Adolescent social defeat alters [ 3 H]-MK801 binding to NMDA receptors in rat brain Lili T. Belcastro, Yan Lin Lei and Shanaz Tejani-Butt Department of Pharmaceutical Sciences, University of the Sciences in Philadelphia, Philadelphia PA ABSTRACT Many human teenagers experience bullying, which is correlated with a greater incidence of stress-related psychiatric and substance abuse disorders in later life. We have used a rat model to investigate how prior experience of chronic social stress during adolescence may affect neural systems associated with such disorders in adulthood. Here we examined whether NMDA receptors are compromised by adolescent experience of social defeat. Male adolescent rats (P35) were defeated daily by larger aggressive males for 5 days, with controls being placed into empty novel cages at matched times. All rats then matured undisturbed to early adulthood (P56-61). Adult rats were given a single injection of amphetamine (2.5 mg/kg, i.p.), with brains collected 30 min later to assess regional changes in monoamine levels. Autoradiographic analysis with [ 3 H]-MK801 was used to measure NMDA receptors in discrete areas of the brain. Rats defeated in adolescence showed a down regulation of NMDA sites in both the infralimbic cortex of the medial prefrontal cortex and the central nucleus of the amygdala compared to control rats. Previously defeated rats exhibited higher NMDA receptor binding in the CA3 region of the hippocampus in comparison to controls. No differences in NMDA receptor binding were found in other brain regions measured. Our results indicate that adolescent defeat may be causing regionally specific changes to the adult glutamate system, which may contribute to behavioral alterations and increased drug seeking. INTRODUCTION Social defeat is a very potent stressor and can lead to a variety of behavioral effects, like social withdrawal, emotional disorders, and abuse of drugs and alcohol. 1 Social defeat is defined as losing a confrontation among conspecific animals, or any kind of hostile dispute among humans, in either a dyadic or in a group-individual context, generating very significant consequences in terms of control over resources, access to mates and social positions. One major concern regarding an adolescent's exposure to social stress is the later acquisition of compulsive drug abuse that can occur during adulthood. 2 Several GABAergic, glutamatergic, monoaminergic and peptidergic mechanisms are activated by anxiety--provoking situations in rats, such as social defeat and maternal separation; they represent important targets for pharmacological interventions and appear to be important in altering vulnerability to abuse of alcohol and other drugs. 2 As the major excitatory neurotransmitter in the brain, glutamate and the N-methyl-d-aspartate (NMDA) receptor plays a large role in anxiety and mood disorders. 3 A study by Takahashi et al. showed that MK-801 (dizocilpine), an NMDA receptor antagonist, dose-dependently reduced distress calling in maternally separated pups. 2 In another study by Covingtion III et al., it was found that social stress stimulates NMDA receptors in the ventral tegmental area (VTA), and this neural action of defeat may be essential for prompting a later increase in cocaine intake during binges. 4 In yet another study, direct infusion of MK-801 into rat amygdala stopped submissive behaviors in conditioned fear and defeat situations. 5 These studies suggest that anxiety-provoking incidents, like social defeat, are related to activation of the glutamatergic neural system. Given the implications of the glutamatergic system in social defeat and subsequent drug-seeking behavior, the present study mapped the distribution of NMDA receptors in discrete areas of the brain in socially defeated adolescent Sprague-Dawley rats given amphetamine in adulthood. NMDA receptor distribution varies in response to amphetamine between socially defeated and control rats. METHODS 20 adolescent (P35) Sprague-Dawley rats were used in this study. Rats were separated into control and social defeat groups. For the social defeat group, rats were subjected daily for 5 days to social defeat in the home cage of a larger, highly aggressive adult male (resident-intruder paradigm). The adolescent and adult are left in the cage separated by a mesh barrier for 35min. Control rats were not subjected to defeat and placed in a novel cage for the duration of each defeat trial. After 5 consecutive defeat sessions, rats were left undisturbed in their home cages and allowed to reach early adulthood (P56-61). Previously defeated and control adult rats were given a single injection of amphetamine (2.5mg/kg i.p.). 30 min. later, defeat and control rats were sacrificed by decapitation and the brains were removed immediately and stored at -80°C until use. Brain tissue sections (16  m) were cut at -18°C in a cryostat microtome according to the Brain Atlas of Paxinos and Watson 6 and mounted on (gelatin-coated) microscope slides. Sections from plates 12, 30, and 42 were used for this study. NMDA receptor autoradiography was performed according to the method of Sakurai et al. 7 with minor modifications. Brain sections were thawed and dried at room temp., pre-washed in 50mM Tris-HCl buffer (pH 7.4) for 30min. at room temp. Then the sections were incubated in 50mM Tris-HCl buffer containing 15nM [ 3 H] MK-801and 30  M glutamate/15  M glycine for 120 min. at room temp., rinsed for 30 min. with cold 50mM Tris-HCl buffer,dipped once in ice-cold distilled water, and immediately dried in a stream of cool air. Nonspecific binding was measured in the presence of 50  M non-radioactive MK-801(?and was less than 10% of total binding?). Dried tissue sections were placed ajascent to autoradiography film. Following a 4-week exposure at 4°C, the film was developed in Kodak D19 at room temp. The autoradiography films were analyzed with ImageJ (ImageJ 1.41 version). Nonspecific binding was subtracted from the total binding to provide the specific binding in the regions of interest and expressed as mean ± S.E.M. specific binding (fmol/mg brain protein). Statistical analysis was performed with Sigma Stat for Windows. The data were analyzed using t-test. RESULTS Figure 2 : Effect of social defeat on the binding of [ 3 H] MK-801 to NMDA receptor in the infralimbic cortex of the medial prefrontal cortex (mPFC). Data are expressed as the mean ± S.E.M. of measurements from 7-8 rats in each group. * Significant difference between groups, t-test, P < 0.05. Figure 1 : Effect of social defeat on the binding of [ 3 H] MK-801 to NMDA receptors in the caudate putamen. No significant difference was seen. Data are expressed as the mean ± S.E.M. of measurements from 9-10 rats in each group. The results of the present study indicate a significant difference in the binding of [ 3 H] MK-801 to NMDA receptors in socially defeated rats compared to non-defeated rats in the mPFC, amygdala, and hippocampus. Socially defeated rats had significantly decreased binding in the infralimbic cortex/ventromedial area of the mPFC (vmPFC) and the central nucleus of the amygdala (CEA), and a significant increase in binding in the CA2 and CA3 regions of the hippocampus compared to controls. The mPFC plays a role in fear-related processes and contributes to the interplay between emotions and memory formation. 8 Evidence from rodent studies implicates the vmPFC in the regulation of conditioned fear. 9 Electrolytic lesion or inactivation of the mPFC facilitates fear-related behavior including freezing, whereas electrical stimulation of the mPFC with conditioned tone suppresses freezing behaviors. 8 In addition, spontaneous activity of the mPFC neurons was reduced by fear conditioning. In contrast, inactivation of the mPFC with tetrodotoxin decreases freezing by conditioned fear, 9 and in humans, an NMDA agonist facilitates fear memory consolidation. 10 Our study showed significantly decreased binding of [ 3 H] MK-801 to NMDA receptors in the vmPFC in socially defeated rats consistent with the theory, that the vmPFC plays a role in the fear response. Decreased binding of [ 3 H] MK-801 to NMDA receptors in the CEA may be attributed to a social defeat response in our rats. It is well known that NMDA receptors in the amygdala play a role in conditioned fear and conditioned defeat. 5,11,12 In contrast, NMDA antagonists in the amygdala stop submissive behaviors in conditioned defeat. Increased binding of [ 3 H] MK-801 to NMDA receptors in the CA2 and CA3 regions of the hippocampus indicates an upregulation of NMDA receptors. The hippocampus plays a critical role in memory and learning processes that involve an emotional component, especially for associative fear memories. 8 In a study by Krugers et al., there was significant increased binding of NMDA antagonist in the CA3 region in rat brain hippocampi after a single inescapable social defeat. 13 Our findings are consistent with this after multiple social defeats. Stress-induced hippocampal response is known to be under the control of the amygdala, and the primary role of the mPFC is inhibitory in mediating hippocampal responses underlying fear-related processes. 8 If the mPFC is inhibited in some way, perhaps by downregulation of its NMDA receptors, this could in theory cause an increase in hippocampal response since the hippocampus is usually inhibited by the mPFC. Given that decreased binding was found in both the amygdala and mPFC, two limbic regions that are known to play a role in mediating hippocampal responses, an increase in binding in the hippocampus follows this logic. 1) Rygula, R., N. Abumaria, G. Fl ugg e, E. Fuchs, E. R uth er, U. Havemann-Reinecke. “A n hedonia and motivational deficits in rats: impact of chronic social stress. ” Behav Brain Res. 162 (2005): 127-134. 2) Miczek, K., J. Yap, H. Covington III. “ Social stress, therapeutics and drug abuse: Preclinical models of escalated and depressed intake. ” Pharmacology & Therapeutics . 120 (2008): 102-128. 3) Mathew, S., J. Coplan, D. Schoepp, E. Smith , L. Rosenblum, J. Gorman. “ Glutamate-hypothalamic-pituitary-adrenal axis interactions: implications for mood and anxiety disorders. ” 7 (2001): 561-564. 4) Covington III, H., T. Tropea, A. Rajadhyaksha, B. Kosofsky, K. Miczek. “N M DA receptors in the rat VTA: a critical site for social stress to intensify cocaine taking. ” Psychopharmacology (Berl) . 197 (2008): 203-216. 5) Jasnow, A., M. Cooper, K. Huhman. “ N - methyl-D-aspartate receptors in the amygdala are necessary for the acquisition and expression of conditioned defeat. ” Neuroscience . 123 (2004): 625-634. 6) Paxinos, G., C. Watson. The Rat Brain in Stereotaxic Coordinates . 4 th ed. 7) Sakurai, S., J. Penney, A. Young. “R e gionally distinct N-methyl-D-aspartate receptors distinguished by quantitative autoradiography of [ 3 H] MK-801 binding in rat brain. ” J Neurochem . 60 (1993): 1344-1353. 8) Hirata, R., M. Matsumoto, C. Judo, T. Yamaguchi, T. Izumi, M. Yoshioka, H. Togashi. “ P o ssible relationship between the stress-induced synaptic response and metaplasticity in the hippocampal CA1 field of freely moving rats. ” Synapse . 63(7) (2009): 549-556. 9) Sierra-Mercado Jr., D., K. Corcoran, K. Lebr on- Milad, G. Quirk. “I n activation of the ventromedial prefrontal cortex reduces expression of conditioned fear and impairs subsequent recall of extinction. ” Eur J Neurosci . 24(6) (2006): 1751-1758. 10) Kalisch, R., B. Holt, P. Petrovic, B. De Martino, S. Kl opp el, C. B uch el, R. Dolan. “ T h e NMDA agonist D-cycloserine facilitates fear memory consolidation in humans. ” Cereb Cortex . 19(1) (2009): 187-196. 11) Laurent, V., R. Westbrook. “I n fusion of the NMDA receptor antagonist, DL-APV, into the basolateral amygdala disrupts learning to fear a novel and a familiar context as well as relearning to fear an extinguished context. ” Learn Mem . 16(1) (2009): 96-105. 12) Goosens, K., S. Maren. “P r etraining NMDA receptor blockade in the basolateral complex, but not the central nucleus, of the amygdala prevents savings of conditional fear. ” Behav Neurosci . 117(4) (2003): 738-750. 13) Krugers, H., J. Koolhaas, B. Bohus, J. Korf. “A single social stress-experience alters glutamate receptor-binding in rat hippocampal CA3 area. ” Neurosci Lett . 154(1-2) (1993): 73-77. Figure 4 : Effect of social defeat on the binding of [ 3 H] MK-801 to NMDA receptors in the central nucleus of the amygdala. Data are expressed as the mean ± S.E.M. of measurements from 6 rats in each group. * Significant difference between groups, t-test, P < 0.05. Figure 7 : Effect of social defeat on the binding of [ 3 H] MK-801 binding in the CA2 region of the hippocampus in Sprague-Dawley rats. Data are expressed as the mean ± S.E.M. of measurements from 6 rats in each group. * Significant difference between groups, t-test, P < 0.05. Figure 6 : Effect of social defeat on the binding of [ 3 H] MK-801 binding in the CA2 region of the hippocampus in Sprague-Dawley rats. Data are expressed as the mean ± S.E.M. of measurements from 6 rats in each group. * Significant difference between groups, t-test, P < 0.05. REFERENCES SUMMARY AND CONCLUSION Figure 3 : Effect of social defeat on the binding of [ 3 H] MK-801 to NMDA receptors in the basolateral nucleus of the amygdala. No significant difference was observed. Data are expressed as the mean ± S.E.M. of measurements from 6 rats in each group. Figure 5 : Effect of social defeat on the binding of [ 3 H] MK-801 to NMDA receptors in the CA1 region of the hippocampus. No significant difference was observed. Data are expressed as the mean ± S.E.M.of measurements from 6 rats in each group. We would like to acknowledge our collaborators in this study. Drs. Gina Forster and Michael Watt for conducting the behavior studies in the Division of Basic Biomedical Sciences, Sanford School of Medicine, and 3Department of Biology, Neuroscience Group, University of South Dakota, Vermillion SD USA. We also acknowledge Andrew Novick, undergraduate student in our lab for assisting inthis study. This work was supported by research funds from USPHS Grants NIH P20 RR15567 & NIDA RO1 DA019921 to Gina Forster and Michael Watt and NIAAA 015921 to Shanaz Tejani-Butt. ACKNOWLEDGEMENTS

Many human teenagers experience bullying, which is correlated with a greater incidence of stress-related psychiatric and substance abuse disorders in later life. We have used a rat model to investigate how prior experience of chronic social stress during adolescence may affect neural systems associated with such disorders in adulthood. Here we examined whether NMDA receptors are compromised by adolescent experience of social defeat. Male adolescent rats (P35) were defeated daily by larger aggressive males for 5 days, with controls being placed into empty novel cages at matched times. All rats then matured undisturbed to early adulthood (P56-61). Adult rats were given a single injection of amphetamine (2.5 mg/kg, i.p.), with brains collected 30 min later to assess regional changes in monoamine levels. Autoradiographic analysis with [ 3 H]-MK801 was used to measure NMDA receptors in discrete areas of the brain. Rats defeated in adolescence showed a down regulation of NMDA sites in both the infralimbic cortex of the medial prefrontal cortex and the central nucleus of the amygdala compared to control rats. Previously defeated rats exhibited higher NMDA receptor binding in the CA3 region of the hippocampus in comparison to controls. No differences in NMDA receptor binding were found in other brain regions measured. Our results indicate that adolescent defeat may be causing regionally specific changes to the adult glutamate system, which may contribute to behavioral alterations and increased drug seeking.

Social defeat is a very potent stressor and can lead to a variety of behavioral effects, like social withdrawal, emotional disorders, and abuse of drugs and alcohol. 1 Social defeat is defined as losing a confrontation among conspecific animals, or any kind of hostile dispute among humans, in either a dyadic or in a group-individual context, generating very significant consequences in terms of control over resources, access to mates and social positions. One major concern regarding an adolescent's exposure to social stress is the later acquisition of compulsive drug abuse that can occur during adulthood. 2 Several GABAergic, glutamatergic, monoaminergic and peptidergic mechanisms are activated by anxiety--provoking situations in rats, such as social defeat and maternal separation; they represent important targets for pharmacological interventions and appear to be important in altering vulnerability to abuse of alcohol and other drugs. 2 As the major excitatory neurotransmitter in the brain, glutamate and the N-methyl-d-aspartate (NMDA) receptor plays a large role in anxiety and mood disorders. 3 A study by Takahashi et al. showed that MK-801 (dizocilpine), an NMDA receptor antagonist, dose-dependently reduced distress calling in maternally separated pups. 2 In another study by Covingtion III et al., it was found that social stress stimulates NMDA receptors in the ventral tegmental area (VTA), and this neural action of defeat may be essential for prompting a later increase in cocaine intake during binges. 4 In yet another study, direct infusion of MK-801 into rat amygdala stopped submissive behaviors in conditioned fear and defeat situations. 5 These studies suggest that anxiety-provoking incidents, like social defeat, are related to activation of the glutamatergic neural system.

Given the implications of the glutamatergic system in social defeat and subsequent drug-seeking behavior, the present study mapped the distribution of NMDA receptors in discrete areas of the brain in socially defeated adolescent Sprague-Dawley rats given amphetamine in adulthood. NMDA receptor distribution varies in response to amphetamine between socially defeated and control rats.

20 adolescent (P35) Sprague-Dawley rats were used in this study. Rats were separated into control and social defeat groups. For the social defeat group, rats were subjected daily for 5 days to social defeat in the home cage of a larger, highly aggressive adult male (resident-intruder paradigm). The adolescent and adult are left in the cage separated by a mesh barrier for 35min. Control rats were not subjected to defeat and placed in a novel cage for the duration of each defeat trial. After 5 consecutive defeat sessions, rats were left undisturbed in their home cages and allowed to reach early adulthood (P56-61). Previously defeated and control adult rats were given a single injection of amphetamine (2.5mg/kg i.p.). 30 min. later, defeat and control rats were sacrificed by decapitation and the brains were removed immediately and stored at -80°C until use. Brain tissue sections (16  m) were cut at -18°C in a cryostat microtome according to the Brain Atlas of Paxinos and Watson 6 and mounted on (gelatin-coated) microscope slides. Sections from plates 12, 30, and 42 were used for this study.

NMDA receptor autoradiography was performed according to the method of Sakurai et al. 7 with minor modifications. Brain sections were thawed and dried at room temp., pre-washed in 50mM Tris-HCl buffer (pH 7.4) for 30min. at room temp. Then the sections were incubated in 50mM Tris-HCl buffer containing 15nM [ 3 H] MK-801and 30  M glutamate/15  M glycine for 120 min. at room temp., rinsed for 30 min. with cold 50mM Tris-HCl buffer,dipped once in ice-cold distilled water, and immediately dried in a stream of cool air. Nonspecific binding was measured in the presence of 50  M non-radioactive MK-801(?and was less than 10% of total binding?). Dried tissue sections were placed ajascent to autoradiography film. Following a 4-week exposure at 4°C, the film was developed in Kodak D19 at room temp.

The autoradiography films were analyzed with ImageJ (ImageJ 1.41 version). Nonspecific binding was subtracted from the total binding to provide the specific binding in the regions of interest and expressed as mean ± S.E.M. specific binding (fmol/mg brain protein). Statistical analysis was performed with Sigma Stat for Windows. The data were analyzed using t-test.

The results of the present study indicate a significant difference in the binding of [ 3 H] MK-801 to NMDA receptors in socially defeated rats compared to non-defeated rats in the mPFC, amygdala, and hippocampus. Socially defeated rats had significantly decreased binding in the infralimbic cortex/ventromedial area of the mPFC (vmPFC) and the central nucleus of the amygdala (CEA), and a significant increase in binding in the CA2 and CA3 regions of the hippocampus compared to controls.

The mPFC plays a role in fear-related processes and contributes to the interplay between emotions and memory formation. 8 Evidence from rodent studies implicates the vmPFC in the regulation of conditioned fear. 9 Electrolytic lesion or inactivation of the mPFC facilitates fear-related behavior including freezing, whereas electrical stimulation of the mPFC with conditioned tone suppresses freezing behaviors. 8 In addition, spontaneous activity of the mPFC neurons was reduced by fear conditioning. In contrast, inactivation of the mPFC with tetrodotoxin decreases freezing by conditioned fear, 9 and in humans, an NMDA agonist facilitates fear memory consolidation. 10 Our study showed significantly decreased binding of [ 3 H] MK-801 to NMDA receptors in the vmPFC in socially defeated rats consistent with the theory, that the vmPFC plays a role in the fear response.

Decreased binding of [ 3 H] MK-801 to NMDA receptors in the CEA may be attributed to a social defeat response in our rats. It is well known that NMDA receptors in the amygdala play a role in conditioned fear and conditioned defeat. 5,11,12 In contrast, NMDA antagonists in the amygdala stop submissive behaviors in conditioned defeat.

Increased binding of [ 3 H] MK-801 to NMDA receptors in the CA2 and CA3 regions of the hippocampus indicates an upregulation of NMDA receptors. The hippocampus plays a critical role in memory and learning processes that involve an emotional component, especially for associative fear memories. 8 In a study by Krugers et al., there was significant increased binding of NMDA antagonist in the CA3 region in rat brain hippocampi after a single inescapable social defeat. 13 Our findings are consistent with this after multiple social defeats.

Stress-induced hippocampal response is known to be under the control of the amygdala, and the primary role of the mPFC is inhibitory in mediating hippocampal responses underlying fear-related processes. 8 If the mPFC is inhibited in some way, perhaps by downregulation of its NMDA receptors, this could in theory cause an increase in hippocampal response since the hippocampus is usually inhibited by the mPFC. Given that decreased binding was found in both the amygdala and mPFC, two limbic regions that are known to play a role in mediating hippocampal responses, an increase in binding in the hippocampus follows this logic.