no interaction. Thus, this result showed that neither treatment with PCP nor FGL affected the rats’ performance in the visible-platform task. It was possible that the rats used spatial information to locate the platform due to the training received in the previous tasks where the animals must utilize extramaze cues to navigate in the water. However, normal visible-platform performance coinciding with impaired hidden-platform performance, as is the case in the present experiment, indicated that motivational and sensorimotor defects did not contribute significantly to hidden-platform deficits. 3.5. Swimming speed The swimming speed during the three water maze tasks is shown in Fig. 5. Swimming speed was on average between 24 and 27 cm/s. Mixed-model analyses with repeated measures (factors: PCP, FGL, and Day) demonstrated that there was no difference in swimming speed among the treatment groups during the reference and working memory tasks. However, during the reversal task, the two PCP-treated groups were swimming slightly faster than the two VEH-I groups (F1,50 = 8.6, P = 0.005). The difference in swimming speed was small (26.4 ± 0.3 vs. 24.2 ± 0.3) and this parameter does not affect the animals swim distances, which was our chosen performance measure.

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Fig. 5. Swimming speed in the Morris water maze. Mean swimming speeds in the (A) reference memory, (B) reversal learning, and (C) working memory tasks. An average was computed across the four daily trials for each rat. There was no difference in swimming speed in the reference memory and working memory tasks. However, the PCP-treated rats were swimming slightly faster in the reversal learning task (P = 0.005). Swimming speed is expressed in cm/s. All data are expressed as mean ± S.E.M. There were between 12 and 15 animals per treatment group.

3.6. Body weight Neonatal PCP treatment was associated with a decrease in the rate of weight gain (not weight loss) during the treatment period. Weight data are shown in Fig. 6B and a detailed graph from PND4–21 is shown in Fig. 6A. It is evident from Fig. 5A that the PCP-treated rats gained weight at a slower rate during the week PCP was administered (PND7–12). A linear regression analysis revealed that the growth rate was 0.4 g/day for the PCP-treated groups (PCP/VEH-II and PCP/FGL) and 2.2 g/day for the control groups (VEH-I/VEH-II and VEH-I/FGL) during this period. Subsequently, after the PCP treatment had finished, the PCP-treated rats gained weight at a normal rate compared to the control groups in the following PND12–21 period: 2.1 g/day vs. 2.3 g/day. However, they never completely “caught up” to the control groups and a 7% weight difference remained on PND81 after the behavioral tests had ended (PCP/VEH-II and PCP/FGL vs. VEH-I/VEH-II and VEH-I/FGL).

The weight data were analyzed with a linear mixed-model analysis with repeated measures (factors: PND, PCP, and FGL). The animals were not individually marked until the behavioral testing began, but housed first in litters (PND0–23), then in cages of three to four juveniles (PND23–39), and finally in cages of two adults (PND39–81). Thus, the weight data were split into three categories (pups, juveniles, and adults) with litter or cage as the statistical units. The statistical analysis was performed on each time-period separately. In all cases, there was a significant effect of PND (pups: F10,163.4 = 707.5, P < 0.001; juveniles: F3,32.8 = 5255.5, P < 0.001; adults: F4,83.4 = 2094.1, P < 0.001) and PCP (pups: F1,15.9 = 52.2, P < 0.001; juveniles: F1,11.1 = 55.0, P < 0.001; adults: F1,20.6 = 27.4, P < 0.001) but no effect of FGL. There was also a significant PND × PCP interaction in the first two time-periods (pups: F10,163.4 = 35.9, P < 0.001; juveniles: F3,32.8 = 12.0, P < 0.001), which disappeared in the adults (F4,83.4 = 0.4, P = 0.814), possibly reflecting that the percent weight difference between PCP and con-

Fig. 6. Body weight. (A) Detailed weight data from the pre-weaning period. The PCP-treated pups gained weight at a slower rate during the PCP treatment period (PND7–12). However, their growth rate returned to normal after the treatment had finished. The PCP-treated animals weighed significantly less than the control animals in the preweaning period (P < 0.001). (B) Weight data from the entire experiment that ended on PND81–82. Although, the PCP-treated groups gained weight at a normal rate after the PCP treatment had ended, they never reached the level of the two control groups. The adult PCP-treated animals weighed significantly less than the control animals (P < 0.001) and a 7% weight difference remained at the end of the experiment. FGL did not affect body weight. Weight data are presented in g and expressed as mean ± S.E.M. for each treatment group.

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trol groups became smaller with time as the PCP animals “caught up”; eventually the percent weight difference stabilized in the adult time-period. In summary, these results show that the rats gained weight over time and that pups neonatally treated with PCP had a reduced growth rate during the treatment period that resulted in a significantly lower body weight throughout the experiment. Even though, the PCP-treated animals gained weight at a normal rate after the PCP treatment had finished a weight difference of 7% remained at the end of the experiment. FGL had no effect body weight. 4. Discussion Results from the present study clearly show that neonatal PCP treatment produces long-lasting impairment in all three water maze tasks. The water maze task contains several components that contribute to the rats’ ultimate escape from the water. For example, in the reference memory task, there is a long-term memory component, from which the rats can benefit. The memory of the platform’s position from previous days aids performance. Trials 2–4 have an additional short-term memory component because there is an immediate benefit from the memory formed within the daily session [2]. It follows that trial 1 is unique because it is the only trial without a short-term memory component. The results from the reference memory task show that PCPtreated rats have similar trial 1 swim distances compared to the VEH-I-treated groups, and the PCP-treated rats perform equally well in the probe test. Trial 1 and the probe test have no shortterm memory component because it is the first trial performed on a particular day, and in the case of the probe test it is the only trial performed. The PCP rats showed decreased performance in the subsequent trials 2–4 when the short-term memory component was present. This suggests that the neonatal PCP treatment produces impairment in short-term memory and not long-term, consolidated memory. This inference is supported by the results from the working memory task that showed decreased performance of the PCP-treated groups. Deficits in this task were very robust. Lower performance was seen across all trials, in absolute trial 2 performance, and in savings. The working memory task has no longterm memory component because the platform position is changed every testing day, and thus the rats cannot benefit from the memory formed on the previous days. It specifically tests the short-term, one-trial type of memory. The reversal learning task is slightly different. It is performed the same way as the reference memory task, but with the platform located in a novel position. Success in this test requires the rats to inhibit the previous learned response and start swimming to the new platform position; a function that has been described as response inhibition or behavioral flexibility. The test clearly showed decreased performance in the PCP-treated animals, indicating that neonatal PCP treatment produces impairment in this task. Taken together, the results from the three tasks in the water maze showed that neonatal treatment with 30 mg/kg PCP produced robust impairment in short-term memory and response inhibition whereas long-term memory in the reference memory task remained intact. This points toward a more selective frontal cortical dysfunction rather than a more widespread memory impairment and is in line with the acute neurodegenerative profile reported for this model [46]. Also, the results demonstrated that using PCP in a higher dose of 30 mg/kg produced schizophrenia relevant cognitive impairment. Comparable deficits have been reported in behavioral paradigms that tests response inhibition and behavioral flexibility, and working memory [1,5,45]. Interestingly, Broberg et al. [5] demonstrated that neonatal treatment with 23 mg/kg PCP resulted in significantly lower performance in an attentional set-shifting paradigm than a lower 11.5 mg/kg dose, suggesting a dose–response relationship between PCP dose and cognitive impairment.

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Several modes of action can be theorized from the FGL dosage regimen used. First, FGL has growth factor-like and neuroprotective properties and is expected to provide protection and growth support for neurons when the neonatal PCP treatment is given. FGL treatment given before and between PCP injections leads to the loss of fewer neurons, and the PCP effect on the development and integration of neuronal networks that are formed at this time will be less severe. Second, continuous administration of FGL after PCP treatment provides trophic neuronal support. The ability to induce neurite outgrowth and synapse formation ensures faster and better recovery of affected neurons and networks. Third, FGL administered shortly before behavioral testing will positively affect the rats. FGL is known to function as a memory enhancer even many days after administration [6,39]. The present experimental design did not allow for distinguishing between these modes of action, but the effects may be due to a combination of the three modes mentioned above. In the reference memory task, there was a trend toward FGLtreated groups performing better than VEH-II-treated groups across days, but the performance differences were too small to reach statistical significance. However, FGL significantly improved performance in the probe test indicating that the peptide was able to enhance the long-term reference memory of the animals. This finding corroborates previous studies, demonstrating that the peptide have memory enhancing properties even when given several days before testing [6,39]. FGL did not have any effect in the reversal learning task, and thus no effect on response inhibition. There was an effect on savings in the working memory task. The PCP/FGL group had statistically better savings than the PCP/VEH-II group, showing near-normalization of performance to indicate an effect on working memory. This is an important finding, because cognitive deficits, including deficits in working memory, are believed to represent a core feature of schizophrenia and to be putative endophenotypes, which are genetically determined. The cognitive deficits also are present before disease symptoms develop and in first-degree relatives [4,49] and are more important for the patients’ functional outcome than the positive symptoms [16]. However, they are in general poorly treated by current antipsychotic drugs [13,18,29,30], which underlines the need for more adequate treatment alternatives that addresses these deficits. In the current study, neonatal treatment with a high dose of 30 mg/kg PCP was used to create pharmacological lesion. PCP is a NMDA receptor antagonist and as discussed in Section 1 neonatal PCP treatment results in acute neuronal degeneration. However, PCP also interacts with a number of other neurotransmitter and receptor systems, e.g. noradrenalin, dopamine, serotonin, sigma opiod receptors, and others [22,23,42]. Thus, it was possible that interaction between PCP and these systems added to the neurotoxicity. Another undesirable side effect of the neonatal PCP treatment was a reduced growth rate during the treatment period. Although, the growth rate reduction was short (5 days) it was in a crucial period in brain development. Therefore, the reduced neonatal growth rate might have contributed a little to the early insult. However, neonatal treatment with 30 mg/kg PCP still resulted in specific cognitive deficits in adult animals with relevance to schizophrenia, some of which could be manipulated by FGL treatment. Neonatal PCP treatment did not appear to affect visual or motor functions of the animals. Visible-platform acquisition was normal although the rats were impaired in hidden-platform performance in the three memory tasks, which indicated that motivational and sensorimotor defects did not contribute significantly to hiddenplatform deficits. With regard to swim speed, the rats were swimming at the same speeds in the reference and working memory tasks, but the PCP-treated rats were swimming slightly faster in the reversal learning task. It is unclear why such differences

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