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Tryptophan depletion impairs object-recognition memory in the rat: Reversal by risperidone
Behavioural Brain Research 208 (2010) 479–483
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Tryptophan depletion impairs object-recognition memory in the rat: Reversal by risperidone Trisha A. Jenkins a,c,∗ , Jennifer J. Elliott b , Tara C. Ardis a , Marie Cahir a , Gavin P. Reynolds a , Robert Bell b , Stephen J. Cooper a a b c
Division of Psychiatry and Neuroscience, Queen’s University Belfast, Northern Ireland, United Kingdom School of Psychology, Queen’s University Belfast, Northern Ireland, United Kingdom Health Innovations Research Institute and School of Medical Sciences, RMIT University, Victoria, Australia
a r t i c l e
i n f o
Article history: Received 19 October 2009 Received in revised form 14 December 2009 Accepted 17 December 2009 Available online 31 December 2009 Keywords: Tryptophan depletion Serotonin Object-recognition memory Cognition Antipsychotic Risperidone
a b s t r a c t Tryptophan depletion techniques are effective in reducing central serotonergic function and have been used to investigate its role in mood and cognition. In the present study a tryptophan-free diet was fed to Lister-hooded male rats chronically for 21 days to investigate the effect of lowering central serotonin concentration on cognition using the novel object-recognition paradigm. Chronically tryptophan-depleted rats had impaired object-recognition memory; this was accompanied by a reduction in central serotonin of 40–50% in the hippocampus, frontal cortex and striatum. In a subsequent experiment, the atypical antipsychotic, risperidone (0.2 mg/kg), but not the typical antipsychotic, haloperidol (0.1 mg/kg), administered i.p. 30 min prior to the retention test, significantly attenuated the chronic tryptophan depletion impairment. These data show that chronic lowering of central serotonin is associated with impaired cognitive performance, and that this can be reversed by the atypical antipsychotic, risperidone. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Serotonin (5-HT) is known to regulate many physiological and behavioural functions including aspects of mood, anxiety and cognition. Synthesized from its amino acid precursor tryptophan (TRP), 5-HT levels in plasma and brain can be influenced by manipulating dietary TRP intake [8,16,18]. Dietary TRP depletion is a non-pharmacological method to reduce central 5-HT and has been widely used as a tool to assess the role of 5-HT in cognitive and affective functioning in both humans and animals [7,9,23,34,41]. Acute tryptophan depletion (ATD) in rats can be induced by oral administration of a TRP− free (TRP−) amino acid mixture [30] or feeding of a TRP− diet [3,11]. Both protocols lead to a robust reduction in plasma TRP and a reduction in central 5-HT concentrations. Moreover ATD produces deficits in object-recognition memory [25,32,43,51], but not reversal or spatial memories [32,50].
∗ Corresponding author at: Health Innovations Research Institute and School of Medical Sciences, PO Box 71, RMIT University, Bundoora, Victoria 3083, Australia. Tel.: +61 3 9925 6523; fax: +61 28 9925 7063. E-mail address: [email protected] (T.A. Jenkins). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.12.030
Chronic TRP depletion (CTD) allows the investigation of the consequences in animals of a long term reduction in central serotonergic function. Along with reducing plasma and brain TRP, and brain 5-HT [11], CTD alters 5-HT receptor binding in the brain with decreases in 5-HT1A receptors and increases in 5-HT2A receptors being reported [11,27]. Moreover, increasing the sensitivity of rats to external and stressful stimuli [47] and contextual fear memory deficits [49] have been reported after CTD. Effects on object-recognition memory have not been determined in this model, however other studies concerning long term reduction in 5-HT function such as injection of serotonergic antagonists [39] and lesioning of the dorsal raphe nucleus [31], produce object memory deficits. Atypical antipsychotics exhibit both dopaminergic, primarily D2, and serotonergic pharmacology as compared to typical antipsychotics which are primarily dopamine-specific [33,40]. Effects of atypical antipsychotics on the brain 5-HT system are varied but extracellular 5-HT concentrations are increased [21,24] while 5-HT2A receptor expression is down regulated [14,45]. In addition, atypical antipsychotics exhibit positive effects on memory in animal models of cognitive dysfunction [10,19]. Typical antipsychotics, however, which primarily blocking brain dopamine receptors, may exhibit indirect effects on the 5-HT sys-
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Table 1 Composition of the TRP diets. g/kg Sucrose Corn oil Zein Gelatin Mineral mix Calcium phosphate, dibasic Vitamin mix Choline chloride Ammonium citrate, dibasic Glycine l-Lysine HCl l-Histidine dl-Methionine l-Phenylalanine l-Leucine l-Isoleucine l-Threonine l-Valine l-Tyrosine l-Arginine HCl
655.75 100 20 30 35 4.5 10 1.5 23.4 23.4 30 4.2 5.3 7.1 14.2 6.2 9.7 11.1 7.0 8.7
l-Tryptophan (TRP− group) l-Tryptophan (TRP+ group)
0.0 7.0
Diets were obtained from Harlan (UK).
tem, as there is a considerable amount of interaction between the dopaminergic and serotonergic neurotransmitter systems [55]. The present study examined the effect of 21 days of CTD on object-recognition memory performance. In addition we investigated the ability of the typical antipsychotic, haloperidol, and the atypical risperidone, at a dose previously used to modify an objectrecognition memory impairment [19], to reverse a CTD-induced disruption of object-recognition memory. Brain changes in 5-HT were also investigated in regions associated with higher order brain function. 2. Experimental procedures
heavy enough not to be displaced by the animals. The rat was then removed and returned to its homecage. One hour later the rat was returned to the arena and exposed to one familiar object and a novel object for 3 min in the test phase. Objects were placed approximately 10 cm apart in the centre of the arena and both were cleaned with 70% alcohol between trials. Familiar and novel objects were alternated between the left and right position to prevent bias for a particular location. Trial and test were video recorded and time spent exploring each object was scored manually by an investigator blinded to the treatment groups. Object exploration was defined as sniffing or touching the object with the nose and/or forepaws. Sitting on the object was not considered exploratory behaviour. Ability to discriminate in the test was expressed as discrimination index (DI) = (time exploring novel object − time exploring familiar object/total exploration time). 2.3.2. Locomotor activity One hour after object-recognition testing locomotor activity was assessed in both groups. Activity was performed under 320 lx light conditions (lighting uniform in all areas of the arena) in a room (185 cm × 305 cm × 285 cm) using a Med Associates (USA) open field test chamber (44.5 cm × 44.5 cm × 30.5 cm) with four pairs of photocells spaced evenly along the length of the cage. Rats were placed in the box for 10 min and analysis performed by Med Associates (USA) activity monitor software, version 4. 2.3.3. Collection and preparation of blood and tissue samples All animals were sacrificed immediately after locomotor testing. Animals were killed with CO2 , cervically dislocated and then blood (approximately 5 ml) was removed by cardiac puncture for plasma TRP measurement. Blood was collected into EDTA tubes and kept on ice prior to centrifugation at 1600 g for 15 min at 4 ◦ C. The resulting plasma supernatant was centrifuged at 12,000 g for 30 min at 20 ◦ C through YM-10 Amicon filters to remove plasma protein and filtrate was frozen at −80 ◦ C until TRP analysis. After blood sampling brains were removed immediately and dissected into frontal cortex, hippocampus and striatum for determination of 5-HT and 5-HIAA. Brain tissue was deprotonised on ice, with 10 volumes 0.1 M perchloric acid and homogenized by hand. Samples were centrifuged at 12,000 g for 15 min at 4 ◦ C, and supernatant was removed and frozen at −80 ◦ C until analysis. 2.3.4. Chromatographic analysis of plasma TRP and central monoamines Free plasma TRP and central monoamines were separately determined by separation on a Hypersil 5 m ODS C18 (150 mm × 4.6 mm) column coupled with an ESA Coulochem II electrochemical detector. The analytical potentials for plasma TRP were set at E1 +300 mV and E2 +575 mV. The mobile phase (pH 4.05) consisted of 0.1 M NaH2 PO4 , 0.1 M citric acid, 0.75 mM octane sulphonate acid, 0.1 mM EDTA and 16% methanol. The analytical potentials for monoamines were set at E1 +50 mV and E2 +500 mV. The mobile phase (pH 3.0) consisted of 0.1 M sodium dihydrogen phosphate monohydrate, 2.5 mM octane sulphonate acid, 0.5 mM EDTA, 20.5 ml glacial acetic acid and 12% methanol. Quantification was obtained by comparing peak height of current E2 with standard concentrations.
2.1. Animals 2.4. Experiment 2 Adult male Lister-hooded rats (Harlan, UK), weighing 200–300 g at the beginning of the experiment, were maintained on a reversed 12 h light dark cycle (lights off 6 a.m.). Room temperature (21 ± 2 ◦ C) and humidity (45–55%) were kept constant throughout. Experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986. 2.2. Diet TRP− free (TRP−) and equivalent control diet (TRP+) were obtained from Harlan (UK). The TRP+ diet contained 0.7% TRP and the TRP− diet 0% TRP. The composition of the two diets is shown in Table 1. Rats had free access to either TRP+ or TRP− diet for 21 days; water was available ad libitum. CTD animals were group housed (3–4 rats/cage) and were weighed each day, as were controls. Behavioural testing was performed at the end of dietary manipulation (day 21).
2.4.1. Antipsychotic drugs In a second series of animals, the effect of antipsychotic drugs on CTD memory in the object-recognition task was investigated. CTD and object recognition was performed as described in experiment 1. Compounds were freshly prepared on the day of the experiment, and administered 30 min prior to the retention test. Groups received vehicle (0.9% saline, i.p.), risperidone (0.2 mg/kg, i.p.), or haloperidol (0.1 mg/kg, i.p.). Rat numbers per group were TRP+ n = 10, TRP− n = 8, TRP− and HAL n = 8, TRP− and RIS n = 9. 2.4.2. Data analysis Data was normally distributed. All data were analysed using SPSS for Windows version 17.0. Student’s paired t-test was performed to compare the effect of tryptophan depletion on the time spent exploring identical objects in the trial and the familiar versus the novel object in the test. Analysis of the DI data was performed using one way ANOVA followed by posthoc Dunnett’s t-tests.
2.3. Experiment 1 2.3.1. Object-recognition testing The object-recognition [15,57] apparatus consisted of a black plastic square arena (45 cm × 63 cm × 63 cm). The light intensity of the testing room (185 cm × 305 cm × 285 cm) was 360 lx and lighting was equal in the different parts of the apparatus. Five different sets of objects made of inert material such as glass, plastic and ceramic, each available in triplicate and that could not be displaced by a rat were used for discrimination. Rats were exposed to the object-recognition task between the 8th and 9th hour of the dark phase of the reverse light cycle. Animals received two sessions of 3 min duration in the empty box over the two days prior to testing to allow habituation to the arena and test room environment. The object-recognition task comprised a trial and test session, both of 3 min duration. In the trial the rat was placed into the arena and exposed to two identical objects for 3 min. The objects to be discriminated were made of glass, plastic, metal, or ceramic and were approximately equal heights and
3. Results 3.1. Effect of CTD on object recognition In the trial phase no significant difference in exploration time of the two objects was observed (total exploration time of both objects: TRP+ 73 ± 5 s; TRP− 82 ± 9 s; p = 0.46; n = 10/group). During the test, no significant difference in total exploration time of the two objects was observed (total exploration time of both objects: TRP+ 77 ± 5 s; TRP− 85 ± 8 s; p = 0.49; n = 10/group). Rats chronically fed the control TRP+ diet spent significantly longer exploring the novel object compared to the familiar object
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Fig. 1. (A) The effect of chronic tryptophan depletion on exploration time (s) of a familiar object and a novel object in the 3-min test. Data are expressed as the mean ± S.E.M. (n = 10/group), significant difference in time spent exploring the novel compared to the familiar object. *** p < 0.001 familiar cf novel. (B) The effect of chronic tryptophan depletion on the discrimination index (DI). Data are expressed as the mean ± S.E.M. (n = 10/group) ** p < 0.01 cf control TRP+.
(p < 0.001). In contrast there was no significant difference between the time spent exploring the novel and familiar object in the TRP− group (p = 0.99; Fig. 1a). Consequently, there was a significant difference in DI level between the TRP+ and TRP− groups (p < 0.01; Fig. 1b). 3.2. Effect of CTD on rat body weight and locomotor activity Analysis of the final weights of the CTD study showed that in the three weeks of feeding TRP+ animals increased their bodyweight by approximately 9% (274.1 ± 2.8 g to 301 ± 5.3 g) while TRP− animals lost weight (22%; 273.1 ± 2.9 g to 215.5 ± 3.6 g). The final weight difference between TRP+ and TRP− is significantly different (p < 0.001). Locomotor activity was assessed at the end of the three week feeding regime. There was no significant difference in distance travelled between TRP+ and TRP− animals (TRP+ 2400 ± 237 cm; TRP− 2350 ± 83 cm; p = 0.84). 3.3. Effect of CTD on free plasma TRP, central 5-HT and 5-HIAA CTD significantly reduced free plasma TRP. Feeding rats a TRP− diet chronically for 21 days reduced TRP by 83% (TRP+ 4.67 ± 0.30 g tryptophan/ml plasma; TRP− 0.78 ± 0.06 g tryptophan/ml plasma; p < 0.001; n = 10/group). TRP− rats showed significant decreases in 5-HT and 5-HIAA concentrations in the frontal cortex, hippocampus and striatum, as compared to TRP+ group (Table 2). 3.4. Effect of antipsychotic drugs on CTD-impaired object-recognition memory All groups of rats spent equivalent time exploring the identical objects (left and right) in the trial phase, with no significant differences in exploration times within groups (TRP+ 65 ± 6 s, TRP− 67 ± 4 s, TRP− and HAL 58 ± 7 s, TRP− and RIS 48 ± 5 s; F(3,34) = 2.4, p = 0.09).
In the test phase, all groups of rats spent equivalent total time exploring the objects, with no significant differences in exploration times within groups (TRP+ 74 ± 5 s, TRP− 76 ± 6 s, TRP− and HAL 58 ± 7 s, TRP− and RIS 73 ± 5 s; F(4,34) = 1.4, p = 0.26). Rats chronically fed the control diet spent significantly longer exploring the novel object compared to the familiar object (p < 0.001). The ability to discriminate familiar and novel objects was abolished following CTD, whereby there was no significant difference in exploration of the objects (p = 0.28). Haloperidol failed to improve the CTD-induced impairment, no difference in time exploring novel and object objects was observed (p = 0.28), while TRP− rats pre-treated with risperidone spent significantly longer exploring the novel object compared with the familiar object (p < 0.001) (Fig. 2a). These findings were reflected by the DI which showed an overall significant effect of group (F(3,34) = 12.0, p < 0.001). Posthoc analysis demonstrated that, as previously shown, CTD significantly reduced the DI compared to control (p < 0.001). This was not affected by pretreatment with haloperidol (p < 0.001 compared to control; p = 1.0 compared to TRP-). Conversely, risperidone pretreatment reversed the TRP− object-recognition memory impairment (p = 0.99, compared to control) (Fig. 2b).
4. Discussion This study demonstrates that a dietary regime of chronic TRP depletion can induce a profound deficit in object-recognition memory, which could be reversed by atypical, but not typical, antipsychotic treatment. Concurrently, CTD induced a substantial reduction in free plasma TRP and a consistent decrease in central 5HT and 5-HIAA in all brain regions measured as compared to control TRP+ fed rats. Tryptophan depletion for twenty-one days produced a significant impairment in object-recognition memory. This is the first report of a deficit using the object-recognition paradigm with a
Table 2 Effect of acute and chronic tryptophan depletion on central 5-HT and 5-HIAA in the rat frontal cortex, striatum and hippocampus. Frontal cortex TRP+ CTD % Change from TRP+
5-HT (ng/g)
112.0 ± 15.83
5-HIAA (ng/g)
522.7 ± 30.03
% Change from TRP+ Data expressed as mean ± S.E.M. (ng/g). * p < 0.05, ** p < 0.01, *** p < 0.001.
Striatum TRP− 57.7 ± 5.93* 49% 261.8 ± 37.83*** 50%
Hippocampus
TRP+
TRP−
TRP+
181.8 ± 14.57
109.1 ± 9.154** 40%
62.10 ± 9.402
TRP− 28.15 ± 3.15* 55%
541.2 ± 35.42
164.0 ± 17.88*** 70%
580.0 ± 35.46
152.7 ± 13.63*** 74%
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Fig. 2. (A) The effect of chronic tryptophan depletion on exploration time (s) of a familiar object and a novel object in the 3-min test. Data are expressed as the mean ± S.E.M., significant difference in time spent exploring the novel compared to the familiar object. *** p < 0.001 familiar cf novel. (B) The effect of antipsychotic pre-treatment on chronic tryptophan depletion on the discrimination index (DI). Data are expressed as the mean ± S.E.M. (n = 10/group) *** p < 0.001 cf control TRP+.
chronic depletion model. Memory deficits have been demonstrated in chronically TRP-depleted mice, showing impaired formation of contextual fear memory [49]. However spatial and conditioned taste aversion memories appear to remain unaffected [49]. Other examples of long-term 5-HT brain deficits in rodents include lesion studies which cause a hyposerotonergic state in localised projection areas [6,26]. Serotonergic lesions of the dorsal raphe nucleus also produce object memory deficits [31]. This effect of 5-HT depletion and object-recognition memory may be somewhat specific as memory deficits after a loss of brain 5-HT are not universal: serotonergic depletion of the dorsal or ventral hippocampus did not show significant changes in the Y-maze, water maze, or Tmaze delayed alternation test [1]; lesions of the raphe nucleus do not affect inhibitory avoidance memory [4]; while forebrain 5-HT depletion actually facilitates the acquisition and performance of a conditional visual discrimination task [53]. Generally it is accepted that ATD also produces objectrecognition deficits: in normal albino rats [25,32,43,51], and in 5-HT transporter knockout rats [38]. Our results show that a long-term reduction in central serotonergic function produces a similar result, but without the other conflicting effects of brain lesions. In the present study feeding rats a TRP− diet chronically reduced free plasma TRP levels by 80–90% from controls. This peripheral TRP depletion was associated with a 40–60% reduction in central 5-HT concentrations in the different brain structures measures. These results are comparable to those reported in our previous study using the TRP− diet [11]. Human tryptophan depletion studies report changes in cognitive functioning associated with 70–90% reductions in peripheral plasma TRP levels [12,46,58]. Therefore achieving a similar degree of plasma TRP depletion in rats allows a better inter-species comparison of behavioural effects. In the present study feeding rats a TRP− diet chronically reduced free plasma TRP levels by 80–90% from controls. This peripheral TRP depletion was associated with a 40–60% reduction in central 5-HT concentrations in the different brain structures measures. These results are comparable to those reported in our previous study using the TRP− diet [11]. The specific brain areas and neural processes involved in objectrecognition memory are still to be elucidated. However it is generally considered that regions within the medial temporal lobe such as parahippocampal cortices, particularly the perirhinal cortex, which is important for memory encoding, consolidation, and retrieval processes [13,54]. Object-recognition memory is suggested not to be hippocampal dependant [2,13,37]. The role of 5-HT in object-recognition memory is complex: 5-HT6 receptor antagonists have been shown to reverse object-recognition memory deficits [17] in scopolamine [29,56] and aged [22,35] animals; as has the 5-HT7 receptor where the selective 5-HT7 antagonist, SB269.970, significantly reduces object recognition in low-responding rats [5]; while the 5-HT4 receptor has been implicated in increased acquisition in the object-recognition task as rats
treated with the agonist, RS 67333, perform better than controls [28]. The second major finding of this work is that the CTD-induced cognitive deficit was reversed by the atypical antipsychotic, risperidone, but not the classical typical antipsychotic, haloperidol. Only one dose was used, however these doses were selected from a previous study by Neill and colleagues as the optimum doses to reverse phencyclidine-induced object-recognition memory deficits [19]. While both antipsychotics exhibit dopamine D2 receptor antagonism, atypical antipsychotics also bind with high affinity to 5-HT2A receptors [36]. Typical agents such as haloperidol only weakly occupy serotonergic receptors [44]. This suggests that 5HT2A receptors may play a role in CTD-induced object-recognition memory deficits and indeed we have shown increased cortical 5HT2A-receptor binding after CTD [11]. Atypical antipsychotics such as risperidone have previously been demonstrated to reverse object-recognition deficits in other models of memory impairment such as the phencyclidine model both acutely [19] and chronically [20]. However this is the first time they have been shown to have an effect in tryptophan depletion. Thus the action of risperidone on object recognition appears independent of the mechanism of its disruption. The specific mechanism underlying the ability of risperidone to reverse the effect of CTD on object-recognition memory remains unclear. It would seem likely that the effect is not D2 receptormediated, given the finding with haloperidol, but via one or more of the other receptors that risperidone influences: particularly 5HT2A, but conceivably 5-HT1B, 5-HT7 or the alpha2C adrenoceptor [42]. Research within the patient population has suggested that 5HT2A affinity plays an important role in the cognitive effects of the atypical antipsychotics, independent of a dopaminergic influence [48]. However comparison of the antipsychotic amisulpiride, which only binds to D2/D3 receptors, and second-generation antipsychotics showed no difference in cognitive improvements, leading the researchers to surmise that combined 5-HT(2A)/D2 receptor blockade is probably not necessary for cognitive improvement by atypical antipsychotics [52]. In summary, CTD produces an animal model of lowered central 5-HT concentration, which exhibits impaired cognitive performance. In addition this memory deficit was reversed by the atypical antipsychotic, risperidone, suggesting in part a role for 5-HT2A receptors. Further studies to investigate whether CTD impairs the formation or retrieval of memory, as well as a dose response study of the antipsychotics used, along with other compounds with 5HT2A effects to ascertain the specificity of this effect will continue. Acknowledgements This research was funded by the Northern Ireland Research and Development Office, Department of Health and Social Services and Public Safety.
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Research report
Tryptophan depletion impairs object-recognition memory in the rat: Reversal by risperidone Trisha A. Jenkins a,c,∗ , Jennifer J. Elliott b , Tara C. Ardis a , Marie Cahir a , Gavin P. Reynolds a , Robert Bell b , Stephen J. Cooper a a b c
Division of Psychiatry and Neuroscience, Queen’s University Belfast, Northern Ireland, United Kingdom School of Psychology, Queen’s University Belfast, Northern Ireland, United Kingdom Health Innovations Research Institute and School of Medical Sciences, RMIT University, Victoria, Australia
a r t i c l e
i n f o
Article history: Received 19 October 2009 Received in revised form 14 December 2009 Accepted 17 December 2009 Available online 31 December 2009 Keywords: Tryptophan depletion Serotonin Object-recognition memory Cognition Antipsychotic Risperidone
a b s t r a c t Tryptophan depletion techniques are effective in reducing central serotonergic function and have been used to investigate its role in mood and cognition. In the present study a tryptophan-free diet was fed to Lister-hooded male rats chronically for 21 days to investigate the effect of lowering central serotonin concentration on cognition using the novel object-recognition paradigm. Chronically tryptophan-depleted rats had impaired object-recognition memory; this was accompanied by a reduction in central serotonin of 40–50% in the hippocampus, frontal cortex and striatum. In a subsequent experiment, the atypical antipsychotic, risperidone (0.2 mg/kg), but not the typical antipsychotic, haloperidol (0.1 mg/kg), administered i.p. 30 min prior to the retention test, significantly attenuated the chronic tryptophan depletion impairment. These data show that chronic lowering of central serotonin is associated with impaired cognitive performance, and that this can be reversed by the atypical antipsychotic, risperidone. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Serotonin (5-HT) is known to regulate many physiological and behavioural functions including aspects of mood, anxiety and cognition. Synthesized from its amino acid precursor tryptophan (TRP), 5-HT levels in plasma and brain can be influenced by manipulating dietary TRP intake [8,16,18]. Dietary TRP depletion is a non-pharmacological method to reduce central 5-HT and has been widely used as a tool to assess the role of 5-HT in cognitive and affective functioning in both humans and animals [7,9,23,34,41]. Acute tryptophan depletion (ATD) in rats can be induced by oral administration of a TRP− free (TRP−) amino acid mixture [30] or feeding of a TRP− diet [3,11]. Both protocols lead to a robust reduction in plasma TRP and a reduction in central 5-HT concentrations. Moreover ATD produces deficits in object-recognition memory [25,32,43,51], but not reversal or spatial memories [32,50].
∗ Corresponding author at: Health Innovations Research Institute and School of Medical Sciences, PO Box 71, RMIT University, Bundoora, Victoria 3083, Australia. Tel.: +61 3 9925 6523; fax: +61 28 9925 7063. E-mail address: [email protected] (T.A. Jenkins). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.12.030
Chronic TRP depletion (CTD) allows the investigation of the consequences in animals of a long term reduction in central serotonergic function. Along with reducing plasma and brain TRP, and brain 5-HT [11], CTD alters 5-HT receptor binding in the brain with decreases in 5-HT1A receptors and increases in 5-HT2A receptors being reported [11,27]. Moreover, increasing the sensitivity of rats to external and stressful stimuli [47] and contextual fear memory deficits [49] have been reported after CTD. Effects on object-recognition memory have not been determined in this model, however other studies concerning long term reduction in 5-HT function such as injection of serotonergic antagonists [39] and lesioning of the dorsal raphe nucleus [31], produce object memory deficits. Atypical antipsychotics exhibit both dopaminergic, primarily D2, and serotonergic pharmacology as compared to typical antipsychotics which are primarily dopamine-specific [33,40]. Effects of atypical antipsychotics on the brain 5-HT system are varied but extracellular 5-HT concentrations are increased [21,24] while 5-HT2A receptor expression is down regulated [14,45]. In addition, atypical antipsychotics exhibit positive effects on memory in animal models of cognitive dysfunction [10,19]. Typical antipsychotics, however, which primarily blocking brain dopamine receptors, may exhibit indirect effects on the 5-HT sys-
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Table 1 Composition of the TRP diets. g/kg Sucrose Corn oil Zein Gelatin Mineral mix Calcium phosphate, dibasic Vitamin mix Choline chloride Ammonium citrate, dibasic Glycine l-Lysine HCl l-Histidine dl-Methionine l-Phenylalanine l-Leucine l-Isoleucine l-Threonine l-Valine l-Tyrosine l-Arginine HCl
655.75 100 20 30 35 4.5 10 1.5 23.4 23.4 30 4.2 5.3 7.1 14.2 6.2 9.7 11.1 7.0 8.7
l-Tryptophan (TRP− group) l-Tryptophan (TRP+ group)
0.0 7.0
Diets were obtained from Harlan (UK).
tem, as there is a considerable amount of interaction between the dopaminergic and serotonergic neurotransmitter systems [55]. The present study examined the effect of 21 days of CTD on object-recognition memory performance. In addition we investigated the ability of the typical antipsychotic, haloperidol, and the atypical risperidone, at a dose previously used to modify an objectrecognition memory impairment [19], to reverse a CTD-induced disruption of object-recognition memory. Brain changes in 5-HT were also investigated in regions associated with higher order brain function. 2. Experimental procedures
heavy enough not to be displaced by the animals. The rat was then removed and returned to its homecage. One hour later the rat was returned to the arena and exposed to one familiar object and a novel object for 3 min in the test phase. Objects were placed approximately 10 cm apart in the centre of the arena and both were cleaned with 70% alcohol between trials. Familiar and novel objects were alternated between the left and right position to prevent bias for a particular location. Trial and test were video recorded and time spent exploring each object was scored manually by an investigator blinded to the treatment groups. Object exploration was defined as sniffing or touching the object with the nose and/or forepaws. Sitting on the object was not considered exploratory behaviour. Ability to discriminate in the test was expressed as discrimination index (DI) = (time exploring novel object − time exploring familiar object/total exploration time). 2.3.2. Locomotor activity One hour after object-recognition testing locomotor activity was assessed in both groups. Activity was performed under 320 lx light conditions (lighting uniform in all areas of the arena) in a room (185 cm × 305 cm × 285 cm) using a Med Associates (USA) open field test chamber (44.5 cm × 44.5 cm × 30.5 cm) with four pairs of photocells spaced evenly along the length of the cage. Rats were placed in the box for 10 min and analysis performed by Med Associates (USA) activity monitor software, version 4. 2.3.3. Collection and preparation of blood and tissue samples All animals were sacrificed immediately after locomotor testing. Animals were killed with CO2 , cervically dislocated and then blood (approximately 5 ml) was removed by cardiac puncture for plasma TRP measurement. Blood was collected into EDTA tubes and kept on ice prior to centrifugation at 1600 g for 15 min at 4 ◦ C. The resulting plasma supernatant was centrifuged at 12,000 g for 30 min at 20 ◦ C through YM-10 Amicon filters to remove plasma protein and filtrate was frozen at −80 ◦ C until TRP analysis. After blood sampling brains were removed immediately and dissected into frontal cortex, hippocampus and striatum for determination of 5-HT and 5-HIAA. Brain tissue was deprotonised on ice, with 10 volumes 0.1 M perchloric acid and homogenized by hand. Samples were centrifuged at 12,000 g for 15 min at 4 ◦ C, and supernatant was removed and frozen at −80 ◦ C until analysis. 2.3.4. Chromatographic analysis of plasma TRP and central monoamines Free plasma TRP and central monoamines were separately determined by separation on a Hypersil 5 m ODS C18 (150 mm × 4.6 mm) column coupled with an ESA Coulochem II electrochemical detector. The analytical potentials for plasma TRP were set at E1 +300 mV and E2 +575 mV. The mobile phase (pH 4.05) consisted of 0.1 M NaH2 PO4 , 0.1 M citric acid, 0.75 mM octane sulphonate acid, 0.1 mM EDTA and 16% methanol. The analytical potentials for monoamines were set at E1 +50 mV and E2 +500 mV. The mobile phase (pH 3.0) consisted of 0.1 M sodium dihydrogen phosphate monohydrate, 2.5 mM octane sulphonate acid, 0.5 mM EDTA, 20.5 ml glacial acetic acid and 12% methanol. Quantification was obtained by comparing peak height of current E2 with standard concentrations.
2.1. Animals 2.4. Experiment 2 Adult male Lister-hooded rats (Harlan, UK), weighing 200–300 g at the beginning of the experiment, were maintained on a reversed 12 h light dark cycle (lights off 6 a.m.). Room temperature (21 ± 2 ◦ C) and humidity (45–55%) were kept constant throughout. Experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986. 2.2. Diet TRP− free (TRP−) and equivalent control diet (TRP+) were obtained from Harlan (UK). The TRP+ diet contained 0.7% TRP and the TRP− diet 0% TRP. The composition of the two diets is shown in Table 1. Rats had free access to either TRP+ or TRP− diet for 21 days; water was available ad libitum. CTD animals were group housed (3–4 rats/cage) and were weighed each day, as were controls. Behavioural testing was performed at the end of dietary manipulation (day 21).
2.4.1. Antipsychotic drugs In a second series of animals, the effect of antipsychotic drugs on CTD memory in the object-recognition task was investigated. CTD and object recognition was performed as described in experiment 1. Compounds were freshly prepared on the day of the experiment, and administered 30 min prior to the retention test. Groups received vehicle (0.9% saline, i.p.), risperidone (0.2 mg/kg, i.p.), or haloperidol (0.1 mg/kg, i.p.). Rat numbers per group were TRP+ n = 10, TRP− n = 8, TRP− and HAL n = 8, TRP− and RIS n = 9. 2.4.2. Data analysis Data was normally distributed. All data were analysed using SPSS for Windows version 17.0. Student’s paired t-test was performed to compare the effect of tryptophan depletion on the time spent exploring identical objects in the trial and the familiar versus the novel object in the test. Analysis of the DI data was performed using one way ANOVA followed by posthoc Dunnett’s t-tests.
2.3. Experiment 1 2.3.1. Object-recognition testing The object-recognition [15,57] apparatus consisted of a black plastic square arena (45 cm × 63 cm × 63 cm). The light intensity of the testing room (185 cm × 305 cm × 285 cm) was 360 lx and lighting was equal in the different parts of the apparatus. Five different sets of objects made of inert material such as glass, plastic and ceramic, each available in triplicate and that could not be displaced by a rat were used for discrimination. Rats were exposed to the object-recognition task between the 8th and 9th hour of the dark phase of the reverse light cycle. Animals received two sessions of 3 min duration in the empty box over the two days prior to testing to allow habituation to the arena and test room environment. The object-recognition task comprised a trial and test session, both of 3 min duration. In the trial the rat was placed into the arena and exposed to two identical objects for 3 min. The objects to be discriminated were made of glass, plastic, metal, or ceramic and were approximately equal heights and
3. Results 3.1. Effect of CTD on object recognition In the trial phase no significant difference in exploration time of the two objects was observed (total exploration time of both objects: TRP+ 73 ± 5 s; TRP− 82 ± 9 s; p = 0.46; n = 10/group). During the test, no significant difference in total exploration time of the two objects was observed (total exploration time of both objects: TRP+ 77 ± 5 s; TRP− 85 ± 8 s; p = 0.49; n = 10/group). Rats chronically fed the control TRP+ diet spent significantly longer exploring the novel object compared to the familiar object
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Fig. 1. (A) The effect of chronic tryptophan depletion on exploration time (s) of a familiar object and a novel object in the 3-min test. Data are expressed as the mean ± S.E.M. (n = 10/group), significant difference in time spent exploring the novel compared to the familiar object. *** p < 0.001 familiar cf novel. (B) The effect of chronic tryptophan depletion on the discrimination index (DI). Data are expressed as the mean ± S.E.M. (n = 10/group) ** p < 0.01 cf control TRP+.
(p < 0.001). In contrast there was no significant difference between the time spent exploring the novel and familiar object in the TRP− group (p = 0.99; Fig. 1a). Consequently, there was a significant difference in DI level between the TRP+ and TRP− groups (p < 0.01; Fig. 1b). 3.2. Effect of CTD on rat body weight and locomotor activity Analysis of the final weights of the CTD study showed that in the three weeks of feeding TRP+ animals increased their bodyweight by approximately 9% (274.1 ± 2.8 g to 301 ± 5.3 g) while TRP− animals lost weight (22%; 273.1 ± 2.9 g to 215.5 ± 3.6 g). The final weight difference between TRP+ and TRP− is significantly different (p < 0.001). Locomotor activity was assessed at the end of the three week feeding regime. There was no significant difference in distance travelled between TRP+ and TRP− animals (TRP+ 2400 ± 237 cm; TRP− 2350 ± 83 cm; p = 0.84). 3.3. Effect of CTD on free plasma TRP, central 5-HT and 5-HIAA CTD significantly reduced free plasma TRP. Feeding rats a TRP− diet chronically for 21 days reduced TRP by 83% (TRP+ 4.67 ± 0.30 g tryptophan/ml plasma; TRP− 0.78 ± 0.06 g tryptophan/ml plasma; p < 0.001; n = 10/group). TRP− rats showed significant decreases in 5-HT and 5-HIAA concentrations in the frontal cortex, hippocampus and striatum, as compared to TRP+ group (Table 2). 3.4. Effect of antipsychotic drugs on CTD-impaired object-recognition memory All groups of rats spent equivalent time exploring the identical objects (left and right) in the trial phase, with no significant differences in exploration times within groups (TRP+ 65 ± 6 s, TRP− 67 ± 4 s, TRP− and HAL 58 ± 7 s, TRP− and RIS 48 ± 5 s; F(3,34) = 2.4, p = 0.09).
In the test phase, all groups of rats spent equivalent total time exploring the objects, with no significant differences in exploration times within groups (TRP+ 74 ± 5 s, TRP− 76 ± 6 s, TRP− and HAL 58 ± 7 s, TRP− and RIS 73 ± 5 s; F(4,34) = 1.4, p = 0.26). Rats chronically fed the control diet spent significantly longer exploring the novel object compared to the familiar object (p < 0.001). The ability to discriminate familiar and novel objects was abolished following CTD, whereby there was no significant difference in exploration of the objects (p = 0.28). Haloperidol failed to improve the CTD-induced impairment, no difference in time exploring novel and object objects was observed (p = 0.28), while TRP− rats pre-treated with risperidone spent significantly longer exploring the novel object compared with the familiar object (p < 0.001) (Fig. 2a). These findings were reflected by the DI which showed an overall significant effect of group (F(3,34) = 12.0, p < 0.001). Posthoc analysis demonstrated that, as previously shown, CTD significantly reduced the DI compared to control (p < 0.001). This was not affected by pretreatment with haloperidol (p < 0.001 compared to control; p = 1.0 compared to TRP-). Conversely, risperidone pretreatment reversed the TRP− object-recognition memory impairment (p = 0.99, compared to control) (Fig. 2b).
4. Discussion This study demonstrates that a dietary regime of chronic TRP depletion can induce a profound deficit in object-recognition memory, which could be reversed by atypical, but not typical, antipsychotic treatment. Concurrently, CTD induced a substantial reduction in free plasma TRP and a consistent decrease in central 5HT and 5-HIAA in all brain regions measured as compared to control TRP+ fed rats. Tryptophan depletion for twenty-one days produced a significant impairment in object-recognition memory. This is the first report of a deficit using the object-recognition paradigm with a
Table 2 Effect of acute and chronic tryptophan depletion on central 5-HT and 5-HIAA in the rat frontal cortex, striatum and hippocampus. Frontal cortex TRP+ CTD % Change from TRP+
5-HT (ng/g)
112.0 ± 15.83
5-HIAA (ng/g)
522.7 ± 30.03
% Change from TRP+ Data expressed as mean ± S.E.M. (ng/g). * p < 0.05, ** p < 0.01, *** p < 0.001.
Striatum TRP− 57.7 ± 5.93* 49% 261.8 ± 37.83*** 50%
Hippocampus
TRP+
TRP−
TRP+
181.8 ± 14.57
109.1 ± 9.154** 40%
62.10 ± 9.402
TRP− 28.15 ± 3.15* 55%
541.2 ± 35.42
164.0 ± 17.88*** 70%
580.0 ± 35.46
152.7 ± 13.63*** 74%
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Fig. 2. (A) The effect of chronic tryptophan depletion on exploration time (s) of a familiar object and a novel object in the 3-min test. Data are expressed as the mean ± S.E.M., significant difference in time spent exploring the novel compared to the familiar object. *** p < 0.001 familiar cf novel. (B) The effect of antipsychotic pre-treatment on chronic tryptophan depletion on the discrimination index (DI). Data are expressed as the mean ± S.E.M. (n = 10/group) *** p < 0.001 cf control TRP+.
chronic depletion model. Memory deficits have been demonstrated in chronically TRP-depleted mice, showing impaired formation of contextual fear memory [49]. However spatial and conditioned taste aversion memories appear to remain unaffected [49]. Other examples of long-term 5-HT brain deficits in rodents include lesion studies which cause a hyposerotonergic state in localised projection areas [6,26]. Serotonergic lesions of the dorsal raphe nucleus also produce object memory deficits [31]. This effect of 5-HT depletion and object-recognition memory may be somewhat specific as memory deficits after a loss of brain 5-HT are not universal: serotonergic depletion of the dorsal or ventral hippocampus did not show significant changes in the Y-maze, water maze, or Tmaze delayed alternation test [1]; lesions of the raphe nucleus do not affect inhibitory avoidance memory [4]; while forebrain 5-HT depletion actually facilitates the acquisition and performance of a conditional visual discrimination task [53]. Generally it is accepted that ATD also produces objectrecognition deficits: in normal albino rats [25,32,43,51], and in 5-HT transporter knockout rats [38]. Our results show that a long-term reduction in central serotonergic function produces a similar result, but without the other conflicting effects of brain lesions. In the present study feeding rats a TRP− diet chronically reduced free plasma TRP levels by 80–90% from controls. This peripheral TRP depletion was associated with a 40–60% reduction in central 5-HT concentrations in the different brain structures measures. These results are comparable to those reported in our previous study using the TRP− diet [11]. Human tryptophan depletion studies report changes in cognitive functioning associated with 70–90% reductions in peripheral plasma TRP levels [12,46,58]. Therefore achieving a similar degree of plasma TRP depletion in rats allows a better inter-species comparison of behavioural effects. In the present study feeding rats a TRP− diet chronically reduced free plasma TRP levels by 80–90% from controls. This peripheral TRP depletion was associated with a 40–60% reduction in central 5-HT concentrations in the different brain structures measures. These results are comparable to those reported in our previous study using the TRP− diet [11]. The specific brain areas and neural processes involved in objectrecognition memory are still to be elucidated. However it is generally considered that regions within the medial temporal lobe such as parahippocampal cortices, particularly the perirhinal cortex, which is important for memory encoding, consolidation, and retrieval processes [13,54]. Object-recognition memory is suggested not to be hippocampal dependant [2,13,37]. The role of 5-HT in object-recognition memory is complex: 5-HT6 receptor antagonists have been shown to reverse object-recognition memory deficits [17] in scopolamine [29,56] and aged [22,35] animals; as has the 5-HT7 receptor where the selective 5-HT7 antagonist, SB269.970, significantly reduces object recognition in low-responding rats [5]; while the 5-HT4 receptor has been implicated in increased acquisition in the object-recognition task as rats
treated with the agonist, RS 67333, perform better than controls [28]. The second major finding of this work is that the CTD-induced cognitive deficit was reversed by the atypical antipsychotic, risperidone, but not the classical typical antipsychotic, haloperidol. Only one dose was used, however these doses were selected from a previous study by Neill and colleagues as the optimum doses to reverse phencyclidine-induced object-recognition memory deficits [19]. While both antipsychotics exhibit dopamine D2 receptor antagonism, atypical antipsychotics also bind with high affinity to 5-HT2A receptors [36]. Typical agents such as haloperidol only weakly occupy serotonergic receptors [44]. This suggests that 5HT2A receptors may play a role in CTD-induced object-recognition memory deficits and indeed we have shown increased cortical 5HT2A-receptor binding after CTD [11]. Atypical antipsychotics such as risperidone have previously been demonstrated to reverse object-recognition deficits in other models of memory impairment such as the phencyclidine model both acutely [19] and chronically [20]. However this is the first time they have been shown to have an effect in tryptophan depletion. Thus the action of risperidone on object recognition appears independent of the mechanism of its disruption. The specific mechanism underlying the ability of risperidone to reverse the effect of CTD on object-recognition memory remains unclear. It would seem likely that the effect is not D2 receptormediated, given the finding with haloperidol, but via one or more of the other receptors that risperidone influences: particularly 5HT2A, but conceivably 5-HT1B, 5-HT7 or the alpha2C adrenoceptor [42]. Research within the patient population has suggested that 5HT2A affinity plays an important role in the cognitive effects of the atypical antipsychotics, independent of a dopaminergic influence [48]. However comparison of the antipsychotic amisulpiride, which only binds to D2/D3 receptors, and second-generation antipsychotics showed no difference in cognitive improvements, leading the researchers to surmise that combined 5-HT(2A)/D2 receptor blockade is probably not necessary for cognitive improvement by atypical antipsychotics [52]. In summary, CTD produces an animal model of lowered central 5-HT concentration, which exhibits impaired cognitive performance. In addition this memory deficit was reversed by the atypical antipsychotic, risperidone, suggesting in part a role for 5-HT2A receptors. Further studies to investigate whether CTD impairs the formation or retrieval of memory, as well as a dose response study of the antipsychotics used, along with other compounds with 5HT2A effects to ascertain the specificity of this effect will continue. Acknowledgements This research was funded by the Northern Ireland Research and Development Office, Department of Health and Social Services and Public Safety.
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