Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit

Carlos M. Contreras; Tania Molina-Jiménez; Ana G. Gutiérrez-García

doi:doi:10.11648/j.ajpn.20140206.11

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Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit

Carlos M. Contreras, Tania Molina-Jiménez, Ana G. Gutiérrez-García

Published in American Journal of Psychiatry and Neuroscience (Volume 2, Issue 6)

Received: 11 November 2014 Accepted: 19 November 2014 Published: 23 November 2014

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Abstract

Alarm substances are released under stressful situations and may constitute signals that prevent other members of the group from encountering dangerous situations by producing fear. 2-Heptanone is an alarm pheromone that increases the neuronal firing rate in temporal lobe structures that are related to fear in the rat, such as the basal amygdala. A single stress session of unavoidable electric footshock or 2-heptanone sniffing increases the responsivity of the medial amygdala-hippocampus circuit, but unknown is the timing of action of simultaneous exposure to both stressors on the firing rate and responsivity of CA1-CA3 neurons identified by their connections with the medial amygdala nucleus. Twenty-four or 48 h after a single stress session, we obtained single-unit extracellular recordings. The firing rate was higher in the 48 h group. The peristimulus histogram showed an increase in the responsivity of amygdala-hippocampus neurons, which was more pronounced 48 h after a single stress session. The present results suggest an increase in the sensitivity of this circuit after a single stress session, seemingly representing a first step in the formation of emotional memories related to a conditioned response to fear.

Published in	American Journal of Psychiatry and Neuroscience (Volume 2, Issue 6)
DOI	10.11648/j.ajpn.20140206.11
Page(s)	83-89
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

2-heptanone, Fear, Footshock, Amygdala, Hippocampus

References

[1]	K. Ackerl, M. Atzmueller, K. Grammer. “The scent of fear”. Neuroendocrinol Lett, 23:79-84, 2002.
[2]	J. Albrecht, M. Demmel, V Schöpf, A.M. Kleemann, R. Kopietz, J. May, T. Schereder, R. Zernecke, H. Brückmann, M. Wiesmann. “Smelling chemosensory signals of males in anxious versus nonaxious condition increases state anxiety of female subjects”. Chem Senses, 36:19-27, 2010.
[3]	R.H. Pastel. “Collective behaviors: mass panic and outbreaks of multiple unexplained symptoms”. Mil Med, 166:44-46, 2001.
[4]	A.R. Mawson. “Understanding mass panic and other collective responses to threat and disaster”. Psychiatry, 68:95-113, 2005.
[5]	J.S. Morris, C.D. Frith, D.I. Perrett, D. Rowland, A.W. Young, A.J. Calder, R.J. Dolan. “A differential neural response in the human amygdala to fearful and happy facial expressions”. Nature, 383:812-815, 1996.
[6]	E.A. Krusemark, L.R. Novak, D.R. Gitelman, W. Li. “When the sense of smell meets emotion: anxiety-state-dependent olfactory processing and neural circuitry adaptation”. J Neurosci, 33:15324-15332, 2013.
[7]	E.A. Phelps. “Human emotional and memory: interaction of the amygdala and hippocampal complex”. Curr Opin Neurobiol, 14:198-202, 2002.
[8]	R. Paz, D. Pare. “Physiological basis for emotional modulation of memory circuits by the amygdala”. Curr Opin Neurobiol, 23:381-386, 2013.
[9]	N. Gutiérrez-Castellanos, A. Martínez-Marcos, F. Martínez-García, E. Lanuza. “Chemosensory function of the amygdala”. Vitam Horm, 83:165-196, 2010.
[10]	R. Hauser, M. Wiergowski, M. Kaliszan, T. Gos, G. Kernbach-Wighton, M. Studniarek, Z. Jankowski, J. Namieśnik. “Olfactory and tissue markers of fear in mammals including humans”. Med Hypotheses, 77:1062-1067, 2011.
[11]	B. de Lacy Costello, A. Amann, H. Al-Kateb, C. Flynn, W. Filipiak, T. Khalid, D. Osborne, N.M. Ratcliffe. “A review of the volatiles from the healthy human body”. J Breath Res, 8:014001, 2014.
[12]	AG. Gutiérrez-García, C.M Contreras, R. Mendoza-López, S. Cruz-Sánchez, O. García-Barradas, J.F. Rodríguez-Landa, B. Bernal-Morales. “A single session of emotional stress produces anxiety in Wistar rats”. Behav Brain Res, 167:30-35, 2006.
[13]	A.G. Gutiérrez-García, C.M. Contreras, R. Mendoza-López, O. García-Barradas, S. Cruz-Sánchez. “Urine from stressed rats increases immobility in receptor rats forced to swim: role of 2-heptanone”. Physiol Behav, 91:166-172, 2007.
[14]	C.M. Contreras, A.G. Gutiérrez-García, T. Molina-Jiménez, R. Mendoza-López. “2-Heptanone increases the firing rate of the basal amygdala: role of anterior olfactory epithelial organs”. Neuropsychobiology, 66:167-173, 2012.
[15]	C.M. Contreras, A.G. Gutiérrez-García, T. Molina-Jiménez. “Anterior olfactory organ removal produces anxiety-like behavior and increases spontaneous neuronal firing rate in basal amygdala”. Behav Brain Res, 252:101-119, 2013.
[16]	G. Richter-Levin, I. Akirav I. “Amygdala-hippocampus dynamic interaction in relation to memory”. Mol Neurobiol, 22:11-20, 2000.
[17]	K. Abe. “Modulation of hippocampal long-term potentiation by the amygdala: a synaptic mechanism linking emotion and memory”. Jpn J Pharmacol, 86:18-22, 2001.
[18]	G. Richter-Levin, I. Akirav I. “Emotional tagging of memory formation: in the search for neural mechanisms”. Brain Res Brain Res Rev, 43:247-256, 2003.
[19]	C.I. Li, T.L. Maglinao, L.K. Takahashi. “Medial amygdala modulation of predator odor-induced unconditioned fear in the rat”. Behav Neurosci, 118:324-332, 2004.
[20]	M. Müller, M. Fendt. “Temporary inactivation of the medial and basolateral amygdala differentially affects TMT-induced fear behavior in rats”. Behav Brain Res, 167:57-62, 2006.
[21]	S. Campeau, T.J. Nyhuis, S.K. Sasse, H.E. Day, C.V. Masini. “Acute and chronic effects of ferret odor exposure in Sprague-Dawley rats”. Neurosci Biobehav Rev, 32:1277-1286, 2008.
[22]	P. Blier, C. de Montigny C. “Serotoninergic but not noradrenergic neurons in rat central nervous system adapt to long-term treatment with monoamine oxidase inhibitors”. Neuroscience, 16:949-955, 1985.
[23]	T. Molina-Jiménez, A.G. Gutiérrez-García, C.M. Contreras. “An alarm pheromone increases the responsivity of amygdaline-hippocampal neurons”. Salud Mental, 36:279-284, 2013.
[24]	National Research Council. Guide for the Care and Use of Laboratory Animals (publication no. 80-23). Washington DC: National Academy Press; 1996.
[25]	T. Leinders-Zufall, A.P. Lane, A.C. Puce, W. Ma, M. Novotny, M.T. Shipley, F. Zufall. “Ultrasensitive pheromone detection by mammalian vomeronasal neurons”. Nature, 405:792-796, 2000.
[26]	R.G. Phillips, J.E. LeDoux. “Differential contributions of amygdala and hippocampus to cued and contextual fear conditioning”. Behav Neurosci, 106:274-285, 1992.
[27]	J.M. Santos, A.C. Gárgaro, A.R. Oliveira, S. Masson, M.L. Brandão. “Pharmacological dissociation of moderate and high contextual fear as assessed by freezing behavior and fear-potentiated startle”. Eur Neuropsychopharmacol, 15:239-246, 2005.
[28]	M. Sánchez-Alvarez, M. León-Olea, M. Condés-Lara, M. Briones, A. Fernández-Guardiola. “Localization of the microelectrode tip combining a rapid procedure method and marking with pontamine sky blue”. Bol Estud Med Biol, 36:55-59, 1988.
[29]	G. Paxinos, C. Watson. “The rat brain in stereotaxic coordinates”, 4th edition. San Diego: Academic Press; 1998.
[30]	E. Thomas, M. DeWolfe, F. Sancar, N. Todi, E. Yadin. “Electrophysiological analysis of the interaction between the lateral septum and the central nucleus of the amygdala”. Neurosci Lett, 524:79-83, 2012.
[31]	S. Frey, J. Bergado-Rosado, T. Seidenbecker, H.C. Pape, J.U. Frey. “Reinforcement of early long-term potentiation (early-LTP) in dentate gyrus by stimulation of the basolateral amygdala: heterosynaptic induction mechanisms of late-LTP”. J Neurosci, 21:3697-3703, 2001.
[32]	F. Scalia, S.S. Winans. “The differential projections of the olfactory bulb and accessory olfactory bulb in mammals”. J Comp Neurol, 161:31-55, 1975.
[33]	N.S. Canteras, R.B. Simerly, L.W. Swanson. “Organization of projections from the medial nucleus of the amygdala: a PHAL study in the rat”. J Comp Neurol, 360:213-245, 1995.
[34]	V.R. Heale, C.H. Vanderwolf, M. Kavaliers. “Components of weasel and fox odors elicit fast wave bursts in the dentate gyrus of rats”. Behav Brain Res, 63:159-165, 1994.
[35]	H.C. Dringenberg, D. Oliveira, D. Habib. “Predator (cat hair)-induced enhancement of hippocampal long-term potentiation in rats: involvement of acetylcholine”. Learn Mem, 15:112-116, 2008.
[36]	J.E. LeDoux. “Evolution of human emotion: a view through fear”. Prog Brain Res, 195:431-442, 2012.
[37]	A.J. McDonald. “Cortical phatways to the mammalian amygdala”. Prog Neurobiol, 55:257-332, 1998.
[38]	D.C. Blanchard, G. Griebel, R.J. Blanchard. “Conditioning and residual emotionality effects of predator stimuli: some reflections on stress and emotion”. Prog Neuropsychopharmacol Biol Psychiatry, 27:1177-1185, 2003.
[39]	R. Hauser, M. Marczak, B. Karaszewski, M. Wiergowski, M. Kaliszan, M. Penkowski, G. Kernbach-Wighton, Z. Jankowski, J. Namieśnik. “A preliminary study for identifying olfactory markers of fear in the rat”. Lab Anim, 37:76-80, 2008.
[40]	Y. Kiyokawa, T. Kikusui, Y. Takeuchi, Y. Mori. “Mapping the neural circuit activated by alarm pheromone perception by c-Fos immunohistochemistry”. Brain Res, 1043:145-154, 2005.
[41]	N. Maggio, M. Segal. “Differential modulation of long-term depression by acute stress in the rat dorsal and ventral hippocampus”. J Neurosci, 29:8633-8638, 2009.
[42]	D. Caudal, B.P. Godsil, F. Mailliet, D. Bergerot, T.M. Jay. “Acute stress induces contrasting changes in AMPA receptor subunit phosphorylation within the prefrontal cortex, amygdala and hippocampus”. PLoS One, 5:e1528, 2010.
[43]	C. Weiss, E. Sametsky, A. Sasse, J. Spiess, J.F. Disterhoft. “Acute stress facilitates trace eyeblink conditioning in C57BL/6 male mice and increases the excitability of their CA1 pyramidal neurons”. Learn Mem, 12:138-143, 2005.
[44]	Z. Wang, RD. Pang, M. Hernandez, MA Ocampo, D.P. Holschneider. “Anxiolytic-like effect of pregabalin on unconditioned fear in the rat: an autoradiographic brain perfusion mapping and functional connectivity study”. Neuroimage, 59:4168-4188, 2012.
[45]	M.S. Fanselow, H.W. Dong. “Are the dorsal and ventral hippocampus functionally distinct structures” Neuron, 65:7-19, 2010.
[46]	D.M. Bannerman, J.N. Rawlins, S.B. McHugh, R.M. Deacon, B.K. Yee, T. Bast, W.N. Zhang, H.H. Pothuizen, J. Feldon. “Regional dissociations within the hippocampus: memory and anxiety”. Neurosci Biobehav Rev, 28:273-283, 2004.
[47]	F. Zufall, S.D. Munger. “From odor and pheromone transduction to the organization of the sense of smell”. Trends Neurosci, 24:191-193, 2001.
[48]	G.Y. Paschall, M. Davis. “Second-order olfactory-mediated fear-potentiated startle”. Learn Mem, 9:395-401, 2002.
[49]	N. Sakai, S. Imada. “Bilateral lesions of the insular cortex or of the prefrontal cortex block the association between taste and odor in the rat”. Neurobiol Learn Mem, 80:24-31, 2003.
[50]	J.A. Kroon, A.P. Carobrez. “Olfactory fear conditioning paradigm in rats: effects of midazolam, propranolol or scopolamine”. Neurobiol Learn Mem, 91:32-40, 2009.
[51]	L.K. Takahashi, D.T. Hubbard, I. Lee, Y. Dar, S.M. Sipes. “Predator odor-induced conditioned fear involves the basolateral and medial amygdala”. Behav Neurosci, 121:100-110, 2007.
[52]	L.K. Takahashi, M.M Chan, M.L. Pilar. “Predator odor fear conditioning: current perspectives and new directions”. Neurosci Biobehav, 32:1218-1227, 2008.
[53]	S.K. Baisley, C.L. Cloninger, V.P. Bakshi. “Fos expression following regimens of predator stress versus footshock that differentially affect prepulse inhibition in rats”. Physiol Behav, 104:796-803, 2011.
[54]	T.V. Bliss, G.L. Collingridge. “A synaptic model of memory: long-term potentiation in the hippocampus”. Nature, 361:31-39, 1993.
[55]	U. Frey, Y.Y. Huang, E.R. Kandel. “Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons”. Science, 260:1661-1664, 1993.
[56]	T.V. Bliss, T. Lomo. “Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path”. J Physiol , 232:331-356, 1973.
[57]	T.W Berger. “Long-term potentiation of hippocampal synaptic transmission affects rate of behavioral learning”. Science, 224:627-630, 1984.

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APA Style

Carlos M. Contreras, Tania Molina-Jiménez, Ana G. Gutiérrez-García. (2014). Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit. American Journal of Psychiatry and Neuroscience, 2(6), 83-89. https://doi.org/10.11648/j.ajpn.20140206.11

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ACS Style

Carlos M. Contreras; Tania Molina-Jiménez; Ana G. Gutiérrez-García. Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit. Am. J. Psychiatry Neurosci. 2014, 2(6), 83-89. doi: 10.11648/j.ajpn.20140206.11

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AMA Style

Carlos M. Contreras, Tania Molina-Jiménez, Ana G. Gutiérrez-García. Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit. Am J Psychiatry Neurosci. 2014;2(6):83-89. doi: 10.11648/j.ajpn.20140206.11

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@article{10.11648/j.ajpn.20140206.11,
  author = {Carlos M. Contreras and Tania Molina-Jiménez and Ana G. Gutiérrez-García},
  title = {Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit},
  journal = {American Journal of Psychiatry and Neuroscience},
  volume = {2},
  number = {6},
  pages = {83-89},
  doi = {10.11648/j.ajpn.20140206.11},
  url = {https://doi.org/10.11648/j.ajpn.20140206.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajpn.20140206.11},
  abstract = {Alarm substances are released under stressful situations and may constitute signals that prevent other members of the group from encountering dangerous situations by producing fear. 2-Heptanone is an alarm pheromone that increases the neuronal firing rate in temporal lobe structures that are related to fear in the rat, such as the basal amygdala. A single stress session of unavoidable electric footshock or 2-heptanone sniffing increases the responsivity of the medial amygdala-hippocampus circuit, but unknown is the timing of action of simultaneous exposure to both stressors on the firing rate and responsivity of CA1-CA3 neurons identified by their connections with the medial amygdala nucleus. Twenty-four or 48 h after a single stress session, we obtained single-unit extracellular recordings. The firing rate was higher in the 48 h group. The peristimulus histogram showed an increase in the responsivity of amygdala-hippocampus neurons, which was more pronounced 48 h after a single stress session. The present results suggest an increase in the sensitivity of this circuit after a single stress session, seemingly representing a first step in the formation of emotional memories related to a conditioned response to fear.},
 year = {2014}
}

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TY  - JOUR
T1  - Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit
AU  - Carlos M. Contreras
AU  - Tania Molina-Jiménez
AU  - Ana G. Gutiérrez-García
Y1  - 2014/11/23
PY  - 2014
N1  - https://doi.org/10.11648/j.ajpn.20140206.11
DO  - 10.11648/j.ajpn.20140206.11
T2  - American Journal of Psychiatry and Neuroscience
JF  - American Journal of Psychiatry and Neuroscience
JO  - American Journal of Psychiatry and Neuroscience
SP  - 83
EP  - 89
PB  - Science Publishing Group
SN  - 2330-426X
UR  - https://doi.org/10.11648/j.ajpn.20140206.11
AB  - Alarm substances are released under stressful situations and may constitute signals that prevent other members of the group from encountering dangerous situations by producing fear. 2-Heptanone is an alarm pheromone that increases the neuronal firing rate in temporal lobe structures that are related to fear in the rat, such as the basal amygdala. A single stress session of unavoidable electric footshock or 2-heptanone sniffing increases the responsivity of the medial amygdala-hippocampus circuit, but unknown is the timing of action of simultaneous exposure to both stressors on the firing rate and responsivity of CA1-CA3 neurons identified by their connections with the medial amygdala nucleus. Twenty-four or 48 h after a single stress session, we obtained single-unit extracellular recordings. The firing rate was higher in the 48 h group. The peristimulus histogram showed an increase in the responsivity of amygdala-hippocampus neurons, which was more pronounced 48 h after a single stress session. The present results suggest an increase in the sensitivity of this circuit after a single stress session, seemingly representing a first step in the formation of emotional memories related to a conditioned response to fear.
VL  - 2
IS  - 6
ER  -

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Author Information

Carlos M. Contreras

Unidad Periférica del Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Xalapa, Veracruz, 91190, México
Tania Molina-Jiménez

Laboratorio de Neurofarmacología, Instituto de Neuroetología, Universidad Veracruzana, Xalapa, Veracruz, 91190, México
Ana G. Gutiérrez-García

Laboratorio de Neurofarmacología, Instituto de Neuroetología, Universidad Veracruzana, Xalapa, Veracruz, 91190, México

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Carlos M. Contreras, Tania Molina-Jiménez, Ana G. Gutiérrez-García. (2014). Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit. American Journal of Psychiatry and Neuroscience, 2(6), 83-89. https://doi.org/10.11648/j.ajpn.20140206.11

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ACS Style

Carlos M. Contreras; Tania Molina-Jiménez; Ana G. Gutiérrez-García. Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit. Am. J. Psychiatry Neurosci. 2014, 2(6), 83-89. doi: 10.11648/j.ajpn.20140206.11

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AMA Style

Carlos M. Contreras, Tania Molina-Jiménez, Ana G. Gutiérrez-García. Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit. Am J Psychiatry Neurosci. 2014;2(6):83-89. doi: 10.11648/j.ajpn.20140206.11

Copy | Download

@article{10.11648/j.ajpn.20140206.11,
  author = {Carlos M. Contreras and Tania Molina-Jiménez and Ana G. Gutiérrez-García},
  title = {Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit},
  journal = {American Journal of Psychiatry and Neuroscience},
  volume = {2},
  number = {6},
  pages = {83-89},
  doi = {10.11648/j.ajpn.20140206.11},
  url = {https://doi.org/10.11648/j.ajpn.20140206.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajpn.20140206.11},
  abstract = {Alarm substances are released under stressful situations and may constitute signals that prevent other members of the group from encountering dangerous situations by producing fear. 2-Heptanone is an alarm pheromone that increases the neuronal firing rate in temporal lobe structures that are related to fear in the rat, such as the basal amygdala. A single stress session of unavoidable electric footshock or 2-heptanone sniffing increases the responsivity of the medial amygdala-hippocampus circuit, but unknown is the timing of action of simultaneous exposure to both stressors on the firing rate and responsivity of CA1-CA3 neurons identified by their connections with the medial amygdala nucleus. Twenty-four or 48 h after a single stress session, we obtained single-unit extracellular recordings. The firing rate was higher in the 48 h group. The peristimulus histogram showed an increase in the responsivity of amygdala-hippocampus neurons, which was more pronounced 48 h after a single stress session. The present results suggest an increase in the sensitivity of this circuit after a single stress session, seemingly representing a first step in the formation of emotional memories related to a conditioned response to fear.},
 year = {2014}
}

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TY  - JOUR
T1  - Exposure to an Alarm Pheromone Combined with Footshock Stress Enhances Responsivity of the Medial Amygdala-Hippocampus Circuit
AU  - Carlos M. Contreras
AU  - Tania Molina-Jiménez
AU  - Ana G. Gutiérrez-García
Y1  - 2014/11/23
PY  - 2014
N1  - https://doi.org/10.11648/j.ajpn.20140206.11
DO  - 10.11648/j.ajpn.20140206.11
T2  - American Journal of Psychiatry and Neuroscience
JF  - American Journal of Psychiatry and Neuroscience
JO  - American Journal of Psychiatry and Neuroscience
SP  - 83
EP  - 89
PB  - Science Publishing Group
SN  - 2330-426X
UR  - https://doi.org/10.11648/j.ajpn.20140206.11
AB  - Alarm substances are released under stressful situations and may constitute signals that prevent other members of the group from encountering dangerous situations by producing fear. 2-Heptanone is an alarm pheromone that increases the neuronal firing rate in temporal lobe structures that are related to fear in the rat, such as the basal amygdala. A single stress session of unavoidable electric footshock or 2-heptanone sniffing increases the responsivity of the medial amygdala-hippocampus circuit, but unknown is the timing of action of simultaneous exposure to both stressors on the firing rate and responsivity of CA1-CA3 neurons identified by their connections with the medial amygdala nucleus. Twenty-four or 48 h after a single stress session, we obtained single-unit extracellular recordings. The firing rate was higher in the 48 h group. The peristimulus histogram showed an increase in the responsivity of amygdala-hippocampus neurons, which was more pronounced 48 h after a single stress session. The present results suggest an increase in the sensitivity of this circuit after a single stress session, seemingly representing a first step in the formation of emotional memories related to a conditioned response to fear.
VL  - 2
IS  - 6
ER  -

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