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Brain Research Bulletin 85 (2011) 141–144
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Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
d-Cycloserine enhances social exploration in the Balb/c mouse Luis F. Jacome, Jessica A. Burket, Amy L. Herndon, Stephen I. Deutsch ∗ Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, 825 Fairfax Avenue, Suite 710, Norfolk, VA 23507-1912, United States
a r t i c l e
i n f o
Article history: Received 25 January 2011 Received in revised form 28 February 2011 Accepted 3 March 2011 Available online 9 March 2011 Keywords: Balb/c mouse strain Sociability Anxiety d-Cycloserine Autism spectrum disorders
a b s t r a c t Inbred Balb/c mice show deficits of sociability. The endogenous tone of NMDA receptor-mediated neurotransmission is altered in Balb/c mice, which may explain the beneficial effect of d-cycloserine on impaired sociability. In the current study, Balb/c mice spent more time than the Swiss Webster comparator strain in the open arms of an elevated plus maze (EPM), suggesting that they are not more anxious or fearful in the absence of a social stimulus mouse. Moreover, Balb/c and Swiss Webster mice did not differ in the amount of time they spent exploring an inanimate object in an open field. Differences in exploratory activity between strains emerged only when a salient social stimulus mouse was enclosed in the open field. d-Cycloserine increased the amount of time Balb/c mice spent exploring the enclosed stimulus mouse to levels observed in vehicle-treated Swiss Webster mice. Finally, irrespective of strain, d-cycloserine increased exploratory activity as measured in open arm entries in the EPM, when no enclosed stimulus mouse was present. The data show that mouse strain influences d-cycloserine’s effect on exploration in the presence of a salient social stimulus mouse. In the absence of an enclosed stimulus mouse, d-cycloserine increased open arm entries significantly in both the sociability-impaired Balb/c and comparator Swiss Webster strains. Thus, d-cycloserine positively affects exploratory activity in general, but strain differences emerge when the stimulus eliciting exploration is a salient social stimulus mouse versus an inanimate object. Further, the sociability deficit of the Balb/c mouse is not an epiphenomenon of increased generalized anxiety. © 2011 Elsevier Inc. All rights reserved.
1. Introduction In a standard paradigm, genetically inbred Balb/c mice show deficits on several quantitative measures of sociability, relative to the outbred Swiss Webster comparator strain [3,5,9]. For example, Balb/c mice show reduced locomotor activity in the presence of an enclosed stimulus mouse and when they interact freely with the stimulus mouse [3]. Moreover, Balb/c mice spend less time exploring and in the vicinity of the stimulus mouse when it is either enclosed or allowed to move freely [5,9]. The Balb/c mouse is thought to have altered endogenous tone of NMDA receptor-mediated neurotransmission because it is more sensitive to several behavioral effects of MK-801 (dizocilpine), a noncompetitive NMDA receptor antagonist, including the ability of MK-801 to elicit irregular episodes of intense jumping behavior [7], antagonize electrically precipitated seizures [6], and elicit circling behavior [2], relative to outbred and other genetically inbred strains. Further, the ability of d-cycloserine and D-serine, a partial and full glycine agonist at the obligatory glycine co-agonist binding site on the NMDA receptor, to improve measures of sociability in the Balb/c strain
[5,9] supports the existence of altered endogenous tone of NMDA receptor-mediated neurotransmission and its involvement in the pathophysiology of impaired sociability in this mouse strain. The current investigation compared the exploratory behavior of Balb/c and Swiss Webster mice elicited by the presence of an enclosed social stimulus mouse or an inanimate object in an open field apparatus and examined strain differences in the effect of d-cycloserine on exploration under these two different stimulus conditions. Further, after testing mice in the open field apparatus, we studied exploratory behavior in an elevated plus maze (EPM), in the absence of an enclosed stimulus mouse, in order to determine if the strains differed on standard measures of generalized anxiety and fearfulness. Importantly, in the EPM, d-cycloserine significantly increased exploratory activity in both strains, as reflected in open arm entries, suggesting that the strains are differentially affected by the presence of an enclosed salient social stimulus mouse in their response to d-cycloserine. 2. Methods 2.1. Subjects
∗ Corresponding author. Tel.: +1 757 446 5888; fax: +1 757 446 5918. E-mail address: [email protected] (S.I. Deutsch). 0361-9230/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2011.03.004
Experimentally naïve, 8-week old male, outbred Swiss Webster and genetically inbred Balb/c test mice (Harlan Laboratories, Frederick, MD) were housed 2 per cage in hanging clear Plexiglas cages with free access to food and water and maintained on a 12 h light/dark cycle. The stimulus mice were 4-week old male ICR mice, housed
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4 per cage. The same mouse (i.e., Balb/c or Swiss Webster) was studied only once in the sociability and EPM paradigms prior to sacrifice. All animal procedures were approved by the Eastern Virginia Medical School Institutional Animal Care and Use Committee and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 2.2. Drugs d-Cycloserine (Sigma–Aldrich Co.; St. Louis, MO) was dissolved in 0.9% saline and prepared each day of the experiment. d-Cycloserine (320 mg/kg or the saline vehicle) was injected intraperitoneally (i.p.) in a volume of 0.01 mg/g of body weight 20 min prior to behavioral testing. 2.3. Open field The open field is a square chamber (18 in. × 18 in.) constructed of black Plexiglas. Briefly, 20 min after i.p. injection of d-cycloserine or saline, test mice (Balb/c or Swiss Webster) were individually placed in the middle of one side of the open field chamber, introduced to an inanimate object (i.e., plastic containers) on the opposite side of the chamber, and allowed to explore the field with the object present for 5 min. Time spent exploring the object was recorded. After each session, both object and test mouse were removed, and the open field chamber was cleaned with Quatricide PV solution. Immediately after, test mice were again individually placed in the middle of one side of the open field, introduced to a salient social ICR stimulus mouse enclosed in an inverted wire cup (Galaxy Cup, Kitchen Plus, http://www.kitchenplus.com) on the opposite side of the chamber, and allowed to explore the field with the enclosed stimulus mouse present for 5 min. Time spent exploring the stimulus mouse was recorded.
Fig. 1. Time spent exploring open arms. Bars represent mean (±SEM) of the time test mice spent (s) exploring open arms in the EPM test. The graph shows that vehicletreated Balb/c mice did not display anxiety-like behaviors in the EPM, compared to Swiss Webster mice. Numbers within bars depict group size. ***(p < 0.001).
2.4. Elevated plus maze The elevated plus maze (EPM) is a test designed to assess anxiety-like behaviors in rodents [4,10,14,17] and consists of four elevated arms (15 in. off the floor): two alternate open arms (12 in. d × 2½ in. w) and two alternate closed arms (12 in. d × 2½ in. w × 6 in. h). Briefly, immediately after testing in the open field, test mice were individually placed in the middle of the four elevated arms, facing one of the open arms, and allowed to explore all four arms for 5 min. An arm was counted as being entered when all four limbs were set on the arm [4,10,14,17]. Time spent and number of entries into the open and closed arms were recorded. All behavioral tests were conducted in dim lighting and videotaped using a Panasonic SDR-S26 SD Video Camera (Panasonic Corp., Osaka, Japan). 2.5. Statistical analyses Data were analyzed using NCSS (NCSS Statistical Software, Kaysville, UT). Twoway ANOVAs were used to examine the effect of strain (Balb/c vs. Swiss Webster) and treatment (d-cycloserine vs. vehicle) on time spent (s) exploring the inanimate object and the salient social stimulus mouse in the open field chamber by the test mouse, and time spent and number of entries into the open arms in the elevated plus maze. Fisher’s LSD Multiple-Comparison post hoc tests were applied, where appropriate.
multiple-comparison test revealed that Balb/c mice treated with d-cycloserine spent significantly more time exploring the social stimulus mouse than vehicle-treated Balb/c mice (p < 0.001; Fig. 3); however, d-cycloserine did not affect the time Swiss Webster mice spent exploring the social stimulus mouse in the open field (p > 0.05). A two-way ANOVA showed a main effect of strain (F[1,45] = 6.00, p < 0.05), treatment (F[1,45] = 85.98, p < 0.0001) and strain by treatment interaction (F[1,45] = 6.99, p < 0.05) on the number of entries into the open arms of the EPM. Fisher’s LSD multiple-comparison tests revealed that Balb/c mice treated with d-cycloserine made significantly more entries into the open arms than vehicle-treated Balb/c mice (p < 0.001; Fig. 4). Similarly, Swiss Webster mice treated with d-cycloserine made significantly more open arm entries than vehicle-treated Swiss Webster mice (p < 0.001).
3. Results A two-way ANOVA showed a significant main effect of strain (F[1,45] = 24.36, p < 0.0001) and strain by treatment interaction (F[1,45] = 10.17, p < 0.01) on time spent exploring open arms in the EPM. Fisher’s LSD multiple-comparison test revealed that vehicletreated Balb/c mice spent significantly more time exploring the open arms than vehicle-treated Swiss Webster mice (p < 0.001; Fig. 1). Interestingly, vehicle-treated Balb/c mice spent significantly more time in the open arms than Balb/c mice treated with dcycloserine (p < 0.05), whereas Swiss Webster mice treated with d-cycloserine spent significantly more time in the open arms than Swiss Webster treated with vehicle (p < 0.05; data not shown). d-Cycloserine did not affect the time Balb/c or Swiss Webster mice spent exploring the inanimate object; moreover, the time spent exploring the inanimate object in the open field did not differ between vehicle-treated Balb/c and vehicle-treated Swiss Webster mice (Fig. 2). A two-way ANOVA showed a significant main effect of strain (F[1,45] = 24.06, p < 0.0001), treatment (F[1,45] = 8.23, p < 0.01) and strain by treatment interaction (F[1,45] = 11.70, p < 0.01) on time spent exploring the salient social stimulus mouse enclosed in the inverted wire cup in the open field. Fisher’s LSD
Fig. 2. Time spent exploring inanimate object in the open field. Bars represent mean (±SEM) of the time test mice spent (s) exploring the inanimate object in the open field test. Numbers within bars depict group size.
L.F. Jacome et al. / Brain Research Bulletin 85 (2011) 141–144
Fig. 3. Time spent exploring stimulus mouse in the open field. Bars represent mean (±SEM) of the time test mice spent (s) exploring the socially salient stimulus mouse enclosed in the inverted wire cup in the open field. Numbers within bars depict group size. ***(p < 0.001).
4. Discussion Deficits in sociability may result from a general diminution of exploratory behavior or relate more specifically to an aversive quality of a salient social stimulus used to elicit exploration, among other possible explanations. In the current study, we confirmed that the Balb/c mouse spent significantly less time exploring an enclosed social stimulus mouse in an open field apparatus than the Swiss Webster comparator strain. However, the Balb/c mouse’s deficit in exploration was specific to its elicitation by a salient social stimulus, as time spent exploring an inanimate object did not differ between the Balb/c and Swiss Webster strains nor did the Balb/c mouse make fewer entries into the open arm of the EPM than the Swiss Webster mouse. Interestingly, anxiety-like behaviors in the Balb/c strain were not observed after these mice had been exposed to an enclosed social stimulus mouse in the open field appara-
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tus, which had a negative emotional valence for this strain, prior to testing in the EPM. Moreover, whereas d-cycloserine increased open arm entries in both strains, suggesting a positive effect on exploratory behavior and anxiety in general, this intervention only increased social exploration by the Balb/c strain in the open field apparatus. Thus, d-cycloserine, a partial glycine agonist at the obligatory co-agonist binding site on the NMDA receptor, may target a fundamental deficit in social exploration manifested by Balb/c mice, in addition to positively affecting exploration in general. Of course, although unlikely, it is possible that d-cycloserine’s ability to increase social exploration in the Balb/c mouse is an indirect effect, resulting from increased exploratory activity and “agitation.” However, “agitation” might be expected to decrease time spent in social exploration by this strain of mouse with impaired sociability [3]. The data are consistent with several prior studies showing that genetically engineered mice with reduced expression of the NR1 subunit of the NMDA receptor (5–10% of normal levels) or 5fold diminished affinity of the glycine binding site on the NR1 subunit for glycine manifest deficits of sociability in a standard paradigm [8,13]. Moreover, d-cycloserine was shown to have salutary effects on social behavior of genetically unaltered mice in the resident-intruder mouse paradigm of social interaction [16]. The data encourage exploration of targeted NMDA receptor agonist interventions on deficits of sociability, especially in the Balb/c mouse that has apparently normal expression of this receptor [18], but altered endogenous tone of NMDA receptor-mediated neurotransmission, as reflected in its heightened behavioral sensitivity to MK-801 [1,2,6,7]. Cross-talk between histaminergic receptors, especially H1 histaminergic receptors, and NMDA receptors may represent another fruitful in vivo strategy for facilitating NMDA receptor-mediated neurotransmission [15]. Moreover, H3 histaminergic receptors located on presynaptic glutamatergic terminals contribute to the regulation of glutamate release and availability in the synaptic cleft [15]. Because normal NMDA receptor function is necessary for memory consolidation, we are interested in exploring possible associations between impaired sociability and impaired memory performance in Balb/c mice [19]. d-Cycloserine dose-dependently improved memory consolidation in mice in the “Plus-Maze Retest Paradigm;” thus, d-cycloserine may be predicted to target sociability and at least some aspects of impaired memory performance [19]. The ability of d-cycloserine to enhance fear extinction, which is an active learning process, has led to translational clinical trials in anxiety disorders; presumably, d-cycloserine is acting at NMDA receptors within circuits linking cerebral cortex, hippocampus and amygdala [11], which are circuits that could also be relevant to the pathophysiology of autism spectrum disorders. Effects of dcycloserine in vivo in man can be attributed to its relatively selective interaction with the glycineB coagonist site on the NMDA receptor [12]. For example, in alcohol dependent men with probable upregulated expression of their NMDA receptors and control subjects, effects of infused glycine (0.3 g/kg over 30 min) and a large orally administered dose of d-cycloserine (1000 mg) on subjective measures of alcohol effects and objective cognitive measures were best explained by their competitive interaction with the glycineB site [12]. Thus, at least some of the effects of orally administered d-cycloserine in man (e.g., to persons with autism spectrum disorders) are likely to be due to its partial glycine agonistic properties.
Acknowledgement Fig. 4. Open arm entries in the EPM. Bars represent mean (±SEM) of the number of entries that test mice made into the open arms of the EPM. Numbers within bars depict group size. ***(p < 0.001).
The authors acknowledge the generous support that they received from the Office of the Dean of the Eastern Virginia Medical School.
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References [1] E.N. Billingslea, J. Mastropaolo, R.B. Rosse, A.S. Bellack, S.I. Deutsch, Interaction of stress and strain on glutamatergic neurotransmission: relevance to schizophrenia, Pharmacology, Biochemistry, and Behavior 74 (2003) 351–356. [2] J.A. Burket, W.R. Cannon, L.F. Jacome, S.I. Deutsch, MK-801, a noncompetitive NMDA receptor antagonist, elicited circling behavior in the genetically-inbred Balb/c mouse strain, Brain Research Bulletin 83 (2010) 337–339. [3] J.A. Burket, A.L. Herndon, S.I. Deutsch, Locomotor activity of the geneticallyinbred mouse strain is suppressed by a socially-salient stimulus, Brain Research Bulletin 83 (2010) 255–256. [4] J.N. Crawley, Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests, Brain Research 835 (1) (1999) 18–26. [5] S.I. Deutsch, J.A. Burket, L.F. Jacome, W.R. Cannon, A.L. Herndon, d-Cycloserine improves the impaired sociability of the Balb/c mouse, Brain Research Bulletin 84 (2010) 8–11. [6] S.I. Deutsch, J. Mastropaolo, D.G. Powell, R.B. Rosse, S.E. Bachus, Inbred mouse strains differ in their sensitivity to an antiseizure effect of MK-801, Clinical Neuropharmacology 21 (1998) 255–257. [7] S.I. Deutsch, R.B. Rosse, S.M. Paul, R.L. Riggs, J. Mastropaolo, Inbred mouse strains differ in sensitivity to popping elicited by MK-801, Pharmacology, Biochemistry, and Behavior 57 (1997) 315–317. [8] T.B. Halene, R.S. Ehrlichman, Y. Liang, E.P. Christian, G.J. Jonak, T.L. Gur, et al., Assessment of NMDA receptor NR1 subunit hypofunction in mice as a model for schizophrenia, Genes, Brain, and Behavior 8 (2009) 661–675. [9] L.F. Jacome, J.A. Burket, A.L. Herndon, W.R. Cannon, S.I. Deutsch, D-serine improves dimensions of the sociability deficit of the genetically-inbred Balb/c mouse strain, Brain Research Bulletin 84 (2010) 12–16. [10] L.F. Jacome, C. Gautreaux, T. Inagaki, G. Mohan, S. Alves, L.S. Lubbers, V.N. Luine, Estradiol and ERb agonists enhance recognition memory, and DPN, an ERb ago-
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nist, alters brain monoamines, Neurobiology of Learning and Memory 94 (2010) 488–498. G.B. Kaplan, K.A. Moore, The use of cognitive enhancers in animal models of fear extinction, Pharmacology, Biochemistry, and Behavior (January (20)) (2011) [Epub ahead of print]. J.H. Krystal, I.L. Petrakis, D. Limoncelli, S.K. Nappi, L. Trevisan, B. Pittman, D.C. D’Souza, Characterization of the interactive effects of glycine and d-cycloserine in men: further evidence for enhanced NMDA receptor function associated with human alcohol dependence, Neuropsychopharmacology 36 (3) (2011) 701–710. V. Labrie, T. Lipina, J.C. Roder, Mice with reduced NMDA receptor glycine affinity model some of the negative and cognitive symptoms of schizophrenia, Psychopharmacology 200 (2) (2008) 217–230. T. Lund, T. Rovis, W. Chung, R. Handa, Novel actions of estrogen receptor- on anxiety-related behaviors, Endocrinology 146 (2) (2005) 797–807. T. Masuoka, C. Kamei, The ameliorating effects of NMDA receptor agonists on histamine H1 antagonist-induced memory and hippocampal theta disruptions are prevented by the H3 receptor agonist in rats, Brain Research Bulletin 79 (6) (2009) 422–425. K.H. McAllister, d-Cycloserine enhances social behavior in individually-housed mice in the resident-intruder test, Psychopharmacology 116 (1994) 317–325. S. Pellow, S. File, Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat, Pharmacology, Biochemistry, and Behavior 24 (3) (1986) 525–529. P.Y. Perera, J.H. Lichy, J. Mastropaolo, R.B. Rosse, S.I. Deutsch, Expression of NR1, NR2A and NR2B NMDA receptor subunits is not altered in the geneticallyinbred Balb/c mouse strain with heightened behavioral sensitivity to MK-801, a noncompetitive NMDA receptor antagonist, European Journal of Neuropsychopharmacology 18 (2008) 814–819. R.J. Rodgers, H. Harvest, C. Hassall, L.A. Kaddour, d-Cycloserine enhances memory consolidation in the plus-maze retest paradigm, Behavioral Neuroscience 125 (1) (2011) 106–116.
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
d-Cycloserine enhances social exploration in the Balb/c mouse Luis F. Jacome, Jessica A. Burket, Amy L. Herndon, Stephen I. Deutsch ∗ Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, 825 Fairfax Avenue, Suite 710, Norfolk, VA 23507-1912, United States
a r t i c l e
i n f o
Article history: Received 25 January 2011 Received in revised form 28 February 2011 Accepted 3 March 2011 Available online 9 March 2011 Keywords: Balb/c mouse strain Sociability Anxiety d-Cycloserine Autism spectrum disorders
a b s t r a c t Inbred Balb/c mice show deficits of sociability. The endogenous tone of NMDA receptor-mediated neurotransmission is altered in Balb/c mice, which may explain the beneficial effect of d-cycloserine on impaired sociability. In the current study, Balb/c mice spent more time than the Swiss Webster comparator strain in the open arms of an elevated plus maze (EPM), suggesting that they are not more anxious or fearful in the absence of a social stimulus mouse. Moreover, Balb/c and Swiss Webster mice did not differ in the amount of time they spent exploring an inanimate object in an open field. Differences in exploratory activity between strains emerged only when a salient social stimulus mouse was enclosed in the open field. d-Cycloserine increased the amount of time Balb/c mice spent exploring the enclosed stimulus mouse to levels observed in vehicle-treated Swiss Webster mice. Finally, irrespective of strain, d-cycloserine increased exploratory activity as measured in open arm entries in the EPM, when no enclosed stimulus mouse was present. The data show that mouse strain influences d-cycloserine’s effect on exploration in the presence of a salient social stimulus mouse. In the absence of an enclosed stimulus mouse, d-cycloserine increased open arm entries significantly in both the sociability-impaired Balb/c and comparator Swiss Webster strains. Thus, d-cycloserine positively affects exploratory activity in general, but strain differences emerge when the stimulus eliciting exploration is a salient social stimulus mouse versus an inanimate object. Further, the sociability deficit of the Balb/c mouse is not an epiphenomenon of increased generalized anxiety. © 2011 Elsevier Inc. All rights reserved.
1. Introduction In a standard paradigm, genetically inbred Balb/c mice show deficits on several quantitative measures of sociability, relative to the outbred Swiss Webster comparator strain [3,5,9]. For example, Balb/c mice show reduced locomotor activity in the presence of an enclosed stimulus mouse and when they interact freely with the stimulus mouse [3]. Moreover, Balb/c mice spend less time exploring and in the vicinity of the stimulus mouse when it is either enclosed or allowed to move freely [5,9]. The Balb/c mouse is thought to have altered endogenous tone of NMDA receptor-mediated neurotransmission because it is more sensitive to several behavioral effects of MK-801 (dizocilpine), a noncompetitive NMDA receptor antagonist, including the ability of MK-801 to elicit irregular episodes of intense jumping behavior [7], antagonize electrically precipitated seizures [6], and elicit circling behavior [2], relative to outbred and other genetically inbred strains. Further, the ability of d-cycloserine and D-serine, a partial and full glycine agonist at the obligatory glycine co-agonist binding site on the NMDA receptor, to improve measures of sociability in the Balb/c strain
[5,9] supports the existence of altered endogenous tone of NMDA receptor-mediated neurotransmission and its involvement in the pathophysiology of impaired sociability in this mouse strain. The current investigation compared the exploratory behavior of Balb/c and Swiss Webster mice elicited by the presence of an enclosed social stimulus mouse or an inanimate object in an open field apparatus and examined strain differences in the effect of d-cycloserine on exploration under these two different stimulus conditions. Further, after testing mice in the open field apparatus, we studied exploratory behavior in an elevated plus maze (EPM), in the absence of an enclosed stimulus mouse, in order to determine if the strains differed on standard measures of generalized anxiety and fearfulness. Importantly, in the EPM, d-cycloserine significantly increased exploratory activity in both strains, as reflected in open arm entries, suggesting that the strains are differentially affected by the presence of an enclosed salient social stimulus mouse in their response to d-cycloserine. 2. Methods 2.1. Subjects
∗ Corresponding author. Tel.: +1 757 446 5888; fax: +1 757 446 5918. E-mail address: [email protected] (S.I. Deutsch). 0361-9230/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2011.03.004
Experimentally naïve, 8-week old male, outbred Swiss Webster and genetically inbred Balb/c test mice (Harlan Laboratories, Frederick, MD) were housed 2 per cage in hanging clear Plexiglas cages with free access to food and water and maintained on a 12 h light/dark cycle. The stimulus mice were 4-week old male ICR mice, housed
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4 per cage. The same mouse (i.e., Balb/c or Swiss Webster) was studied only once in the sociability and EPM paradigms prior to sacrifice. All animal procedures were approved by the Eastern Virginia Medical School Institutional Animal Care and Use Committee and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 2.2. Drugs d-Cycloserine (Sigma–Aldrich Co.; St. Louis, MO) was dissolved in 0.9% saline and prepared each day of the experiment. d-Cycloserine (320 mg/kg or the saline vehicle) was injected intraperitoneally (i.p.) in a volume of 0.01 mg/g of body weight 20 min prior to behavioral testing. 2.3. Open field The open field is a square chamber (18 in. × 18 in.) constructed of black Plexiglas. Briefly, 20 min after i.p. injection of d-cycloserine or saline, test mice (Balb/c or Swiss Webster) were individually placed in the middle of one side of the open field chamber, introduced to an inanimate object (i.e., plastic containers) on the opposite side of the chamber, and allowed to explore the field with the object present for 5 min. Time spent exploring the object was recorded. After each session, both object and test mouse were removed, and the open field chamber was cleaned with Quatricide PV solution. Immediately after, test mice were again individually placed in the middle of one side of the open field, introduced to a salient social ICR stimulus mouse enclosed in an inverted wire cup (Galaxy Cup, Kitchen Plus, http://www.kitchenplus.com) on the opposite side of the chamber, and allowed to explore the field with the enclosed stimulus mouse present for 5 min. Time spent exploring the stimulus mouse was recorded.
Fig. 1. Time spent exploring open arms. Bars represent mean (±SEM) of the time test mice spent (s) exploring open arms in the EPM test. The graph shows that vehicletreated Balb/c mice did not display anxiety-like behaviors in the EPM, compared to Swiss Webster mice. Numbers within bars depict group size. ***(p < 0.001).
2.4. Elevated plus maze The elevated plus maze (EPM) is a test designed to assess anxiety-like behaviors in rodents [4,10,14,17] and consists of four elevated arms (15 in. off the floor): two alternate open arms (12 in. d × 2½ in. w) and two alternate closed arms (12 in. d × 2½ in. w × 6 in. h). Briefly, immediately after testing in the open field, test mice were individually placed in the middle of the four elevated arms, facing one of the open arms, and allowed to explore all four arms for 5 min. An arm was counted as being entered when all four limbs were set on the arm [4,10,14,17]. Time spent and number of entries into the open and closed arms were recorded. All behavioral tests were conducted in dim lighting and videotaped using a Panasonic SDR-S26 SD Video Camera (Panasonic Corp., Osaka, Japan). 2.5. Statistical analyses Data were analyzed using NCSS (NCSS Statistical Software, Kaysville, UT). Twoway ANOVAs were used to examine the effect of strain (Balb/c vs. Swiss Webster) and treatment (d-cycloserine vs. vehicle) on time spent (s) exploring the inanimate object and the salient social stimulus mouse in the open field chamber by the test mouse, and time spent and number of entries into the open arms in the elevated plus maze. Fisher’s LSD Multiple-Comparison post hoc tests were applied, where appropriate.
multiple-comparison test revealed that Balb/c mice treated with d-cycloserine spent significantly more time exploring the social stimulus mouse than vehicle-treated Balb/c mice (p < 0.001; Fig. 3); however, d-cycloserine did not affect the time Swiss Webster mice spent exploring the social stimulus mouse in the open field (p > 0.05). A two-way ANOVA showed a main effect of strain (F[1,45] = 6.00, p < 0.05), treatment (F[1,45] = 85.98, p < 0.0001) and strain by treatment interaction (F[1,45] = 6.99, p < 0.05) on the number of entries into the open arms of the EPM. Fisher’s LSD multiple-comparison tests revealed that Balb/c mice treated with d-cycloserine made significantly more entries into the open arms than vehicle-treated Balb/c mice (p < 0.001; Fig. 4). Similarly, Swiss Webster mice treated with d-cycloserine made significantly more open arm entries than vehicle-treated Swiss Webster mice (p < 0.001).
3. Results A two-way ANOVA showed a significant main effect of strain (F[1,45] = 24.36, p < 0.0001) and strain by treatment interaction (F[1,45] = 10.17, p < 0.01) on time spent exploring open arms in the EPM. Fisher’s LSD multiple-comparison test revealed that vehicletreated Balb/c mice spent significantly more time exploring the open arms than vehicle-treated Swiss Webster mice (p < 0.001; Fig. 1). Interestingly, vehicle-treated Balb/c mice spent significantly more time in the open arms than Balb/c mice treated with dcycloserine (p < 0.05), whereas Swiss Webster mice treated with d-cycloserine spent significantly more time in the open arms than Swiss Webster treated with vehicle (p < 0.05; data not shown). d-Cycloserine did not affect the time Balb/c or Swiss Webster mice spent exploring the inanimate object; moreover, the time spent exploring the inanimate object in the open field did not differ between vehicle-treated Balb/c and vehicle-treated Swiss Webster mice (Fig. 2). A two-way ANOVA showed a significant main effect of strain (F[1,45] = 24.06, p < 0.0001), treatment (F[1,45] = 8.23, p < 0.01) and strain by treatment interaction (F[1,45] = 11.70, p < 0.01) on time spent exploring the salient social stimulus mouse enclosed in the inverted wire cup in the open field. Fisher’s LSD
Fig. 2. Time spent exploring inanimate object in the open field. Bars represent mean (±SEM) of the time test mice spent (s) exploring the inanimate object in the open field test. Numbers within bars depict group size.
L.F. Jacome et al. / Brain Research Bulletin 85 (2011) 141–144
Fig. 3. Time spent exploring stimulus mouse in the open field. Bars represent mean (±SEM) of the time test mice spent (s) exploring the socially salient stimulus mouse enclosed in the inverted wire cup in the open field. Numbers within bars depict group size. ***(p < 0.001).
4. Discussion Deficits in sociability may result from a general diminution of exploratory behavior or relate more specifically to an aversive quality of a salient social stimulus used to elicit exploration, among other possible explanations. In the current study, we confirmed that the Balb/c mouse spent significantly less time exploring an enclosed social stimulus mouse in an open field apparatus than the Swiss Webster comparator strain. However, the Balb/c mouse’s deficit in exploration was specific to its elicitation by a salient social stimulus, as time spent exploring an inanimate object did not differ between the Balb/c and Swiss Webster strains nor did the Balb/c mouse make fewer entries into the open arm of the EPM than the Swiss Webster mouse. Interestingly, anxiety-like behaviors in the Balb/c strain were not observed after these mice had been exposed to an enclosed social stimulus mouse in the open field appara-
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tus, which had a negative emotional valence for this strain, prior to testing in the EPM. Moreover, whereas d-cycloserine increased open arm entries in both strains, suggesting a positive effect on exploratory behavior and anxiety in general, this intervention only increased social exploration by the Balb/c strain in the open field apparatus. Thus, d-cycloserine, a partial glycine agonist at the obligatory co-agonist binding site on the NMDA receptor, may target a fundamental deficit in social exploration manifested by Balb/c mice, in addition to positively affecting exploration in general. Of course, although unlikely, it is possible that d-cycloserine’s ability to increase social exploration in the Balb/c mouse is an indirect effect, resulting from increased exploratory activity and “agitation.” However, “agitation” might be expected to decrease time spent in social exploration by this strain of mouse with impaired sociability [3]. The data are consistent with several prior studies showing that genetically engineered mice with reduced expression of the NR1 subunit of the NMDA receptor (5–10% of normal levels) or 5fold diminished affinity of the glycine binding site on the NR1 subunit for glycine manifest deficits of sociability in a standard paradigm [8,13]. Moreover, d-cycloserine was shown to have salutary effects on social behavior of genetically unaltered mice in the resident-intruder mouse paradigm of social interaction [16]. The data encourage exploration of targeted NMDA receptor agonist interventions on deficits of sociability, especially in the Balb/c mouse that has apparently normal expression of this receptor [18], but altered endogenous tone of NMDA receptor-mediated neurotransmission, as reflected in its heightened behavioral sensitivity to MK-801 [1,2,6,7]. Cross-talk between histaminergic receptors, especially H1 histaminergic receptors, and NMDA receptors may represent another fruitful in vivo strategy for facilitating NMDA receptor-mediated neurotransmission [15]. Moreover, H3 histaminergic receptors located on presynaptic glutamatergic terminals contribute to the regulation of glutamate release and availability in the synaptic cleft [15]. Because normal NMDA receptor function is necessary for memory consolidation, we are interested in exploring possible associations between impaired sociability and impaired memory performance in Balb/c mice [19]. d-Cycloserine dose-dependently improved memory consolidation in mice in the “Plus-Maze Retest Paradigm;” thus, d-cycloserine may be predicted to target sociability and at least some aspects of impaired memory performance [19]. The ability of d-cycloserine to enhance fear extinction, which is an active learning process, has led to translational clinical trials in anxiety disorders; presumably, d-cycloserine is acting at NMDA receptors within circuits linking cerebral cortex, hippocampus and amygdala [11], which are circuits that could also be relevant to the pathophysiology of autism spectrum disorders. Effects of dcycloserine in vivo in man can be attributed to its relatively selective interaction with the glycineB coagonist site on the NMDA receptor [12]. For example, in alcohol dependent men with probable upregulated expression of their NMDA receptors and control subjects, effects of infused glycine (0.3 g/kg over 30 min) and a large orally administered dose of d-cycloserine (1000 mg) on subjective measures of alcohol effects and objective cognitive measures were best explained by their competitive interaction with the glycineB site [12]. Thus, at least some of the effects of orally administered d-cycloserine in man (e.g., to persons with autism spectrum disorders) are likely to be due to its partial glycine agonistic properties.
Acknowledgement Fig. 4. Open arm entries in the EPM. Bars represent mean (±SEM) of the number of entries that test mice made into the open arms of the EPM. Numbers within bars depict group size. ***(p < 0.001).
The authors acknowledge the generous support that they received from the Office of the Dean of the Eastern Virginia Medical School.
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