Quantitative Assessment of Anti-Gravity Reflexes to Evaluate Vestibular Dysfunction in Rats

  • Vanessa Martins-Lopes
  • Anna Bellmunt
  • Erin A. Greguske
  • Alberto F. Maroto
  • Pere Boadas-Vaello
  • Jordi LlorensEmail author
Research Article


The tail-lift reflex and the air-righting reflex are anti-gravity reflexes in rats that depend on vestibular function. To obtain objective and quantitative measures of performance, we recorded these reflexes with slow-motion video in two experiments. In the first experiment, vestibular dysfunction was elicited by acute exposure to 0 (control), 400, 600, or 1000 mg/kg of 3,3′-iminodipropionitrile (IDPN), which causes dose-dependent hair cell degeneration. In the second, rats were exposed to sub-chronic IDPN in the drinking water for 0 (control), 4, or 8 weeks; this causes reversible or irreversible loss of vestibular function depending on exposure time. In the tail-lift test, we obtained the minimum angle defined during the lift and descent maneuver by the nose, the back of the neck, and the base of the tail. In the air-righting test, we obtained the time to right the head. We also obtained vestibular dysfunction ratings (VDRs) using a previously validated behavioral test battery. Each measure, VDR, tail-lift angle, and air-righting time demonstrated dose-dependent loss of vestibular function after acute IDPN and time-dependent loss of vestibular function after sub-chronic IDPN. All measures showed high correlations between each other, and maximal correlation coefficients were found between VDRs and tail-lift angles. In scanning electron microscopy evaluation of the vestibular sensory epithelia, the utricle and the saccule showed diverse pathological outcomes, suggesting that they have a different role in these reflexes. We conclude that these anti-gravity reflexes provide useful objective and quantitative measures of vestibular function in rats that are open to further development.


vestibular assessment tail-lift reflex test air-righting reflex test rat ototoxicity 3,3′-iminodipropionitrile 



The scanning electron microscopy studies were performed at the Scientific and Technological Centers of the University of Barcelona (CCiT-UB). We thank Josep M. Rebled and Eva Parts for technical assistance. We also thank Meritxell Deulofeu, Sílvia Prades and Adrià Ricarte for their contributions to the study as part of their final degree projects.

Funding Information

This study was supported by grants BFU2015-66109-R (Ministerio de Economia y Competitividad, MINECO/FEDER, EU) and 2017 SGR 621 (Agència de Gestió d’Ajuts Universitaris i de Recerca, Generalitat de Catalunya). E.A.G. was supported by the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (FI-DGR 2015 Program) and by the Ministerio de Educación, Cultura y Deporte de España (FPU 2015).

Compliance with Ethical Standards

The use of the animals was in accordance with EU Directive 2010/63 as implemented by Law 5/1995 and Act 214/1997 of the Generalitat de Catalunya, and Law 6/2013 and Act 53/2013 of the Gobierno de España. The experiments were approved by the Ethics Committee on Animal Experimentation of the Universitat de Barcelona.

Supplementary material

10162_2019_730_MOESM6_ESM.docx (18 kb)
ESM 6 (DOCX 16 kb)
10162_2019_730_MOESM7_ESM.docx (35 kb)
ESM 7 (DOCX 27 kb)
10162_2019_730_MOESM8_ESM.r (12 kb)
ESM 8 (R 12 kb)


  1. Allum JHJ, Carpenter MG (2013) Postural control and the vestibulospinal system. In: In: Bronstein AM (ed) Oxford textbook of vertigo and imbalance. Oxford University Press, Oxford, pp 35–48Google Scholar
  2. Basaldella E, Takeoka A, Sigrist M, Arber S (2015) Multisensory signaling shapes vestibulo-motor circuit specificity. Cell 163:301–312PubMedGoogle Scholar
  3. Beraneck M, Bojados M, Le Séac'h A, Jamon M, Vidal PP (2012) Ontogeny of mouse vestibulo-ocular reflex following genetic or environmental alteration of gravity sensing. PLoS One 7:e40414PubMedPubMedCentralGoogle Scholar
  4. Besnard S, Lopez C, Brandt T, Denise P, Smith PF (2015) Editorial: the vestibular system in cognitive and memory processes in mammalians. Front Integr Neurosci 9:55PubMedPubMedCentralGoogle Scholar
  5. Boadas-Vaello P, Riera J, Llorens J (2005) Behavioral and pathological effects in the rat define two groups of neurotoxic nitriles. Toxicol Sci 88:456–466PubMedGoogle Scholar
  6. Brown DJ, Pastras CJ, Curthoys IS (2017) Electrophysiological measurements of peripheral vestibular function-a review of electrovestibulography. Front Syst Neurosci 11:34PubMedPubMedCentralGoogle Scholar
  7. Chalansonnet M, Carreres-Pons M, Venet T, Thomas A, Merlen L, Seidel C, Cosnier F, Nunge H, Pouyatos B, Llorens J, Campo P (2018) Combined exposure to carbon disulfide and low-frequency noise reversibly affects vestibular function. Neurotoxicology 67:270–278PubMedGoogle Scholar
  8. Crofton KM, Janssen R, Prazma J, Pulver S, Barone S Jr (1994) The ototoxicity of 3,3′-iminodipropionitrile: functional and morphological evidence of cochlear damage. Hear Res 80:129–140PubMedGoogle Scholar
  9. Curthoys IS, Grant JW, Burgess AM, Pastras CJ, Brown DJ, Manzari L (2018) Otolithic receptor mechanisms for vestibular-evoked myogenic potentials: a review. Front Neurol 9:366PubMedPubMedCentralGoogle Scholar
  10. Curthoys IS, MacDougall HG, Vidal P-P, de Waele C (2017) Sustained and transient vestibular systems: a physiological basis for interpreting vestibular function. Front Neurol 8:117PubMedPubMedCentralGoogle Scholar
  11. De Jeu M, De Zeeuw CI (2012) Video-oculography in mice. J Vis Exp 65:e3971Google Scholar
  12. Dyhrfjeld-Johnsen J, Gaboyard-Niay S, Broussy A, Saleur A, Brugeaud A, Chabbert C (2013) Ondansetron reduces lasting vestibular deficits in a model of severe peripheral excitotoxic injury. J Vestib Res 23:177–186PubMedGoogle Scholar
  13. Gaboyard-Niay S, Travo C, Saleur A, Broussy A, Brugeaud A, Chabbert C (2016) Correlation between afferent rearrangements and behavioral deficits after local excitotoxic insult in the mammalian vestibule: a rat model of vertigo symptoms. Dis Model Mech 9:1181–1192PubMedPubMedCentralGoogle Scholar
  14. Greguske EA, Carreres-Pons M, Cutillas B, Boadas-Vaello P, Llorens J (2019) Calyx junction dismantlement and synaptic uncoupling precede hair cell extrusion in the vestibular sensory epithelium during sub-chronic 3,3′-iminodipropionitrile ototoxicity in the mouse. Arch Toxicol 93:417–434PubMedGoogle Scholar
  15. Hunt MA, Miller SW, Nielson HC, Horn KM (1987) Intratympanic injections of sodium arsanilate (atoxil) solution results in postural changes consistent with changes described for labyrinthectomized rats. Behav Neurosci 101:427–428PubMedGoogle Scholar
  16. Imai T, Takimoto Y, Takeda N, Uno A, Inohara H, Shimada S (2016) High-speed video-oculography for measuring three-dimensional rotation vectors of eye movements in mice. PLoS One 11:e0152307PubMedPubMedCentralGoogle Scholar
  17. Jones SM, Jones TA (2014) Genetics of peripheral vestibular dysfunction: lessons from mutant mouse strains. J Am Acad Audiol 25:289–301PubMedPubMedCentralGoogle Scholar
  18. Jones TA, Jones SM, Vijayakumar S, Brugeaud A, Bothwell M, Chabbert C (2011) The adequate stimulus for mammalian linear vestibular evoked potentials (VsEPs). Hear Res 280:133–140PubMedGoogle Scholar
  19. King EB, Shepherd RK, Brown DJ, Fallon JB (2017) Gentamicin applied to the oval window suppresses vestibular function in Guinea pigs. J Assoc Res Otolaryngol 18:291–299PubMedPubMedCentralGoogle Scholar
  20. Llorens J, Callejo A, Greguske EA, Maroto AF, Cutillas B, Martins-Lopes V (2018) Physiological assessment of vestibular function and toxicity in humans and animals. Neurotoxicology 66:204–212PubMedGoogle Scholar
  21. Llorens J, Demêmes D (1994) Hair cell degeneration resulting from 3,3′-iminodipropionitrile toxicity in the rat vestibular epithelia. Hear Res 76:78–86PubMedGoogle Scholar
  22. Llorens J, Demêmes D, Sans A (1993) The behavioral syndrome caused by 3,3′-iminodipropionitrile and related nitriles in the rat is associated with degeneration of the vestibular sensory hair cells. Toxicol Appl Pharmacol 123:199–210PubMedGoogle Scholar
  23. Llorens J, Rodríguez-Farré E (1997) Comparison of behavioral, vestibular, and axonal effects of subchronic IDPN in the rat. Neurotoxicol Teratol 19:117–127PubMedGoogle Scholar
  24. Lo WC, Chang CM, Liao LJ, Wang CT, Young YH, Chang YL, Cheng PW (2015) Assessment of D-methionine protecting cisplatin-induced otolith toxicity by vestibular-evoked myogenic potential tests, ATPase activities and oxidative state in Guinea pigs. Neurotoxicol Teratol 51:12–20PubMedGoogle Scholar
  25. Luebke AE, Holt JC, Jordan PM, Wong YS, Caldwell JS, Cullen KE (2014) Loss of α-calcitonin gene-related peptide (αCGRP) reduces the efficacy of the vestibulo-ocular reflex (VOR). J Neurosci 34:10453–10458PubMedPubMedCentralGoogle Scholar
  26. Luxa N, Salanova M, Schiffl G, Gutsmann M, Besnard S, Denise P, Clarke A, Blottner D (2013) Increased myofiber remodelling and NFATc1-myonuclear translocation in rat postural skeletal muscle after experimental vestibular deafferentation. J Vestib Res 23:187–193PubMedGoogle Scholar
  27. Ossenkopp KP, Prkacin A, Hargreaves EL (1990) Sodium arsanilate-induced vestibular dysfunction in rats: effects on open-field behavior and spontaneous activity in the automated digiscan monitoring system. Pharmacol Biochem Behav 36:875–881PubMedGoogle Scholar
  28. Pasquet MO, Tihy M, Gourgeon A, Pompili MN, Godsil BP, Léna C, Dugué GP (2016) Wireless inertial measurement of head kinematics in freely-moving rats. Sci Rep 6:35689PubMedPubMedCentralGoogle Scholar
  29. Pellis SM, Pellis VC, Morrissey TK, Teitelbaum P (1989) Visual modulation of vestibularly-triggered air-righting in the rat. Behav Brain Res 35:23–26PubMedGoogle Scholar
  30. Russell NA, Horii A, Smith PF, Darlington CL, Bilkey DK (2003) Long-term effects of permanent vestibular lesions on hippocampal spatial firing. J Neurosci 23:6490–6498PubMedPubMedCentralGoogle Scholar
  31. Saldaña-Ruíz S, Boadas-Vaello P, Sedó-Cabezón L, Llorens J (2013) Reduced systemic toxicity and preserved vestibular toxicity following co-treatment with nitriles and CYP2E1 inhibitors: a mouse model for hair cell loss. J Assoc Res Otolaryngol 14:661–671PubMedPubMedCentralGoogle Scholar
  32. Schlecker C, Praetorius M, Brough DE, Presler RG Jr, Hsu C, Plinkert PK, Staecker H (2011) Selective atonal gene delivery improves balance function in a mouse model of vestibular disease. Gene Ther 18:884–890PubMedPubMedCentralGoogle Scholar
  33. Sedó-Cabezón L, Jedynak P, Boadas-Vaello P, Llorens J (2015) Transient alteration of the vestibular calyceal junction and synapse in response to chronic ototoxic insult in rats. Dis Model Mech 8:1323–1337PubMedPubMedCentralGoogle Scholar
  34. Seoane A, Demêmes D, Llorens J (2001a) Pathology of the rat vestibular sensory epithelia during subchronic 3,3′-iminodipropionitrile exposure: hair cells may not be the primary target of toxicity. Acta Neuropathol 102:339–348PubMedGoogle Scholar
  35. Seoane A, Demêmes D, Llorens J (2001b) Relationship between insult intensity and mode of hair cell loss in the vestibular system of rats exposed to 3,3′-iminodipropionitrile. J Comp Neurol 439:385–399PubMedGoogle Scholar
  36. Serra A, Salame K, Liao K, Leigh RJ (2013) Eye movements, vision, and the vestibulo-ocular reflexes. In: Bronstein AM (ed) Oxford textbook of vertigo and imbalance. Oxford University Press, Oxford, pp 27–33Google Scholar
  37. Soler-Martín C, Diez-Padrisa N, Boadas-Vaello P, Llorens J (2007) Behavioral disturbances and hair cell loss in the inner ear following nitrile exposure in mice, Guinea pigs, and frogs. Toxicol Sci 96:123–132PubMedGoogle Scholar
  38. Sichel JY, Eliashar R, Plotnick M, Sohmer H, Elidan J (2000) Assessment of vestibular ototoxicity of ear drops by recording of vestibular evoked potentials to acceleration impulses. Am J Otolaryngol 21:192–195Google Scholar
  39. Vignaux G, Chabbert C, Gaboyard-Niay S, Travo C, Machado ML, Denise P, Comoz F, Hitier M, Landemore G, Philoxène B, Besnard S (2012) Evaluation of the chemical model of vestibular lesions induced by arsanilate in rats. Toxicol Appl Pharmacol 258:61–71PubMedGoogle Scholar
  40. Vulovic V, Curthoys IS (2011) Bone conducted vibration activates the vestibulo-ocular reflex in the Guinea pig. Brain Res Bull 86:74–81PubMedGoogle Scholar
  41. Wallace DG, Hines DJ, Pellis SM, Whishaw IQ (2002) Vestibular information is required for dead reckoning in the rat. J Neurosci 22:10009–10017PubMedPubMedCentralGoogle Scholar
  42. Wilson VJ, Yoshida M (1968) Vestibulospinal and reticulospinal effects on hindlimb, forelimb, and neck alpha motoneurons of the cat. Proc Natl Acad Sci U S A 60:836–840PubMedPubMedCentralGoogle Scholar
  43. Yang TH, Liu SH, Young YH (2010) Evaluation of Guinea pig model for ocular and cervical vestibular-evoked myogenic potentials for vestibular function test. Laryngoscope 120:1910–1917PubMedGoogle Scholar

Copyright information

© Association for Research in Otolaryngology 2019

Authors and Affiliations

  1. 1.Departament de Ciències Fisiològiques, Institut de NeurocièncesUniversitat de BarcelonaL’Hospitalet de LlobregatSpain
  2. 2.Institut d’Investigació Biomèdica de Bellvitge, IDIBELLL’Hospitalet de LlobregatSpain
  3. 3.Research Group of Clinical Anatomy, Embryology and Neuroscience (NEOMA), Departament de Ciències Mèdiques, Facultat de MedicinaUniversitat de GironaGironaSpain

Personalised recommendations