Skip to main content

Sex differences in specific aspects of two animal tests of anxiety-like behavior

Abstract

Rationale

Anxiety, a negative state of high arousal and vigilance, is especially prevalent in women, making identification of underlying mechanisms critical for developing effective therapies. With the challenge of disentangling biological and social factors in humans, animal tests can provide valuable insights, although such tests, developed in males, have unclear validity for females.

Objective

To better understand patterns of sex differences across multiple measures within two classical rodent anxiety tests.

Methods

We examined female and male adult Wistar rats (n = 15–18/group) that were single-housed in the novelty suppression of feeding test (NSFT) that involves food under a bright light in food-restricted animals, and light–dark test (LDT), which reflects innate aversion to bright light. To further validate these tests in females, we also examined the impact of 1 mg/kg diazepam.

Results

NSFT measures of the most direct interaction with food, latency to grab food and food consumed, indicated increased anxiety-like behavior in females versus males, with diazepam altering these behaviors in females but not males. Most other measures showed more similar effects of diazepam across the sexes, with some evidence of reduced anxiety-like behavior in LDT for females. Principal component analyses indicated limited relationships across behavioral factors, underscoring previous suggestions of the importance of assessing multiple measures to maximize information and ethological relevance.

Conclusions

Combining our findings and previous studies, we speculate that increased anxiety-like behavior in females manifests especially when there is a specific, life-relevant condition (e.g., food in the NSFT). Our findings also validate NSFT and LDT use in females.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Abramov U, Raud S, Koks S, Innos J, Kurrikoff K, Matsui T, Vasar E (2004) Targeted mutation of CCK(2) receptor gene antagonises behavioural changes induced by social isolation in female, but not in male mice. Behav Brain Res 155:1–11

    CAS  PubMed  Article  Google Scholar 

  • Acevedo MB, Nizhnikov ME, Molina JC, Pautassi RM (2014) Relationship between ethanol-induced activity and anxiolysis in the open field, elevated plus maze, light-dark box, and ethanol intake in adolescent rats. Behav Brain Res 265:203–215

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Albonetti ME, Farabollini F (1995) Effects of single restraint on the defensive behavior of male and female rats. Physiol Behav 57:431–437

    CAS  PubMed  Article  Google Scholar 

  • Albrechet-Souza L, Schratz CL, Gilpin NW (2020) Sex differences in traumatic stress reactivity in rats with and without a history of alcohol drinking. Biol Sex Differ 11:27–38

  • Altemus M, Sarvaiya N, Neill EC (2014) Sex differences in anxiety and depression clinical perspectives. Front Neuroendocrinol 35:320–330

    PubMed  PubMed Central  Article  Google Scholar 

  • Amodeo LR, Wills DN, Sanchez-Alavez M, Nguyen W, Conti B, Ehlers CL (2018) Intermittent voluntary ethanol consumption combined with ethanol vapor exposure during adolescence increases drinking and alters other behaviors in adulthood in female and male rats. Alcohol 73:57–66

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Bangasser DA, Wicks B (2017) Sex-specific mechanisms for responding to stress. J Neurosci Res 95:75–82

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Baratta MV, Leslie NR, Fallon IP, Dolzani SD, Chun LE, Tamalunas AM, Watkins LR, Maier SF (2018) Behavioural and neural sequelae of stressor exposure are not modulated by controllability in females. Eur J Neurosci 47:959–967

    PubMed  PubMed Central  Article  Google Scholar 

  • Becker JB, Chartoff E (2019) Sex differences in neural mechanisms mediating reward and addiction. Neuropsychopharm 44:166–183

    CAS  Article  Google Scholar 

  • Belviranli M, Atalik KE, Okudan N, Gokbel H (2012) Age and sex affect spatial and emotional behaviors in rats: the role of repeated elevated plus maze test. Neuroscience 227:1–9

    CAS  PubMed  Article  Google Scholar 

  • Blasco-Serra A, Gonzalez-Soler EM, Cervera-Ferri A, Teruel-Marti V, Valverde-Navarro AA (2017) A standardization of the novelty-suppressed feeding test protocol in rats. Neurosci Lett 658:73–78

    CAS  PubMed  Article  Google Scholar 

  • Bodnoff SR, Suranyi-Cadotte B, Quirion R, Meaney MJ (1989) A comparison of the effects of diazepam versus several typical and atypical anti-depressant drugs in an animal model of anxiety. Psychopharm 97:277–279

    CAS  Article  Google Scholar 

  • Bourin M, Hascoet M (2003) The mouse light/dark box test. Eur J Pharmacol 463:55–65

    CAS  PubMed  Article  Google Scholar 

  • Chaouloff F, Durand M, Mormede P (1997) Anxiety- and activity-related effects of diazepam and chlordiazepoxide in the rat light/dark and dark/light tests. Behav Brain Res 85:27–35

    CAS  PubMed  Article  Google Scholar 

  • Chisholm D, Sweeny K, Sheehan P, Rasmussen B, Smit F, Cuijpers P, Saxena S (2016) Scaling-up treatment of depression and anxiety: a global return on investment analysis. Lancet Psychiatry 3:415–424

    PubMed  Article  Google Scholar 

  • Dalla C, Antoniou K, Drossopoulou G, Xagoraris M, Kokras N, Sfikakis A, Papadopoulou-Daifoti Z (2005) Chronic mild stress impact: are females more vulnerable? Neuroscience 135:703–714

    CAS  PubMed  Article  Google Scholar 

  • De Jesus-Burgos MI, Gonzalez-Garcia S, Cruz-Santa Y, Perez-Acevedo NL (2016) Amygdalar activation of group I metabotropic glutamate receptors produces anti- and pro-conflict effects depending upon animal sex in a sexually dimorphic conditioned conflict-based anxiety model. Behav Brain Res 302:200–212

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Donner NC, Lowry CA (2013) Sex differences in anxiety and emotional behavior. Pflugers Arch 465:601–626

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Du Preez A, Law T, Onorato D, Lim YM, Eiben P, Musaelyan K, Egeland M, Hye A, Zunszain PA, Thuret S, Pariante CM, Fernandes C (2020) The type of stress matters: repeated injection and permanent social isolation stress in male mice have a differential effect on anxiety- and depressive-like behaviours, and associated biological alterations. Transl Psychiatry 10:325–242

  • Fernandes C, Gonzalez MI, Wilson CA, File SE (1999) Factor analysis shows that female rat behaviour is characterized primarily by activity, male rats are driven by sex and anxiety. Pharmacol Biochem Behav 64:731–738

    CAS  PubMed  Article  Google Scholar 

  • Fleming W, Jones Q, Chandra U, Saini A, Walker D, Francis R, Ocampo G, Kuhn C (2019) Withdrawal from brief repeated alcohol treatment in adolescent and adult male and female rats. Alcohol Clin Exp Res 43:204–211

    CAS  PubMed  Article  Google Scholar 

  • Gobinath AR, Wong S, Chow C, Lieblich SE, Barr AM, Galea LAM (2018) Maternal exercise increases but concurrent maternal fluoxetine prevents the increase in hippocampal neurogenesis of adult offspring. Psychoneuroendo 91:186–197

    CAS  Article  Google Scholar 

  • Gonzalez LE, File SE (1997) A five minute experience in the elevated plus-maze alters the state of the benzodiazepine receptor in the dorsal raphe nucleus. J Neurosci 17:1505–1511

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Griebel G, Perrault G, Sanger DJ (1997) CCK receptor antagonists in animal models of anxiety: comparison between exploration tests, conflict procedures and a model based on defensive behaviours. Behav Pharmacol 8:549–560

    CAS  PubMed  Article  Google Scholar 

  • Heinrich LM, Gullone E (2006) The clinical significance of loneliness: a literature review. Clin Psychol Rev 26:695–718

    PubMed  Article  Google Scholar 

  • Henricks AM, Berger AL, Lugo JM, Baxter-Potter LN, Bieniasz KV, Petrie G, Sticht MA, Hill MN, McLaughlin RJ (2017) Sex- and hormone-dependent alterations in alcohol withdrawal-induced anxiety and corticolimbic endocannabinoid signaling. Neuropharm 124:121–133

    CAS  Article  Google Scholar 

  • Homberg JR, Olivier JD, Blom T, Arentsen T, van Brunschot C, Schipper P, Korte-Bouws G, van Luijtelaar G, Reneman L (2011) Fluoxetine exerts age-dependent effects on behavior and amygdala neuroplasticity in the rat. PLoS One 6:e16646–e16656

  • Hughes RN (2011) Adult anxiety-related behavior of rats following consumption during late adolescence of alcohol alone and in combination with caffeine. Alcohol 45:365–372

    CAS  PubMed  Article  Google Scholar 

  • Jaric I, Rocks D, Cham H, Herchek A, Kundakovic M (2019) Sex and estrous cycle effects on anxiety- and depression-related phenotypes in a two-hit developmental stress model. Front Mol Neurosci 12:74–89

  • Johnston AL, File SE (1991) Sex differences in animal tests of anxiety. Physiol Behav 49:245–250

    CAS  PubMed  Article  Google Scholar 

  • Keeley RJ, Bye C, Trow J, McDonald RJ (2015) Strain and sex differences in brain and behaviour of adult rats: learning and memory, anxiety and volumetric estimates. Behav Brain Res 288:118–131

    CAS  PubMed  Article  Google Scholar 

  • Kokras N, Dioli C, Paravatou R, Sotiropoulos MG, Delis F, Antoniou K, Calogeropoulou T, Charalampopoulos I, Gravanis A, Dalla C (2020) Psychoactive properties of BNN27, a novel neurosteroid derivate, in male and female rats. Psychopharm 237:2435–2449

    CAS  Article  Google Scholar 

  • Kosobud AE, Wetherill L, Plawecki MH, Kareken DA, Liang T, Nurnberger JL, Windisch K, Xuei X, Edenberg HJ, Foroud TM, O’Connor SJ (2015) Adaptation of subjective responses to alcohol is affected by an interaction of GABRA2 genotype and recent drinking. Alcohol Clin Exp Res 39:1148–1157

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Le Moene O, Ramirez-Renteria ML, Agmo A (2020) Male and female immediate fear reaction to white noise in a semi-natural environment: a detailed behavioural analysis of the role of sex and oestrogen receptors. J Neuroendocrinol 32:e12902–e12921

  • Lopez-Aumatell R, Martinez-Membrives E, Vicens-Costa E, Canete T, Blazquez G, Mont-Cardona C, Johannesson M, Flint J, Tobena A, Fernandez-Teruel A (2011) Effects of environmental and physiological covariates on sex differences in unconditioned and conditioned anxiety and fear in a large sample of genetically heterogeneous (N/Nih-HS) rats. Behav Brain Funct 7:48–63

  • Martin AL, Brown RE (2010) The lonely mouse: verification of a separation-induced model of depression in female mice. Behav Brain Res 207:196–207

    CAS  PubMed  Article  Google Scholar 

  • Mead H, Andres E, Katch H, Siegel B, Regenstein M (2010) Gender differences in psychosocial issues affecting low-income, underserved patients’ ability to manage cardiovascular disease. Womens Health Issues 20:308–315

    PubMed  Article  Google Scholar 

  • Miragaia AS, de Oliveira Wertheimer GS, Consoli AC, Cabbia R, Longo BM, Girardi CEN, Suchecki D (2018) Maternal deprivation increases anxiety- and depressive-like behaviors in an age-dependent fashion and reduces neuropeptide Y expression in the amygdala and hippocampus of male and female young adult rats. Front Behav Neurosci 12:159–176

  • Oh JE, Zupan B, Gross S, Toth M (2009) Paradoxical anxiogenic response of juvenile mice to fluoxetine. Neuropsychopharm 34:2197–2207

    CAS  Article  Google Scholar 

  • Olivier JD, Van Der Hart MG, Van Swelm RP, Dederen PJ, Homberg JR, Cremers T, Deen PM, Cuppen E, Cools AR, Ellenbroek BA (2008) A study in male and female 5-HT transporter knockout rats: an animal model for anxiety and depression disorders. Neuroscience 152:573–584

    CAS  PubMed  Article  Google Scholar 

  • Olvera-Hernandez S, Fernandez-Guasti A (2011) Sex differences in the burying behavior test in middle-aged rats: effects of diazepam. Pharmacol Biochem Behav 99:532–539

    CAS  PubMed  Article  Google Scholar 

  • Ou C, Dringenberg HC, Soutar CN (2019) Is hippocampal theta frequency related to individual and sex differences in anxiety-like behaviour? An analysis in male and female Long-Evans rats. Behav Brain Res 364:366–373

    PubMed  Article  Google Scholar 

  • Palanza P, Gioiosa L, Parmigiani S (2001) Social stress in mice: gender differences and effects of estrous cycle and social dominance. Physiol Behav 73:411–420

    CAS  PubMed  Article  Google Scholar 

  • Palanza P, Parmigiani S (2017) How does sex matter? Behavior, stress and animal models of neurobehavioral disorders. Neurosci Biobehav Rev 76:134–143

    PubMed  Article  Google Scholar 

  • Pereira JK, Vieira RJ, Konishi CT, Ribeiro RA, Frussa-Filho R (1999) The phenomenon of “one-trial tolerance” to the anxiolytic effect of chlordiazepoxide in the elevated plus-maze is abolished by the introduction of a motivational conflict situation. Life Sci 65:PL101–7.

  • Pisu MG, Garau A, Boero G, Biggio F, Pibiri V, Dore R, Locci V, Paci E, Porcu P, Serra M (2016) Sex differences in the outcome of juvenile social isolation on HPA axis function in rats. Neuroscience 320:172–182

    CAS  PubMed  Article  Google Scholar 

  • Pittman DW, McGinnis MR, Richardson LM, Miller EJ, Alimohamed ML, Baird JP (2012) Multiple processes underlie benzodiazepine-mediated increases in the consumption of accepted and avoided stimuli. Chem Senses 37:431–444

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Pomrenze MB, Millan EZ, Hopf FW, Keiflin R, Maiya R, Blasio A, Dadgar J, Kharazia V, De Guglielmo G, Crawford E, Janak PH, George O, Rice KC, Messing RO (2015) A transgenic rat for investigating the anatomy and function of corticotrophin releasing factor circuits. Front Neurosci 9:487–501

  • Ramaker MJ, Dulawa SC (2017) Identifying fast-onset antidepressants using rodent models. Mol Psychiatry 22:656–665

    CAS  PubMed  Article  Google Scholar 

  • Ramos A (2008) Animal models of anxiety: do I need multiple tests? Trends Pharmacol Sci 29:493–498

    CAS  PubMed  Article  Google Scholar 

  • Roman E, Arborelius L (2009) Male but not female Wistar rats show increased anxiety-like behaviour in response to bright light in the defensive withdrawal test. Behav Brain Res 202:303–307

    PubMed  Article  Google Scholar 

  • Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, Weisstaub N, Lee J, Duman R, Arancio O, Belzung C, Hen R (2003) Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 301:805–809

    CAS  PubMed  Article  Google Scholar 

  • Scholl JL, Afzal A, Fox LC, Watt MJ, Forster GL (2019) Sex differences in anxiety-like behaviors in rats. Physiol Behav 211:112670–112678

  • Shephard RA, Broadhurst PL (1982) Effects of diazepam and picrotoxin on hyponeophagia in rats. Neuropharm 21:771–773

    CAS  Article  Google Scholar 

  • Shepherd JK, Flores T, Rodgers RJ, Blanchard RJ, Blanchard DC (1992) The anxiety/defense test battery: influence of gender and ritanserin treatment on antipredator defensive behavior. Physiol Behav 51:277–285

    CAS  PubMed  Article  Google Scholar 

  • Sheynin J, Beck KD, Pang KC, Servatius RJ, Shikari S, Ostovich J, Myers CE (2014) Behaviourally inhibited temperament and female sex, two vulnerability factors for anxiety disorders, facilitate conditioned avoidance (also) in humans. Behav Processes 103:228–235

    PubMed  PubMed Central  Article  Google Scholar 

  • Slavich GM, Sacher J (2019) Stress, sex hormones, inflammation, and major depressive disorder: Extending Social Signal Transduction Theory of Depression to account for sex differences in mood disorders. Psychopharm 236:3063–3079

    CAS  Article  Google Scholar 

  • Vicente MA, Zangrossi H Jr (2014) Involvement of 5-HT2C and 5-HT1A receptors of the basolateral nucleus of the amygdala in the anxiolytic effect of chronic antidepressant treatment. Neuropharm 79:127–135

    CAS  Article  Google Scholar 

  • Wegner SA, Hu B, De Oliveira Sergio T, Darevsky D, Kwok CC, Lei K, Hopf FW (2019) A novel NMDA receptor-based intervention to suppress compulsion-like alcohol drinking. Neuropharm 157:107681–107688

Download references

Acknowledgements

Thanks to Molly Sazer-Hopf for assistance with Fig. 1.

Funding

Work was supported by National Institutes of Health (R01 AA024109 to FWH).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Frederic W. Hopf.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary File 1 (PDF 999 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

De Oliveira Sergio, T., Wetherill, L., Kwok, C. et al. Sex differences in specific aspects of two animal tests of anxiety-like behavior. Psychopharmacology 238, 2775–2787 (2021). https://doi.org/10.1007/s00213-021-05893-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00213-021-05893-w

Keywords

  • Sex difference
  • Anxiety
  • Females
  • Novelty-suppressed feeding
  • Light–dark box
  • Diazepam