Inactivation of the dorsolateral periaqueductal gray matter impairs the promoting influence of stress on fear memory during retrieval

  • Marcelo Giachero
  • Eloisa Pavesi
  • Gastón Calfa
  • Simone C. Motta
  • Newton S. Canteras
  • Víctor A. Molina
  • Antonio P. CarobrezEmail author
Original Article


Exposure to stressful conditions induces long-lasting neurobiological changes in selected brain areas, which could be associated with the emergence of negative emotional responses. Moreover, the interaction of a stressful experience and the retrieval of an established fear memory trace enhance both fear expression and fear retention. Related to this, the stimulation of the dorsolateral part of the mesencephalic periaqueductal gray matter (dlPAG) prior to retrieval potentiates a fear memory trace previously acquired. Therefore, the question that arises is whether the dlPAG mediates the increased fear expression and fear retention after retrieval. Rats were subjected to a contextual fear conditioning paradigm using a single footshock, and 1 day later, rats were subjected to a stressful situation. As previously reported, there was an increase of freezing response only in those rodents that were re-exposed to the associated context at 1 and 5 days after stress exposure. Muscimol intra-dlPAG prior to the restraint event prevented such increase. Conversely, Muscimol intra-dlPAG infusion immediately after the stress experience had no effect on the resulting fear memory. When the neuroendocrine response to stress was explored, intra-dlPAG infusion of muscimol prior to stress decreased Fos expression in the paraventricular nucleus and serum corticosterone levels. Moreover, this treatment prevented the enhancement of the density of hippocampal “mature” spines associated with fear memory. In conclusion, the present results suggest that the dlPAG is a key neural site for the negative valence instruction necessary to modulate the promoting influence of stress on fear memory.


Fear memory Stress Retrieval Periaqueductal gray matter Valence instruction 



This research was supported by FAPESP, CAPES, and CNPq from which MG and EP received a post-doctoral fellowship and APC and NSC a research fellowship.

Compliance with ethical standards

Conflict of interest

We have no commercial associations which impact on this work. Funding for this study was provided by Brazilian public agencies CAPES, CNPq, and FAPESP; they had no further role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Ethical approval

The current research was approved by the Federal University of Santa Catarina, Animal Ethics Committee (23080.0055752/2006-64/UFSC), and was performed in accordance with the Brazilian Society of Neuroscience and Behavior Guidelines for the Care and Use of Laboratory Animals.


  1. Bender CL, Giachero M, Comas-Mutis R, Molina VA, Calfa GD (2018) Stress influences the dynamics of hippocampal structural remodeling associated with fear memory extinction. Neurobiol Learn Mem 155:412–421. CrossRefGoogle Scholar
  2. Bittencourt AS, Carobrez AP, Zamprogno LP, Tufik S, Schenberg LC (2004) Organization of single components of defensive behaviors within distinct columns of periaqueductal gray matter of the rat: role of N-methyl-d-aspartic acid glutamate receptors. Neuroscience 125:71–89. CrossRefGoogle Scholar
  3. Blanchard RJ, Blanchard DC (1969) Crouching as an index of fear. J Comp Physiol Psychol 67:370–375CrossRefGoogle Scholar
  4. Calfa G, Chapleau CA, Campbell S, Inoue T, Morse SJ, Lubin FD, Pozzo-Miller L (2012) HDAC activity is required for BDNF to increase quantal neurotransmitter release and dendritic spine density in CA1 pyramidal neurons. Hippocampus 22(7):1493–1500. CrossRefGoogle Scholar
  5. Cameron AA, Khan IA, Westlund KN, Willis WD (1995) The efferent projections of the periaqueductal gray in the rat: a Phaseolus vulgaris-leucoagglutinin study. II. Descending projections. J Comp Neurol. 351:585–601. CrossRefGoogle Scholar
  6. Canteras NS (2002) The medial hypothalamic defensive system: hodological organization and functional implications. Pharmacol Biochem Behav 71:481–491. CrossRefGoogle Scholar
  7. Canteras NS, Pavesi E, Carobrez AP (2015) Olfactory instruction for fear: neural system analysis. Front Neurosci 9:276. CrossRefGoogle Scholar
  8. Carobrez AP, Teixeira KV, Graeff FG (2001) Modulation of defensive behavior by periaqueductal gray NMDA/glycine-B 45 receptor. Neurosci Biobehav Rev 25:697–709. CrossRefGoogle Scholar
  9. Cezario AF, Ribeiro-Barbosa ER, Baldo MV, Canteras NS (2008) Hypothalamic sites responding to predator threats—the role of the dorsal premammillary nucleus in unconditioned and conditioned antipredatory defensive behavior. Eur J Neurosci 28:1003–1015. CrossRefGoogle Scholar
  10. Chapleau CA, Calfa GD, Lane MC, Albertson AJ, Larimore JL, Kudo S, Armstrong DL, Percy AK, Pozzo-Miller L (2009) Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations. Neurobiol Dis 35:219–233. CrossRefGoogle Scholar
  11. Cordero MI, Venero C, Kruyt ND, Sandi C (2003) Prior exposure to a single stress session facilitates subsequent contextual fear conditioning in rats. Evidence for a role of corticosterone. Horm Behav 44:338–345. CrossRefGoogle Scholar
  12. Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ (1995) Pattern and time course of immediate early gene expression in rat brain following acute stress. Neuroscience 64:477–505CrossRefGoogle Scholar
  13. De Kloet ER (2004) Hormones and the stressed brain. Ann N Y Acad Sci 1018:1–15. (Review) CrossRefGoogle Scholar
  14. De Oca BM, DeCola JP, Maren S, Fanselow MS (1998) Distinct regions of the periaqueductal gray are involved in the acquisition and expression of defensive responses. J Neurosci 18(9):3426–3432. CrossRefGoogle Scholar
  15. Di Scala G, Mana MJ, Jacobs WJ, Phillips AG (1987) Evidence of Pavlovian conditioned fear following electrical stimulation of the periaqueductal grey in the rat. Physiol Behav 40(1):55–63. CrossRefGoogle Scholar
  16. Dudai Y (2002) Memory from A to Z. Keywords, concepts and beyond. Oxford University Press, OxfordGoogle Scholar
  17. Faul F, Erdfelder E, Lang AG, Buchner A (2007) G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 39(2):175–191CrossRefGoogle Scholar
  18. Giachero M, Bustos SG, Calfa G, Molina VA (2013a) A BDNF sensitive mechanism is involved in the fear memory resulting from the interaction between stress and the retrieval of an established trace. Learn Mem 20(5):245–255. CrossRefGoogle Scholar
  19. Giachero M, Calfa GD, Molina VA (2013b) Hippocampal structural plasticity accompanies the resulting contextual fear memory following stress and fear conditioning. Learn Mem 20:611–616. CrossRefGoogle Scholar
  20. Giachero M, Calfa GD, Molina VA (2015) Hippocampal dendritic spines remodeling and fear memory are modulated by GABAergic signaling within the basolateral amygdala complex. Hippocampus 00:1–11. Google Scholar
  21. Gisquet-Verrier P, Riccio DC (2012) Memory reactivation effects independent of reconsolidation. Learn Mem 19:401–409. CrossRefGoogle Scholar
  22. Harris KM (1999) Structure, development, and plasticity of dendritic spines. Curr Opin Neurobiol 9:343–348CrossRefGoogle Scholar
  23. Horovitz O, Richter-Levin G (2015) Dorsal periaqueductal gray simultaneously modulates ventral subiculum induced-plasticity in the basolateral amygdala and the nucleus accumbens. Front Behav Neurosci 9:53. CrossRefGoogle Scholar
  24. Horovitz O, Richter-Levin A, Xu L, Jing L, Richter-Levin G (2017) Periaqueductal Grey differential modulation of nucleus accumbens and basolateral amygdala plasticity under controllable and uncontrollable stress. Sci Rep 7(1):487. CrossRefGoogle Scholar
  25. Isoardi NA, Bertotto ME, Martijena ID, Molina VA, Carrer HF (2007) Lack of feedback inhibition on rat basolateral amygdala following stress or withdrawal from sedative-hypnotic drugs. Eur J Neurosci 26:1036–1044. CrossRefGoogle Scholar
  26. Johansen JP, Tarpley JW, LeDoux JE, Blair HT (2010) Neural substrates for expectation modulated fear learning in the amygdala and periaqueductal gray. Nat Neurosci 13:979–986. CrossRefGoogle Scholar
  27. Kandel ER (2001) The molecular biology of memory storage: a dialogue between genes and synapses. Science 294:1030–1038. CrossRefGoogle Scholar
  28. Kasai H, Matsuzaki M, Noguchi J, Yasumatsu N, Nakahara H (2003) Structure–stability–function relationships of dendritic spines. Trends Neurosci 26:360–368. CrossRefGoogle Scholar
  29. Kim JJ, Rison RA, Fanselow MS (1993) Effects of amygdala, hippocampus, and periaqueductal gray lesions on short- and long-term contextual fear. Behav Neurosci 107(6):1093–1098. CrossRefGoogle Scholar
  30. Kim EJ, Horovitz O, Pellman BA, Tan LM, Li Q, Richter-Levin G, Kim JJ (2013) Dorsal periaqueductal gray-amygdala pathway conveys both innate and learned fear responses in rats. Proc Natl Acad Sci USA 110(36):14795–14800. CrossRefGoogle Scholar
  31. Kincheski GC, Mota-Ortiz SR, Pavesi E, Canteras NS, Carobrez AP (2012) The dorsolateral periaqueductal gray and its role in mediating fear learning to life threatening events. PLoS One 7:e50361. CrossRefGoogle Scholar
  32. Koh IY, Lindquist WB, Zito K, Nimchinsky EA, Svoboda K (2002) An image analysis algorithm for dendritic spines. Neural Comput 14:1283–1310. CrossRefGoogle Scholar
  33. LeDoux J (2007) The amygdala. Curr Biol 17:868–874. CrossRefGoogle Scholar
  34. Lee G, Goosens KA (2015) Sampling blood from the lateral tail vein of the rat. J Vis Exp 99:e52766. Google Scholar
  35. Leuner B, Falduto J, Shors TJ (2003) Associative memory formation increases the observation of dendritic spines in the hippocampus. J Neurosci 23:659–665. CrossRefGoogle Scholar
  36. Lewis DJ (1979) Psychobiology of active and inactive memory. Psychol Bull 86:1054–1083CrossRefGoogle Scholar
  37. Maldonado NM, Martijena ID, Molina VA (2011) Facilitating influence of stress on the consolidation of fear memory induced by a weak training: reversal by midazolam pretreatment. Behav Brain Res 225:77–84. CrossRefGoogle Scholar
  38. Maren S, Fanselow MS (1995) Synaptic plasticity in the basolateral amygdala induced by hippocampal formation stimulation in vivo. J Neurosci 15(11):7548–7564. CrossRefGoogle Scholar
  39. Mochny CR, Kincheski GC, Molina VA, Carobrez AP (2012) Dorsolateral periaqueductal gray stimulation prior to retrieval potentiates a contextual fear memory in rats. Behav Brain Res 237C:76–81. Google Scholar
  40. Motta SC, Canteras NS (2015) Restraint stress and social defeat: what they have in common. Physiol Behav 146:105–110. CrossRefGoogle Scholar
  41. Motta SC, Carobrez AP, Canteras NS (2017) The periaqueductal gray and primal emotional processing critical to influence complex defensive responses, fear learning and reward seeking. Neurosci Biobehav Rev 76(Pt A):39–47. CrossRefGoogle Scholar
  42. Murphy DD, Segal M (1996) Regulation of dendritic spine density in cultured rat hippocampal neurons by steroid hormones. J Neurosci 16:4059–4068. CrossRefGoogle Scholar
  43. Nimchinsky EA, Sabatini BL, Svoboda K (2002) Structure and function of dendritic spines. Annu Rev Physiol 64:313–353. CrossRefGoogle Scholar
  44. Pavesi E, Canteras NS, Carobrez AP (2011) Acquisition of Pavlovian fear conditioning using beta-adrenoceptor activation of the dorsal premammillary nucleus as an unconditioned stimulus to mimic live predator-threat exposure. Neuropsychopharmacology 36:926–939. CrossRefGoogle Scholar
  45. Paxinos G, Watson C (2007) The rat brain in stereotaxic coordinates. Academic Press, San DiegoGoogle Scholar
  46. Pozzo-Miller LD, Inoue T, Murphy DD (1999) Estradiol increases spine density and NMDA-dependent Ca2+ transients in spines of CA1 pyramidal neurons from hippocampal slices. J Neurophysiol 81:1404–1411. CrossRefGoogle Scholar
  47. Rau V, Fanselow MS (2009) Exposure to a stressor produces a long-lasting enhancement of fear learning in rats. Stress 12:125–133. CrossRefGoogle Scholar
  48. Restivo L, Vetere G, Bontempi B, Ammassari-Teule M (2009) The formation of recent and remote memory is associated with time-dependent formation of dendritic spines in the hippocampus and anterior cingulate cortex. J Neurosci 29:8206–8214. CrossRefGoogle Scholar
  49. Rodriguez Manzanares PA, Isoardi NA, Carrer HF, Molina VA (2005) Previous stress facilitates fear memory, attenuates GABAergic inhibition, and increases synaptic plasticity in the rat basolateral amygdala. J Neurosci 25:8725–8734. CrossRefGoogle Scholar
  50. Roozendaal B, McEwen BS, Chattarji S (2009) Stress, memory and the amygdala. Nat Rev Neurosci 10:423–433. CrossRefGoogle Scholar
  51. Sandkühler J, Herdegen T (1995) Distinct patterns of activated neurons throughout the rat midbrain periaqueductal gray induced by chemical stimulation within its subdivisions. J Comp Neurol. 357:546–553. CrossRefGoogle Scholar
  52. Segal I, Korkotian I, Murphy DD (2000) Dendritic spine formation and pruning: common cellular mechanisms? Trends Neurosci 23:53–57. CrossRefGoogle Scholar
  53. Shors TJ (2001) Acute stress rapidly and persistently enhances memory formation in the male rat. Neurobiol Learn Mem 75:10–29. CrossRefGoogle Scholar
  54. Souza RR, Carobrez AP (2016) Acquisition and expression of fear memories are distinctly modulated along the dorsolateral periaqueductal gray axis of rats exposed to predator odor. Behav Brain Res 315:160–167. CrossRefGoogle Scholar
  55. Swanson LW (1987) The hypothalamus. In: Bjorklund A, Hokfelt T, Swanson LW (eds) Handbook of chemical neuroanatomy. Integrated systems of the CNS, Part I, vol 5. Elsevier, Amsterdam, pp 1–125Google Scholar
  56. Tulving E (1983) Elements of episodic memory. Clarendon Press, OxfordGoogle Scholar
  57. Tyler WJ, Pozzo-Miller L (2003) Miniature synaptic transmission and BDNF modulate dendritic spine growth and form in rat CA1 neurones. J Physiol 553:497–509. CrossRefGoogle Scholar
  58. Vetere G, Restivo L, Cole CJ, Ross PJ, Ammassari-Teule M, Josselyn SA, Frankland PW (2011) Spine growth in the anterior cingulate cortex is necessary for the consolidation of contextual fear memory. Proc Natl Acad Sci 108:8456–8460. CrossRefGoogle Scholar
  59. Watson TC, Cerminara NL, Lumb BM, Apps R (2016) Neural correlates of fear in the periaqueductal gray. J Neurosci 36(50):12707–12719. CrossRefGoogle Scholar
  60. Yuste R, Majewska A, Holthoff K (2000) From form to function: calcium compartmentalization in dendritic spines. Nat Neurosci 3:653–659. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Departamento de Farmacologia, CCBUniversidade Federal de Santa CatarinaFlorianópolisBrazil
  2. 2.Instituto de Neurociencia Cognitiva y TraslacionalUniversidad Favaloro, INECO, CONICETBuenos AiresArgentina
  3. 3.IFEC-CONICET, Departamento de FarmacologíaFacultad de Ciencias Químicas, Universidad Nacional de CórdobaCórdobaArgentina
  4. 4.Departamento de Anatomia, ICBUniversidade de São PauloSão PauloBrazil

Personalised recommendations