Cyclooxygenase-2 inhibition reduces stress-induced affective pathology
Mood and anxiety disorders including major depression , generalized anxiety disorder and posttraumatic stress disorder are the most common psychiatric disorders ( Kessler et al . , 2005; 2012 ) . Unfortunately , current pharmacological therapies , which act primarily via monoamine reuptake mechanisms , are only partially effective ( Griebel and Holmes , 2013 ) . Recent studies have begun to emphasize novel non-monoaminergic pathophysiological mechanisms underlying mood and anxiety disorders and support immunological ( Miller et al . , 2009; Raison and Miller , 2013 ) , glutamatergic ( Abdallah et al . , 2015; Dutta et al . , 2015 ) , and bioactive lipid-based pharmacological approaches for treatment-resistant affective disorders ( Papakostas and Ionescu , 2015 ) . In this regard , there has been growing interest in cyclooxygenase-2 ( COX-2 ) as a bioactive lipid-mechanism-based target for the treatment of affective disorders ( Akhondzadeh and Jafari , 2010; Fond et al . , 2014; Hermanson et al . , 2014 ) . COX-2 catalyzes synthesis of inflammatory prostaglandins ( PGs ) from free arachidonic acid and is expressed constitutively and in an activity-dependent manner in neurons ( Kaufmann et al . , 1996; Kulmacz and Lands , 1984; Yamagata et al . , 1993 ) . Based on the immune hyperactivity hypothesis of depression ( Felger and Miller , 2014; Haroon et al . , 2012; Miller et al . , 2009; Raison and Miller , 2013 ) , several clinical trials have demonstrated efficacy of COX-2 inhibition as an augmentation strategy to selective-serotonin reuptake inhibitors ( SSRIs ) for the treatment of major depressive disorder ( MDD ) ( Akhondzadeh and Jafari , 2010; Akhondzadeh et al . , 2009; Muller et al . , 2006 ) . A number of preliminary studies also suggest that inhibition of COX-2 could have anxiolytic- and antidepressant-like behavioral effects in animal models . Specifically , chronic Celecoxib treatment prevents the development of anhedonia after chronic unpredictable stress ( Guo et al . , 2009 ) , and chronic COX-2 inhibition reduces immobilization stress-induced hypoactivity , memory deficits ( Kumari et al . , 2007 ) , and anxiety-like behavior in the mirror chamber test ( Dhir et al . , 2006 ) . Interestingly , in addition to generating PGs , COX-2 also metabolizes endogenous cannabinoids ( eCBs; anandamide and 2-arachidonoylglycerol ) ( Kozak et al . , 2000; 2001 ) , and several eCB augmentation strategies , including inhibition of fatty acid amide hydrolase ( FAAH ) and monoacylglycerol lipase ( MAGL ) , have been found to attenuate anxiety- and depression-related behaviors ( Fu et al . , 2003; Griebel and Holmes , 2013; Gunduz-Cinar et al . , 2013; Hill et al . , 2009; Ruehle et al . , 2012 ) . However , little is currently understood about the possible contribution of eCB signaling to the behavioral and therapeutic actions of COX-2 inhibitors in mood and anxiety disorders . We have recently developed a class of COX-2 inhibitors that differentially inhibit COX-2 enzymatic activity based on the substrate: arachidonic acid or eCBs ( Duggan et al . , 2011; Hermanson et al . , 2013 ) . Specifically , these ‘substrate-selective COX-2 inhibitors’ ( SSCIs ) inhibit COX-2 activity when eCBs are used as substrates , without affecting the ability of COX-2 to generate PGs ( Hermanson et al . , 2013 ) . The development of these SSCIs provides a powerful tool for dissecting the mechanisms by which pharmacological COX-2 inhibition can modulate a variety of physiological and pathophysiological processes ( Hermanson et al . , 2014 ) . In the current study , we performed a comparative analysis between the prototypic SSCI , LM-4131 , and two traditional COX-2 inhibitors currently or previously approved for pain and arthritis , Lumiracoxib ( LMX ) and Celecoxib , in a variety of measures of mouse anxiety- , fear- and depression-related behavior . We show that all three COX-2 inhibitors attenuate stress-induced anxiety-like states , with minimal effects on basal anxiety-like , despair-like , and locomotor behaviors . The anxiolytic-like effects of LM-4131 were paralleled by a reduction in anxiety-related BLA neuronal activity ex vivo and in vivo . Collectively , these data support the therapeutic potential of COX-2 inhibition for the treatment of stress-related neuropsychiatric conditions .
We sought to determine the efficacy of pharmacological COX-2 inhibition in preclinical models of affective disorders . Since stress is the major environmental risk factor for the development of mood and anxiety disorders ( Caspi et al . , 2003; Kessler , 1997; McEwen , 2003 ) , we evaluated the behavioral effects of COX-2 inhibition after stress exposure . The novelty-induced hypophagia ( NIH ) assay is a well-validated ethologically relevant measure of both anxiety and depressive-related behaviors , encompassing both conflict anxiety and hedonic motivational processes , and is responsive to chronic ( but not acute ) SSRI treatment ( Cryan and Holmes , 2005; Dulawa and Hen , 2005; Merali et al . , 2003 ) . We utilized this assay to test the efficacy of a newly developed SSCI , LM-4131 , relative to two clinically effective COX-2 inhibitors , LMX and Celecoxib , to reduce basal and stress-induced anxiety-like behavior in mice . Results showed that systemic administration of either LM-4131 or LMX reduced mean feeding latency in ( 5–7 week old ) mice that had been subjected to foot-shock stress 8 or 24 , but not 4 or 72 , hours earlier ( Figure 1B–E ) . Celecoxib , tested at the 8-hr post-stress time point , also reduced mean feeding latency ( Figure 1C ) . In contrast , only LM-4131 , and not LMX or Celecoxib , produced a small reduction in mean feeding latency in non-stressed controls ( Figure 1A ) . It is interesting to note that the anxiety-like effect of foot-shock stress peaked at ~8 hr after foot-shock , and resolved by 72 hr . The delayed onset of the anxiety-like response to foot-shock may be related to time-dependent neuronal plasticity mechanism induced by the foot-shock exposure . Together , these data show that stress causes an increase in feeding latency between 8 and 24 hr , which was abrogated by the acute SSCI and the traditional COX-2 inhibitor treatment ( Figure 1F ) . 10 . 7554/eLife . 14137 . 003Figure 1 . Effects of COX-2 inhibition in the NIH assay . Effects of vehicle , LM-4131 , Lumiracoxib , and Celecoxib administered 2 hr prior to behavioral testing on feeding latency under non-stressed ( control conditions ) ( A ) , and 4 hr ( B ) , 8 hr ( C ) , 24 hr ( D ) , and 72 hr ( E ) after foot-shock stress ( experimental design shown above ) . Cumulative distribution curves depict percentage of mice feeding at each latency time point ( sec ) , while bar graphs represent mean ± S . E . M feeding latency for each group and individual data points . Each point represents 1 mouse . ( F ) Re-plotted data from A-E in time-course format depicting effects of drugs on feeding latency over time . Significant F and P values from one-way ANOVA noted above bar graphs; *p<0 . 05 , **p<0 . 01 , ****p<0 . 0001 by Holm-Sidak post hoc multiple comparisons test in bar graphs . For cumulative frequency distributions , *p<0 . 05 , ****p<0 . 0001 by K-S test . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 00310 . 7554/eLife . 14137 . 004Figure 1—figure supplement 1 . Effects of COX-2 inhibition on feeding latency in the NIH assay in 4-month-old male mice . ( A ) Effects of LM-4131 and Lumiracoxib ( LMX ) on feeding latency in control , non-stressed mice . ( B ) Effects of LM-4131 , LMX , and Celecoxib on feeding latency tested 8 hr after acute foot-shock exposure . Significant F and P values for one-way ANOVA shown above figures . **p<0 . 01 by Holm-Sidak post hoc multiple comparisons test . For cumulative distribution curves , **p<0 . 01 , ****p<0 . 0001 by K-S test . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 00410 . 7554/eLife . 14137 . 005Figure 1—figure supplement 2 . Effects of COX-2 inhibition on feeding latency in the NIH test in female mice . ( A ) Effects of LM-4131 on feeding latency in the NIH assay under control , non-stressed conditions in female mice . ( B ) Effects of LM-4131 on feeding latency 8 hr after foot-shock exposure . ( C ) Effects of Rimonabant + vehicle , or Rimonabant + LM-4131 co-treatment , on feeding latency tested 8 hr after foot-shock exposure . *p<0 . 05 by two-tailed unaired t-test . For cumulative distribution curves , **p<0 . 01 by K-S test . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 00510 . 7554/eLife . 14137 . 006Figure 1—figure supplement 3 . Effects of COX-2 inhibition on feeding latency in the NIH test after sub-chronic stress . ( A ) Effects of LM-4131 and Lumiracoxib ( LMX ) on feeding latency in the NIH assay under control , non-stressed conditions . ( B ) Effects of LM-4131 and LMX on feeding latency in the NIH assay 8 hr after the last of 5 daily foot-shock exposures . Significant F and P values by one-way ANOVA shown above figures . **p<0 . 01 by Holm-Sidak post hoc multiple comparisons test . For cumulative distribution curves , ****p<0 . 0001 by K-S test . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 00610 . 7554/eLife . 14137 . 007Figure 1—figure supplement 4 . Effects of subchronic COX-2 inhibition on feeding latency in the NIH test . ( A ) Effects of 5 consecutive daily administrations of Lumiracoxib ( LMX ) on feeding latency and food consumption in the NIH test , and body weight , in control non-stressed mice . ( B ) Effects of 5 consecutive daily administrations of LMX on feeding latency and food consumption in the NIH test 8 hr after foot-shock exposure . *p<0 . 05 by two-tailed unpaired t-test . For cumulative distribution curves , ****p<0 . 0001 by K-S test . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 00710 . 7554/eLife . 14137 . 008Figure 1—figure supplement 5 . Effects of COX-2 inhibition on locomotor activity in the open field test . ( A ) Effects of LM-4131 on total distance travelled . ( B ) Effects of Lumiracoxib on total distance travelled . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 008 Demonstrating the generalizability of these effects across different subjects , stressor durations and drug treatment conditions , we showed that LM-4131 , LMX , and Celecoxib decreased mean feeding latency in older , 4-month-old mice , 8 hr after foot-shock stress , but not in non-stressed controls ( Figure 1—figure supplement 1 ) . Additionally , LM-4131 also reduced mean feeding latency 8-hr post-stress in female mice ( Figure 1—figure supplement 2 ) . Moreover , to examine whether the SSCI , LM-4131 , retained anxiolytic-like efficacy following a more chronic , severe stressor , male mice were exposed to foot-shock stress for 5 consecutive days . Under these conditions , LM-4131 administered 2 hr prior to testing was still able to reduce mean feeding latency in stressed mice , but not non-stressed controls ( Figure 1—figure supplement 3 ) . Finally , we tested for the effects of repeated daily treatment with LMX ( used instead of LM-4131 due to its long half-life ( Mangold et al . , 2004 ) for 5 consecutive days prior to acute stress exposure . This subchronic treatment regimen reduced feeding latency 8 hr after stress , with no effects in non-stressed controls , thereby mimicking the effects of acute treatment ( Figure 1—figure supplement 4 ) . There were no effects of this subchronic LMX treatment on body weight in either group ( Figure S4 ) . Also of note , neither acute LM-4131 nor LMX treatment affected overall locomotor activity in the open-field assay ( Figure 1—figure supplement 5 ) , indicating drug-induced reductions in feeding latency were not an artifact of hyperactivity . Taken together , these data demonstrate that COX-2 inhibition exerts a robust , preferential attenuation of stress-induced affective dysregulation across a range of experimental conditions . We also examined the effects of COX-2 inhibition in a well-established test for anxiety-like behavior , the elevated-plus maze ( EPM ) . In non-stressed mice , drug treatment increased open arm entries and closed arm latency , without affecting other measures ( Figure 2A ) . In mice stressed 8 hr earlier , treatment with either LM-4131 or LMX reduced the latency to enter an open arm and increased the latency to enter closed arm and to freeze , but did not alter open arm time or entries or total distance traveled ( Figure 2B ) . In a separate cohort of 4-month-old mice , LM-4131 and LMX reduced open-arm latency and latency to freeze regardless of prior stress exposure ( Figure 2—figure supplement 1 ) . These data demonstrate the effects of COX-2 inhibition are evident across behavioral assays , and are not restricted to appetitive motivated behavioral tests such as the NIH assay . 10 . 7554/eLife . 14137 . 009Figure 2 . Effects of COX-2 inhibition in the elevated-plus maze . ( A ) Effects of LM-4131 ( 10 mg/kg ) and Lumiracoxib ( experimental design shown above ) under basal non-stressed conditions . ( B ) Effects of LM-4131 and Lumiracoxib 8 hr after acute foot-shock exposure . Significant F and P values for one-way ANOVA shown above bar graphs . *p<0 . 05 , ****p<0 . 0001 by Holm-Sidak multiple comparisons post hoc test . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 00910 . 7554/eLife . 14137 . 010Figure 2—figure supplement 1 . Effects of COX-2 inhibition in the EPM in ~4-month-old mice . ( A ) Effects of LM-4131 and Lumiracoxib ( LMX ) in the EPM in ~4-month-old mice under non-stressed conditions . ( B ) Effects of LM-4131 and LMX in the EPM in 4-month old mice tested 8 hr after foot-shock stress exposure . Significant F and P values for one-way ANOVA shown above figures . *p<0 . 05 , ** p<0 . 01 , ***p<0 . 001 , ****p<0 . 0001 by Holm-Sidak multiple comparisons post-hoc test . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 010 In addition to the anxiety-like phenotype caused by foot-shock stress exposure , re-exposure to the foot-shock context induces a conditioned fear response in the form of freezing . We therefore tested the effects of COX-2 inhibition on this conditioned fear response by administering LM-4131 , LMX or Celecoxib 6 hr after foot-shock and then testing for contextual freezing 2 hr later ( see Figure 3A for experimental design ) . Acquisition of freezing behavior during foot-shock was not different between treatment groups prior to drug administration ( Figure 3B ) . However , drug treatment reduced expression of contextual fear measured 8 hr post-conditioning ( Figure 3C ) . Freezing levels were low on a subsequent drug-free retrieval test 24 hr after conditioning , indicating extinction or decay of the conditioned-fear response , and no residual drug treatment effect was evident at this time point ( Figure 3D ) . These data extend our stress-induced anxiety-related findings by demonstrating attenuation in the expression of contextual conditioned fear by COX-2 inhibition . 10 . 7554/eLife . 14137 . 011Figure 3 . Effects of COX-2 inhibition on contextual fear conditioning . ( A ) Timeline for contextual fear conditioning . Data from each time point shown in correspondingly labeled panel ( B–D ) . ( B ) No differences in freezing behavior between groups were observed during conditioning . Drugs were administered 6 hr after conditioning ( 2 hr before fear retrieval ) . ( C ) Time course and summary of effects of LM-4131 , Lumiracoxib ( LMX ) , or Celecoxib on conditioned fear expression during context re-exposure . ( D ) Effects of LM-4131 , LMX , and Celecoxib on contextual freezing on a second context re-exposure . Significant F and P values for two-way ANOVA ( C ) and one-way ANOVA ( D ) shown above figure . *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , ****p<0 . 0001 by Holm-Sidak post hoc analysis . Color of * indicates significance level of comparison between each drug and vehicle; red , LM-4131; blue , LMX; green , Celecoxib . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 01110 . 7554/eLife . 14137 . 012Figure 4 . Effects of COX-2 inhibition in the TST . ( A ) Effects of LM-4131 , LMX , and Celecoxib on immobility duration under non-stressed conditions . ( B–C ) Effects of LM-4131 on immobility duration 8 and 24 hr after foot-shock exposure . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 012 The effects of COX-2 inhibition were next tested in two assays sensitive to monoamine antidepressant treatment , the tail suspension test ( TST ) and the sucrose preference test ( Cryan and Holmes , 2005 ) . Interestingly , we found that neither LM-4131 , LMX , nor Celecoxib altered TST immobility time in non-stressed mice ( Figure 4A ) and that LM-4131 also had no effect on this behavior in mice given foot-shock stress 8 or 24 hr previously ( Figure 4B–C ) . In a similar vein , LM-4131 had no effect on sucrose consumption or preference , either under non-stressed conditions or 8 hr following foot-shock ( Figure 5 ) . These data highlight a degree of specificity in the behavioral effects of acute COX-2 inhibition . 10 . 7554/eLife . 14137 . 013Figure 5 . Effects of COX-2 inhibition in the sucrose preference test . ( A–C ) 2 hr cumulative sucrose preference , sucrose consumption , and water consumption after vehicle or LM-4131 treatment in non-stressed mice and 8 hr after foot-shock exposure . Testing was conducted during the light phase of the circadian cycle . ( D–F ) Effects of LM-4131 or vehicle on sucrose preference , sucrose consumption , and water consumption during the light and dark phases of the circadian cycle . Mice were treated with LM-4131 or vehicle 8 hr after foot-shock exposure and tested for 16 hr ( 4 hr during the light cycle and 12 hr during the dark cycle ) . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 013 As we have previously shown that LM-4131 exerts behavioral effects via type-1 cannabinoid receptor ( CB1R ) activation subsequent to anandamide elevation ( Hermanson et al . , 2013 ) , we tested whether the anxiolytic-like effects of LM-4131 after foot-shock stress exposure were mediated via CB1R signaling . Replicating data shown in Figure 1A , under non-stressed control conditions , LM-4131 produced a slight but significant reduction in feeding latency , and this effect was blocked by co-treatment with the CB1R antagonist Rimonabant ( 2 mg/kg ) ( Figure 6—figure supplement 1 ) . By contrast , LM-4131-induced decreases in feeding latency , that were seen 8 hr after stress , were not prevented by co-administering a low ( 2 mg/kg ) or higher ( 5 mg/kg ) dose of Rimonabant ( Figure 6A–B ) . These data show that while the anxiolytic-like effects of LM-4131 are CB1R dependent under non-stressed conditions , in line with previous work ( Hermanson et al . , 2013 ) ; however , following stress these effects become CB1R-insensitive and likely mediated by other mechanisms . 10 . 7554/eLife . 14137 . 014Figure 6 . Receptor mechanisms mediating anxiolytic-like effects of COX-2 inhibition . ( A ) Cumulative feeding latency distribution curves for vehicle , LM-4131 , and Lumiracoxib treated mice in the presence of vehicle , or the CB1R antagonist Rimonabant ( 2 or 5 mg/kg ) co-treatment . ( B ) Mean feeding latency for each group tested 8 hr after stress exposure . ( C ) Effects of the CB2R antagonist , SR144528 ( 3 mg/kg ) , or the TRPV1 antagonist , Capsazapine ( 10 mg/kg ) , on LM-4131-induced reductions in feeding latency tested 8 hr after stress exposure . ( D ) Effects of the SK channel inhibitor , Apamin ( 0 . 4 or 0 . 8 mg/kg; combined data shown ) , on LM-4131 , LMX , and Celecoxib-induced reductions in feeding latency tested 8 hr after stress exposure . ( E ) Effects of the SK channel activator , 1-EBIO ( 5 mg/kg ) , on feeding latency in the presence and absence of LM-4131 tested 8 hr after stress exposure . Significant F and P values from one-way ANOVA noted above bar graphs; *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , ****p<0 . 0001 by Holm-Sidak post hoc comparisons test in bar graphs . For cumulative frequency distributions , ****p<0 . 0001 by K-S test . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 01410 . 7554/eLife . 14137 . 015Figure 6—figure supplement 1 . Effects of CB1R antagonism on LM-4131-induced reductions in feeding latency in the NIH test . ( A ) Under non-stressed conditions , LM-4131 produces a small but significant reduction in feeding latency in the NIH test . ( B ) In the presence of the CB1R antagonist , Rimonabant ( Rim ) LM-4131 has no effect on feeding latency relative to Rimonabant alone . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 01510 . 7554/eLife . 14137 . 016Figure 6—figure supplement 2 . Effects of Apamin and 1-EBIO on locomotor activity . ( A ) Effects of Apamin ( 0 . 2–1 mg/kg ) on locomotor activity . ( B ) Effects of 1-EBIO 1–10 mg/kg on locomotor activity . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 016 Although CB1R activation may not mediate the anxiolytic-like effects of COX-2 inhibition in stressed mice , the eCB anandamide can activate several molecular targets in addition to CB1R , including CB2R , transient receptor potential cation channel , subfamily V , member 1 ( TRPV1 ) receptors , and SK channels ( Di Marzo and De Petrocellis , 2012; Ross , 2003; Wang et al . , 2011 ) . Moreover , LM-4131 exerts preferential effects on anandamide over 2-AG ( Hermanson et al . , 2013 ) . We therefore tested whether blocking these alternate anandamide targets affected the anxiolytic-like effects of LM-4131 after stress . Neither co-treatment with the CBR2 antagonist , SR144528 ( 3 mg/kg ) , nor the TRPV1 antagonist , Capsazepine ( 10 mg/kg ) , blocked LM-4131-induced reductions in feeding latency 8 hr after stress ( Figure 6C ) . However , co-administration of the SK channel inhibitor , Apamin ( 0 . 4 and 0 . 8 mg/kg ) , blocked the ability of LM-4131 , as well as LMX or Celecoxib , to reduce stress-induced anxiety-like behavior ( Figure 6D ) . To further substantiate a role for SK channels in the effects of LM-4131 after foot-shock stress , we tested whether direct activation of SK channels was sufficient to mimic the anxiolytic-like effects of LM-4131 . Consistent with earlier data , LM-4131 reduced feeding latency in mice stressed 8 hr earlier ( Figure 6E ) . The SK channel activator 1-EBIO also reduced feeding latency , and the combined administration of 1-EBIO and LM-4131 did not produce a further decrease in feeding latency relative to either drug alone ( Figure 6E ) . Apamin and 1-EBIO had minimal effect on locomotor activity when administered alone at doses used above ( Figure 6—figure supplement 2 ) , showing these drug effects on feeding latency were not artifacts of locomotor disturbances . We have previously shown that eCB signaling effects on food consumption and feeding latency in the assay are dissociable ( Gamble-George et al . , 2013 ) . Therefore , we extended our analyses to measure drug effects on total food consumption in the NIH assay to clarify whether COX-2 inhibition affects feeding via a CB1R-dependent or independent mechanism . We found that reductions in food consumption produced by testing in a novel cage ( relative to the home cage ) ( Figure 7A ) were partially reversed by LM-4131 in mice with prior foot-shock stress exposure , albeit not in all experiments ( Figure 7B–F ) . In addition , examination of feeding behavior in our CB1R antagonist studies indicated that , in contrast to the negative effects of CB1R blockade on feeding latency after stress exposure , the ability of LM-4131 and LMX to normalize stress-induced deficits in food consumption was completely blocked by either 2 or 5 mg/kg Rimonabant ( Figure 7G–H ) . Thus there appear to be dissociable contributions of CB1R to the stress-related consummatory and anxiolytic-like effects of COX-2 inhibition ( Gamble-George et al . , 2013 ) . 10 . 7554/eLife . 14137 . 017Figure 7 . Effects of COX-2 inhibition on food consumption in the NIH test . ( A ) Cumulative distribution curves depicting proportion of mice consuming a given amount of food , and mean consumption amounts in the home cage and novel cage setting . ( B–F ) Effects of LM-4131 on Ensure consumption compared to vehicle treatment under control ( non-stressed ) conditions ( B ) , 4 hr ( C ) , 8 hr ( D ) , 24 hr ( E ) , and 72 hr ( F ) after foot-shock stress . ( G–H ) Effects of Vehicle , LM-4131 or Lumiracoxib on consumption in the presence of vehicle or Rimonabant ( 2 or 5 mg/kg ) co-treatment . In bar graphs *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 by unpaired two-tailed t-test ( B–F ) or by Holm-Sidak multiple comparisons test ( G ) . For cumulative frequency distributions , ***p<0 . 001 , ****p<0 . 0001 by K-S test . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 017 In order to gain insight into the synaptic and cellular mechanisms by which LM-4131 could affect stress-induced anxiety , we utilized ex vivo whole-cell patch-clamp electrophysiological approaches . Specifically , we determined the effects of LM-4131 on cellular and synaptic physiology in the BLA , given its key role in regulating anxiety and stress-response integration ( Janak and Tye , 2015; Pape and Pare , 2010 ) . In control , non-stressed mice , incubation of brain slices with LM-4131 ( 10 μM ) for 2 hr did not affect intrinsic excitability ( Figure 8A–B ) or current-membrane voltage relationship ( Figure 8C ) of BLA principal neurons , and did not affect resting membrane potential , input resistance , or action potential threshold ( Figure 8D ) . Conversely , in brain slices prepared 24 hr after foot-shock stress , LM-4131 incubation resulted in a significant decrease in intrinsic excitability of BLA neurons ( Figure 8E–F ) . This reduction was not associated with changes in the current-membrane voltage relationship ( Figure 8G ) , or the resting membrane potential , input resistance , or action potential threshold ( Figure 8H ) . These data suggest that one mechanism by which LM-4131 reduced stress-induced behavioral dysregulation is via reductions in the overall activity of BLA neurons via reductions in intrinsic excitability . Consistent with our behavioral data , that the ability of LM-4131 to reduced neuronal excitability after stress exposure was blocked by the SK channel blocker , Apamin , but not the CB1 antagonist SR141716 or the TRPV1 antagonist , Capsazepine ( Figure 8—figure supplement 1A–C ) . 10 . 7554/eLife . 14137 . 018Figure 8 . Effects of COX-2 inhibition on ex vivo BLA neuronal excitability . ( A–B ) Effects of LM-4131 ( 10 μM ) on BLA neuron excitability , ( C ) I/V relationship , ( D ) resting membrane potential ( Vm ) , input resistance ( Ri ) , and action potential ( AP ) threshold in non-stressed mice . ( E–F ) Effects of LM-4131 on BLA neuron excitability , ( G ) I/V relationship , ( H ) resting membrane potential ( Vm ) , input resistance ( Ri ) , and action potential ( AP ) threshold in mice 24 hr after foot-shock stress . F and P values for drug effects by two-way ANOVA shown in ( A and E ) , *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , ****p<0 . 0001 by post hoc Holm-Sidak test in ( A ) and ( E ) . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 01810 . 7554/eLife . 14137 . 019Figure 8—figure supplement 1 . Effects of Rimonabant , Apamin , and Capsazepine on LM-4131-induced reductions in BLA neuron excitability . ( A ) Effects of Rimonabant ( Rim; 5 μM ) on LM-4131 ( 10 μM ) induced reductions in BLA neuronal excitability ex vivo . ( A ) Effects of Capsazepine ( CPZ; 10 μM ) on LM-4131 induced reductions in BLA neuronal excitability ex vivo . ( A ) Effects of Apamin ( 500 nM ) on LM-4131 induced reductions in BLA neuronal excitability ex vivo . Drug effect F and P values by two-way ANOVA shown in figures . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 01910 . 7554/eLife . 14137 . 020Figure 8—figure supplement 2 . Effects of FAAH inhibition on ex vivo BLA neuronal excitability . ( A ) Effects of PF-3845 on BLA neuron excitability and in non-stressed mice , and ( B ) 24 hr after foot-chock stress . F and P values for drug effects by two-way ANOVA shown in A and B . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 020 Given our behavioral data had indicated a role for COX-2-mediated enhancement of anandamide signaling in the anxiolytic effects of LM-4131 , we tested whether directly increasing anandamide levels , via FAAH inhibition , mimicked the effects of LM-4131 on cellular excitability and glutamatergic transmission . Indeed , incubation of brain slices from both control and stressed mice with the FAAH inhibitor , PF-3845 ( 10 μM ) , resulted in a decrease in cellular excitability ( Figure 8—figure supplement 2 ) . These data suggest LM-4131 may reduce stress-induced anxiety-like behavior by dampening neuronal activity in the BLA and , moreover , that these cellular effects can be mimicked by anandamide augmentation via FAAH inhibition . Amygdala neuron activity has been linked to the expression of anxiety-like behaviors ( Janak and Tye , 2015; Rau et al . , 2015 ) . On this basis and given our ex vivo physiological data , we asked whether LM-4131-induced reductions in BLA neuronal excitability were evident in behaving mice by conducting in vivo single unit recordings . We employed the EPM for this experiment because preliminary work indicated robust behavioral profiles and drug effects under the chronic recording conditions . That is , during recordings , mice treated with LM-4131 and tested 8 hr after stress showed increased open arm time and entries and reduced open arm latency , as compared to vehicle treated controls , consistent with an anxiolytic-like effect in this test ( Figure 9A–B ) . It is interesting to note that under these recording conditions mice exhibited high levels of anxiety with open arm time ~10 s , and LM-4131 was able to increase open arm time and decrease latency to open arm entry . In contrast , in previous studies ( Figure 2 ) , LM-4131 only affected open arm latency . Given that open arm time in these studies was ~25–40 s , these data suggest that the open arm latency measure is more sensitive to LM-4131 treatment , but as anxiety levels increase , LM-4131 can affect more conventional measures of anxiety in the EPM including open arm time . 10 . 7554/eLife . 14137 . 021Figure 9 . Effects of COX-2 inhibition on in vivo activity of BLA neuronal activity . ( A ) LM-4131 decreases latency to enter and increases entries into and time spent in the open arms of the EPM 8 hr following stress exposure . ( B ) Representative heat maps of time spent in the closed ( vertical ) versus open ( horizontal ) arms of the EPM . ( C ) Percentage of total cells recorded that were responsive upon exiting a closed arm . ( D ) LM-4131 reduced the maximum firing rate of cells responsive to closed arm exit , but had no effect on cells inhibited by closed-arm exit . ( E ) LM-4131 reduced the increase in average firing rate following closed arm exit in responsive cells . *p<0 . 05 , **p<0 . 01 by two-tailed unpaired t-test . F and P values for two-way ANOVA shown in ( E ) . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 02110 . 7554/eLife . 14137 . 022Figure 9—figure supplement 1 . Schematic diagram of in vivo electrophysiological recording sites . Black dots represent cannula tip locations for vehicle and grey dots represent cannula tip locations for LM-4131 treated mice . Numbers refer to coordinates lateral to bregma for each sagittal brain section . DOI: http://dx . doi . org/10 . 7554/eLife . 14137 . 022 While mice were exploring the EPM , we recorded 62 and 66 single units from vehicle and LM-4131-treated mice , respectively ( n=12 mice per group; recording sites depicted in Figure 9—figure supplement 1 ) . Firing rates of each unit were analyzed during exit from the safety of the closed arms , and were classified as being excitatory ( at least 1 of 10 100-ms time bins following closed arm exit had an average Z-score of >2 , relative to a 1 sec pre-exit ‘baseline’ ) , inhibitory ( converse of excitatory ) , or unaltered ( no significant increase or decrease ) . The proportion of units falling into each of these 3 categories was no different between vehicle and LM-4131 treated groups ( Figure 9C ) . However , the magnitude of the increase in excitatory unit activity on exiting the closed arm was significantly lower in LM-4131 treated mice than vehicle controls ( Figure 9D–E ) . Furthermore , the effect of the drug was specific to excitatory activity , with no change evident for units exhibiting inhibitory activity on closed arm exits ( Figure 9D ) . The finding that COX-2 inhibition blunted BLA unit excitation in vivo fits well with our ex vivo slice results and , taken together , suggest a neurophysiological correlate of the anxiolytic-like actions of LM-4131 .
Here we show that acute treatment with the novel SSCI , LM-4131 , as well as two traditional COX-2-selective inhibitors , LMX and Celecoxib , acutely reduces stress-induced behavioral dysregulation in two separate assays ( the NIH and EPM ) , and reduces expression of conditioned fear , without affecting locomotor activity , sucrose preference , or despair-like behaviors . These effects generalized to male mice of different ages , female mice , and were evident even after repeated stress exposure . Moreover , the behavioral effects of COX-2 inhibition were maintained after subchronic treatment indicating a lack of drug tolerance . Together , these results suggest that SSCIs and traditional COX-2 inhibitors could represent viable therapeutic approaches for stress-related neuropsychiatric disorders ( Muller , 2010 ) . The three COX-2 inhibitors used in the present study represent different chemical scaffolds: LM-4131 is a morpholinoamide of indomethacin , while LMX is an arylacetic acid , and Celecoxib is a diarylheterocycle . LM-4131 is also a weak antagonist of CB2R ( Gaetani et al . , 2003 ) ; however , CB2R activation ( Bahi et al . , 2014; Garcia-Gutierrez and Manzanares , 2011 ) , but not inhibition ( Gamble-George et al . , 2013 ) , has been shown to have anxiolytic-like effects , and we have previously shown that CB2R blockade does not affect anxiety-like behavior in the NIH assay ( Gamble-George et al . , 2013 ) . Indeed , we were unable to prevent the anxiolytic-like effects of LM-4131 with a CB2R antagonist . The similar anxiolytic-like profile of the two other chemically distinct COX-2 inhibitors , which have no known activity at cannabinoid receptors , also argues against a CB2R contribution to the anxiolytic effects of LM-4131 . In principle , COX-2 inhibition could produce anxiolytic and antidepressant actions via one or more mechanisms of action: 1 ) preventing PG synthesis , 2 ) increasing eCB levels , and 3 ) concomitantly reducing levels of prostamides and prostaglandin-glycerol esters ( COX-2 eCB oxygenation products ) . On the one hand , the prior observation that COX-2 inhibition is associated with reduced stress-induced PG levels , cytokine levels , and other markers of neuroinflammation supports the first mechanism ( Dhir et al . , 2006; Kumari et al . , 2007; Munhoz et al . , 2008 ) . On the other hand , the current results provide some support for the alternate mechanisms . For example , the SSCI , LM-4131 , does not affect production of pro-inflammatory PGs ( Hermanson et al . , 2013 ) , yet is as effective as traditional COX-2 inhibitors in reducing stress-induced anxiety-like behavior . In addition , SK channel blockade and to a lesser extent , CB1R antagonism , both of which are molecular targets of the eCB anandamide , block some behavioral effects of LM-4131 , LMX , and Celecoxib . Consistent with our data , systemic and intra-BLA SK channel activation has been shown to reduce stress-induced anxiety and expression of conditioned-fear ( Atchley et al . , 2012; Rau et al . , 2015 ) . Finally , it is also possible that reductions in prostamides or prostaglandin-glycerols contribute to the anxiolytic-like actions of traditional COX-2 inhibitors and especially SSCIs . However , very little is currently known regarding the physiological role of these metabolites in the regulation of stress responses or behavior in general . Future studies will thus be aimed at determining the differential contributions of these three potential mechanisms of action of COX-2 inhibitors to reduce stress-related pathology . At a cellular level , LM-4131 decreased amygdala neuron excitability in stressed , but not control , mice . This effect was mimicked by the FAAH inhibitor , PF-3843 , which causes robust increases in anandamide levels ( Lee et al . , 2015 ) . Taken together , these findings support the notion that LM-4131 affects amygdala cellular physiology indirectly via enhancing anandamide signaling . We also found the decrease in BLA neuron excitability induced by LM-4131 ex vivo was blocked by the SK channel antagonist Apamin , but not the CB1 or TRPV1 antagonists , mirroring our behavioral studies . It is unlikely that that the cellular effects of LM-4131 are mediated via decreases in PG signaling since our in vitro data indicate that LM-4131 is a very weak inhibitor of AA oxygenation by COX-2 ( Hermanson et al . , 2013 ) . However , it is remains possible that the neurophysiological effects of LM-4131 are mediated in part via inhibition of prostamide signaling; a hypothesis that will require further testing . We also investigated the in vivo neurophysiological correlates of LM-4131-induced anxiolytic-like effects . Consistent with our ex vivo electrophysiological results , in vivo single-unit recordings revealed that the anxiolytic effects of LM-4131 were associated with a significantly attenuated increase in BLA neuronal excitability that was evident when mice left a relatively safe location . Given the strong link between amygdala neuron activity and generation of anxiety and fear states ( Janak and Tye , 2015 ) , these data suggest that LM-4131 could reduce anxiety-like behavior by modulating encoding of negative valence by BLA neurons; though further studies will be required to substantiate this idea . Similarly , whether LM-4131 induced AEA augmentation , and subsequent SK channel activation , contributes to the reduction in BLA neuron activity in vivo remains to be determined . Several clinical trials and meta-analyses of trials have now confirmed the efficacy of COX-2 inhibition as an effective augmentation strategy to SSRIs in MDD ( Abdallah et al . , 2015; Akhondzadeh and Jafari , 2010; Akhondzadeh et al . , 2009; Faridhosseini et al . , 2014; Fond et al . , 2014; Muller et al . , 2006 ) . In addition , COX-2 mRNA is increased in the circulation of patients with MDD ( Galecki et al . , 2012 ) . These findings are generally consistent with the known role of COX-2 as an immediate-early gene up-regulated by neurons in response to various types of stress ( Kaufmann et al . , 1996; Kulmacz and Lands , 1984; Yamagata et al . , 1993 ) . On this basis , it has been proposed that stress exposure could increase susceptibility to depression via generation of inflammatory PGs generated by COX-2; a contention in line with the neuroimmune hypothesis of depression ( Felger and Miller , 2014; Haroon et al . , 2012; Miller et al . , 2009; Raison and Miller , 2013 ) and the aforementioned clinical efficacy of COX-2 inhibition in MDD . The current study extends this model by supporting additional mechanisms by which COX-2 could exert therapeutic effects; specifically , via eCB augmentation and possibly reductions in COX-2 eCB oxidative metabolites , which are known to have actions opposing eCBs in some systems ( Ligresti et al . , 2014; Sang et al . , 2006 ) . This alternate hypothesis is consistent with data suggesting eCB deficiencies could contribute to the pathogenesis of some anxiety disorders , including PTSD ( Hill and Patel , 2013; Jenniches et al . , 2015; Shonesy et al . , 2014 ) . Our preclinical data support robust effects of COX-2 inhibition in fear and anxiety-domain behaviors , but preliminary analysis of anhedonia and despair-like behavior were negative . Therefore , although anxiety symptoms are highly reported in patients with MDD ( Fava et al . , 2006; Fried et al . , 2015 ) , and anxiety disorders and MDD are highly co-morbid ( Kessler et al . , 2005 ) , our data suggest clinical trials of COX-2 inhibitors in anxiety and trauma- and stressor-related disorders may also be warranted , and that SSCIs could represent a potentially attractive class of novel therapeutics for mood and anxiety disorders .
Male or female ICR ( CD-1 ) mice between 5–7 weeks of age or ~4 months of age were used for all experiments ( Harlan , Indianapolis , IN , USA ) . All mice were housed on a 12:12 light-dark cycle ( lights on at 6:00 a . m . ) with food and water available ad libitum . All studies were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals , and approved by the Vanderbilt University Institutional Animal Care and Use Committee ( #M10/227 ) and by the NIAAA Animal Care and Use Committee ( #LBGN-AH-41 ) . All behavioral testing was performed during the inactive light phase of the mouse circadian cycle ( between 6:00 am and 6:00 pm ) . The drugs used were the COX-2 inhibitors , LM-4131 ( 10 mg/kg ) , Lumiracoxib ( 1 mg/kg ) , and Celecoxib ( 10 mg/kg ) , the SK channel antagonist , Apamin ( 0 . 4 and 0 . 8 mg/kg ) , the SK channel agonist , 1-EBIO ( 5 mg/kg ) , the CB1R antagonist , Rimonabant ( 2 and 5 mg/kg ) , the CB2R antagonist , SR144528 ( 3 mg/kg ) , and the TRPV1 antagonist , Capsazepine ( 10 mg/kg ) . Rimonabant and SR144528 were gifts from the National Institute of Mental Health Drug Supply Program . LM-4131 was synthesized as previously described ( Hermanson et al . , 2013 ) . Lumiracoxib was obtained from Selleck Chemicals ( Houston , TX , USA ) . Celecoxib and Capsazepine were from Cayman Chemical ( Ann Arbor , MI , USA ) . Apamin and 1-EBIO were from Tocris ( Minneapolis , MN , USA ) . Drugs or vehicle were administered by intraperitoneal injection at a volume of 1 mL/kg in DMSO . Drug pretreatment times were two hours prior to behavioral testing . Mice were moved to the test room for 1 hr habituation and placed in a 30 . 5 x 24 . 1 x 21 . 0 cm ( 175–177 lux ) chamber , which was cleaned in between testing with a chlorine dioxide solution ( Vimoba ) . After a 30-second habituation period , there were 6 x 0 . 7 mA foot-shocks separated by a 30-second interval , with each shock preceded by a 30-s tone ( 80 dB , 3000 Hz ) . Mice were then returned to their home cages and a holding room until further behavioral testing . Detailed methods for all behavioral tests are provided in the Supplemental Materials and methods section . NIH testing was carried out exactly as described previously ( Bluett et al . , 2014; Gamble-George et al . , 2013 ) . Individually housed mice were habituated to a novel , palatable food ( Ensure Homemade Vanilla Shake ) in their home cages for 30 min per day for 3 days under red light conditions ( 40 lux ) . The day after the final habituation session , mice were presented with the shake in their home cages and the latency to begin feeding and the amount consumed was measured . Mice were then exposed to foot-shock stress as described above and tested in the novel cage at 4 , 8 , 24 , or 72 hr after stress exposure depending on the experiment . Novel cage testing was conducted in a new cage , without bedding , and with a white paper placed under the cage using bright ambient lighting conditions ( 295 lux ) . Latency to feed in the novel cage and the amount of Ensure® consumed are reported . Drugs were administered 2 hr prior to novel cage testing . Open-field locomotor activity was analyzed and performed as previously described ( Gamble-George et al . , 2013 ) . The apparatus was elevated 47 cm from the ground , and consisted of 2 open arms ( 30 × 5 cm; 90 lux ) and 2 closed arms ( 30 × 5 × 15 cm; 20 lux ) extending from a 5 × 5 cm central area ( San Diego Instruments , San Diego , CA , USA ) . The walls and floor were made from black ABS ( acrylonitrile butadiene styrene ) plastic . To begin the test , each mouse was placed in the center , facing an open arm . Time spent , latency to enter , and entries into the open and closed arms , latency to closed arm exit , latency to freeze , and total distance traveled were recorded using CinePlex Behavioral Research System ( Plexon Inc , Dallas , TX , USA ) for chronic in vivo BLA unit recordings , or , for all other experiments , Any Maze tracking software . The mouse was considered to be in an arm when all four paws were in the arm . The test was 5 min in duration , with the exception of the chronic in vivo BLA unit recording experiment , which was 10 min in length . Mice were conditioned using the stress exposure procedure described above . Six hours later , mice were administered either vehicle or LM-4131 ( 10 mg/kg ) . Two hours later and again 16 hr later , mice were returned to the conditioned chamber for a 10-minute test . The VideoFreeze system ( Med Associates ) was used to measure freezing: defined as no movement other than breathing and measured when mouse movement fell below a preset motion threshold of 18 . The TST was performed as previously described ( Gamble-George et al . , 2013 ) . Each mouse was given twenty-four hours to choose freely between a 2% sucrose solution and tap water in 50 mL clear plastic conical tubes . To prevent side preference bias , the position of the two tubes was switched at every measurement of the amount of fluid consumed ( e . g . , 2–4 hr ) . No previous water or food deprivation was applied before testing . The consumption of the 2% sucrose solution or water was measured by weighing the conical tubes containing the fluids . Percentage sucrose preference was calculated as the ratio of 2% sucrose consumed over total fluid consumption multiplied by 100 . Mice were anesthetized with 2% Isoflurane ( Baxter Healthcare , Deerfield , IL , USA ) and implanted with 2 × 8 electrode ( 35-µm ) tungsten micro-arrays ( Innovative Neurophysiology Inc , Durham , NC , USA ) bilaterally targeted at the BLA ( 3 . 1 mm lateral , 1 . 2–2 . 2 mm posterior , 4 . 5 mm ventral relative to bregma ) . Following surgery , mice were individually housed and allowed at least 1 week to recover prior to behavioral testing . Animals were exposed to stress as described above . Five hours after stress exposure , mice received a pre-drug baseline recording session in its home cage . Individual units were identified and recorded for 5 min using the Plexon Omniplex Neural Data Acquisition System . One hour later , mice were administered either LM-4131 ( 10 mg/kg ) or vehicle and , 2 hr later , received a 5-minute post-drug recording session in the home cage . Immediately following this test , mice were assessed on the EPM as described above . Neural data was sorted using the Plexon Offline Sorter and analyzed using NeuroExplorer 5 . 0 ( Nex Technologies , Madison , AL , USA ) . For analysis of unit recording data , waveforms were isolated manually using principal component analysis ( Offline Sorter , Plexon Inc ) . To be included in the analyses , spikes had to exhibit a refractory period of at least 1 millisecond . Autocorrelograms from simultaneously recorded units were examined to ensure that no cell was counted twice . Each unit’s activity was sorted into 100 millisecond bins , time-locked to exits from the safety of the closed arm and z-scored to average activity during a 1-s pre-exit baseline . Units were classified as either 1 ) excitatory , if 1 or more post-exit bins during 1-s post-exit exceeded 2 z-scores , 2 ) inhibitory , if 1 or more post-exit bins was less than 2 z-scores , or 3 ) non-responsive , if no post-exit bin was > or < 2 z-scores . Analysis of arm entry responsiveness included 67 cells in vehicle-treated animals , and 72 cells in LM-4131-treated animals . Data reported for raw firing rates included only putative principal neurons; any unit with a baseline firing rate over 7 Hz was excluded ( 5 vehicle and 3 LM-4131; see [Likhtik et al . , 2006] ) . Brain slice preparation and whole-cell patch clamp recordings were performed exactly as described previously ( Ramikie et al . , 2014; Shonesy et al . , 2014; Sumislawski et al . , 2011 ) . Statistical analysis of single unit data is above . For all other experiments , statistical significance was calculated by two-tailed unpaired t-test or Kolmogorov-Smirnov test ( only for frequency distribution plots ) , or one-way or two-way ANOVA with post hoc Holm-Sidak’s multiple comparisons test as noted in figure legends . All statistical analyses were conducted using Prism Graphpad 6 ( San Diego , CA , USA ) . No a priori power analysis was conducted; however , minimum sample sized were based on our published experience with behavioral and physiological assays used . All experiments were performed in 2 or more independent cohorts of animals except in vivo electrophysiological studies . For behavioral studies , all replicates ( n values ) represent biological replicates defined as data derived from a single mouse . Data are presented as mean ± S . E . M . unless otherwise stated in the figure legends . P<0 . 05 was considered significant throughout . Grubb’s test was used to remove outliers .
Mood and anxiety disorders are the most prevalent psychiatric conditions and are exacerbated by stress . Recent studies have suggested cyclooxygenase-2 ( COX-2 ) inhibition could represent a novel treatment approach or augmentation strategy for affective disorders including anxiety disorders and major depression . We show that traditional COX-2 inhibitors and a newly developed substrate-selective COX-2 inhibitor ( SSCI ) reduce a variety of stress-induced behavioral pathologies in mice . We found that these behavioral effects were associated with a dampening of neuronal excitability in the basolateral amygdala ( BLA ) ex vivo and in vivo , and were mediated by small-conductance calcium-activated potassium ( SK ) channel and CB1 cannabinoid receptor activation . Taken together , these data provide further support for the potential utility of SSCIs , as well as traditional COX-2 inhibitors , as novel treatment approaches for stress-related psychiatric disorders .
People may experience a stressful or traumatic event that can cause anxiety in their day-to-day lives . For some people , this anxiety can persist and develop into a disorder , such as posttraumatic stress disorder ( PTSD ) or acute stress disorder . Millions of people worldwide suffer from these disorders , but there are few classes of drug treatments currently available . Some preliminary studies in mice have suggested that anti-inflammatory drugs called cyclooxygenase-2 ( COX-2 ) inhibitors may relieve symptoms of anxiety . COX-2 inhibitors target an enzyme called cyclooxygenase-2 , which is produced in many tissues throughout the body and brain . Some COX-2 inhibitors , such as Celebrex , are already widely used to treat arthritis . Studies in people have also suggested these drugs given in combination with antidepressants might also help people with depression , while a recently developed COX-2 inhibitor , called LM-4131 , stops the breakdown of chemicals produced in the brain that interact with the same brain receptors that marijuana does . In animal studies , this new drug reduced anxious behaviors . However , scientists didn’t know if the drug would also reduce anxious behaviors after animals were exposed to a traumatic or stressful experience . Now , Gamble-George et al . – including some of the same scientists that created LM-4131 – show this drug does reduce anxious behavior in mice after a traumatic or stressful experience . In the experiments , mice were stressed with an electric shock to their feet and then treated with LM-4131 , Celebrex , another COX-2 inhibitor sometimes used in humans , or an inactive control . The mice were then placed in new environments or mazes high off the ground that usually trigger anxious behavior . The mice treated with any of the three COX-2 inhibitors were less likely to act like they were anxious than the mice treated with the control solution . Gamble-George et al . then showed that LM-4131 changes how cells in the brain become excited in both isolated mouse brain cells and live mice . Short-term stress in humans is known to excite cells in an area of the brain called the amygdala , which controls how we respond to stress or trauma . The experiments showed that LM-4131 decreases this activity and decreases anxiety in stressed mice . Future animal and human studies are needed to confirm that COX-2 inhibitors are a useful treatment for stress disorders , and learn more about how they work .