The recruitment of a neuronal ensemble in the CN during the first extinction episode has persistent effects on extinction expression



      Adaptive behaviour depends on the delicate and dynamic balance between acquisition and extinction memories. Disruption of this balance, particularly when the extinction memory loses control over behaviour, is the root of treatment failure of maladaptive behaviours such as substance abuse or anxiety disorders. Understanding this balance requires a better understanding of the underlying neurobiology and its contribution to behavioural regulation.


      We microinjected Daun02 in Fos-lacZ transgenic rats following a single extinction training to delete extinction-recruited neuronal ensembles in the basolateral (BLA) and central (CN) nuclei of the amygdala and examine their contribution to behaviour in an appetitive Pavlovian task. In addition, we used immunohistochemistry and neuronal staining methods to identify the molecular markers of activated neurons in the BLA and CN during extinction learning or retrieval.


      CN neurons were preferentially engaged following extinction and deletion of this extinction-activated ensembles in CN but not BLA impaired the retrieval of extinction even despite additional extinction training, and promoted greater levels of behavioural restoration in spontaneous recovery and reinstatement. Disrupting extinction processing in the CN in turn increased activity in the BLA. Our results also show a specific role for CN PKCδ+ neurons in behavioural inhibition but not during initial extinction learning.


      We show that the initial extinction-recruited CN ensemble is critical to the acquisition-extinction balance, and that greater behavioural restoration does not mean weaker extinction contribution. These findings provide a novel avenue for thinking about the neural mechanisms of extinction and in developing treatments for cue-triggered appetitive behaviours.

      Key words

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Biological Psychiatry
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Rescorla R.A.
        • Heth C.D.
        Reinstatement of fear to an extinguished conditioned stimulus.
        J Exp Psychol Anim Behav Process. 1975; 1: 88-96
      1. Pavlov IP (1927): Conditioned reflexes: an investigation of the physiological activity of the cerebral cortex. Oxford, England: Oxford Univ. Press.

        • Rescorla R.A.
        Spontaneous recovery after Pavlovian conditioning with multiple outcomes.
        Animal Learning & Behavior. 1997; 25: 99-107
        • Conklin C.A.
        • Tiffany S.T.
        Applying extinction research and theory to cue-exposure addiction treatments.
        Addiction. 2002; 97: 155-167
        • Marlatt G.A.
        Cue exposure and relapse prevention in the treatment of addictive behaviors.
        Addict Behav. 1990; 15: 395-399
        • Herry C.
        • Ciocchi S.
        • Senn V.
        • Demmou L.
        • Müller C.
        • Lüthi A.
        Switching on and off fear by distinct neuronal circuits.
        Nature. 2008; 454: 600-606
        • Lacagnina A.F.
        • Brockway E.T.
        • Crovetti C.R.
        • Shue F.
        • McCarty M.J.
        • Sattler K.P.
        • et al.
        Distinct hippocampal engrams control extinction and relapse of fear memory.
        Nature Neuroscience. 2019; 22: 753-761
        • Warren B.L.
        • Kane L.
        • Venniro M.
        • Selvam P.
        • Quintana-Feliciano R.
        • Mendoza M.P.
        • et al.
        Separate vmPFC Ensembles Control Cocaine Self-Administration Versus Extinction in Rats.
        J Neurosci. 2019; 39: 7394-7407
        • Warren B.L.
        • Mendoza M.P.
        • Cruz F.C.
        • Leao R.M.
        • Caprioli D.
        • Rubio F.J.
        • et al.
        Distinct Fos-Expressing Neuronal Ensembles in the Ventromedial Prefrontal Cortex Mediate Food Reward and Extinction Memories.
        J Neurosci. 2016; 36: 6691-6703
        • Suto N.
        • Laque A.
        • De Ness G.L.
        • Wagner G.E.
        • Watry D.
        • Kerr T.
        • et al.
        Distinct memory engrams in the infralimbic cortex of rats control opposing environmental actions on a learned behavior.
        Elife. 2016; 5
        • Laque A.
        • LDN G.
        • Wagner G.E.
        • Nedelescu H.
        • Carroll A.
        • Watry D.
        • et al.
        Anti-relapse neurons in the infralimbic cortex of rats drive relapse-suppression by drug omission cues.
        Nat Commun. 2019; 10: 3934
        • Haubensak W.
        • Kunwar P.S.
        • Cai H.
        • Ciocchi S.
        • Wall N.R.
        • Ponnusamy R.
        • et al.
        Genetic dissection of an amygdala microcircuit that gates conditioned fear.
        Nature. 2010; 468: 270-276
        • Calu D.J.
        • Roesch M.R.
        • Haney R.Z.
        • Holland P.C.
        • Schoenbaum G.
        Neural correlates of variations in event processing during learning in central nucleus of amygdala.
        Neuron. 2010; 68: 991-1001
        • Holland P.C.
        • Gallagher M.
        Effects of amygdala central nucleus lesions on blocking and unblocking.
        Behav Neurosci. 1993; 107: 235-245
        • Iordanova M.D.
        • Deroche M.L.
        • Esber G.R.
        • Schoenbaum G.
        Neural correlates of two different types of extinction learning in the amygdala central nucleus.
        Nat Commun. 2016; 712330
        • Esber G.R.
        • Roesch M.R.
        • Bali S.
        • Trageser J.
        • Bissonette G.B.
        • Puche A.C.
        • et al.
        Attention-related Pearce-Kaye-Hall signals in basolateral amygdala require the midbrain dopaminergic system.
        Biol Psychiatry. 2012; 72: 1012-1019
        • Esber G.R.
        • Holland P.C.
        The basolateral amygdala is necessary for negative prediction errors to enhance cue salience, but not to produce conditioned inhibition.
        Eur J Neurosci. 2014; 40: 3328-3337
      2. Roesch MR, Calu DJ, Esber GR, Schoenbaum G (2010): Neural Correlates of Variations in Event Processing during Learning in Basolateral Amygdala. 30:2464-2471.

        • Tye K.M.
        • Janak P.H.
        Amygdala neurons differentially encode motivation and reinforcement.
        J Neurosci. 2007; 27: 3937-3945
        • Kim J.
        • Zhang X.
        • Muralidhar S.
        • LeBlanc S.A.
        • Tonegawa S.J.N.
        Basolateral to central amygdala neural circuits for appetitive behaviors.
        Neuron. 2017; 93 (e1465): 1464-1479
        • Li H.
        • Penzo M.A.
        • Taniguchi H.
        • Kopec C.D.
        • Huang Z.J.
        • Li B.
        Experience-dependent modification of a central amygdala fear circuit.
        Nature Neuroscience. 2013; 16: 332-339
        • Koya E.
        • Golden S.A.
        • Harvey B.K.
        • Guez-Barber D.H.
        • Berkow A.
        • Simmons D.E.
        • et al.
        Targeted disruption of cocaine-activated nucleus accumbens neurons prevents context-specific sensitization.
        Nat Neurosci. 2009; 12: 1069-1073
        • Barker G.R.
        • Banks P.J.
        • Scott H.
        • Ralph G.S.
        • Mitrophanous K.A.
        • Wong L.F.
        • et al.
        Separate elements of episodic memory subserved by distinct hippocampal-prefrontal connections.
        Nat Neurosci. 2017; 20: 242-250
        • Pfarr S.
        • Meinhardt M.W.
        • Klee M.L.
        • Hansson A.C.
        • Vengeliene V.
        • Schönig K.
        • et al.
        Losing Control: Excessive Alcohol Seeking after Selective Inactivation of Cue-Responsive Neurons in the Infralimbic Cortex.
        The Journal of Neuroscience. 2015; 3510750
        • Cruz F.C.
        • Koya E.
        • Guez-Barber D.H.
        • Bossert J.M.
        • Lupica C.R.
        • Shaham Y.
        • et al.
        New technologies for examining the role of neuronal ensembles in drug addiction and fear.
        Nat Rev Neurosci. 2013; 14: 743-754
        • Lay B.P.P.
        • Pitaru A.A.
        • Boulianne N.
        • Esber G.R.
        • Iordanova M.D.
        Different methods of fear reduction are supported by distinct cortical substrates.
        Elife. 2020; 9
        • Lay B.P.P.
        • Nicolosi M.
        • Usypchuk A.A.
        • Esber G.R.
        • Iordanova M.D.
        Dissociation of Appetitive Overexpectation and Extinction in the Infralimbic Cortex.
        Cereb Cortex. 2019; 29: 3687-3701
        • Burn S.F.
        Detection of β-galactosidase activity: X-gal staining.
        Methods Mol Biol. 2012; 886: 241-250
        • Lee S.-C.
        • Amir A.
        • Headley D.B.
        • Haufler D.
        • Pare D.
        Basolateral amygdala nucleus responses to appetitive conditioned stimuli correlate with variations in conditioned behaviour.
        Nature communications. 2016; 7 (12275): 12275
        • Adke A.P.
        • Khan A.
        • Ahn H.-S.
        • Becker J.J.
        • Wilson T.D.
        • Valdivia S.
        • et al.
        Cell-Type Specificity of Neuronal Excitability and Morphology in the Central Amygdala.
        eNeuro. 2021; 8 (ENEURO.0402-0420.2020)
        • Venniro M.
        • Zhang M.
        • Caprioli D.
        • Hoots J.K.
        • Golden S.A.
        • Heins C.
        • et al.
        Volitional social interaction prevents drug addiction in rat models.
        Nat Neurosci. 2018; 21: 1520-1529
        • Bossert J.M.
        • Stern A.L.
        • Theberge F.R.
        • Cifani C.
        • Koya E.
        • Hope B.T.
        • et al.
        Ventral medial prefrontal cortex neuronal ensembles mediate context-induced relapse to heroin.
        Nat Neurosci. 2011; 14: 420-422
        • Fanous S.
        • Goldart E.M.
        • Theberge F.R.
        • Bossert J.M.
        • Shaham Y.
        • Hope B.T.
        Role of orbitofrontal cortex neuronal ensembles in the expression of incubation of heroin craving.
        J Neurosci. 2012; 32: 11600-11609
        • Funk D.
        • Coen K.
        • Tamadon S.
        • Hope B.T.
        • Shaham Y.
        • Lê A.D.
        Role of Central Amygdala Neuronal Ensembles in Incubation of Nicotine Craving.
        J Neurosci. 2016; 36: 8612-8623
        • Schiffino F.L.
        • Holland P.C.
        Consolidation of altered associability information by amygdala central nucleus.
        Neurobiol Learn Mem. 2016; 133: 204-213
        • Lindgren J.L.
        • Gallagher M.
        • Holland P.C.
        Lesions of basolateral amygdala impair extinction of CS motivational value, but not of explicit conditioned responses, in Pavlovian appetitive second-order conditioning.
        Eur J Neurosci. 2003; 17: 160-166
        • Hatfield T.
        • Han J.S.
        • Conley M.
        • Gallagher M.
        • Holland P.
        Neurotoxic lesions of basolateral, but not central, amygdala interfere with Pavlovian second-order conditioning and reinforcer devaluation effects.
        J Neurosci. 1996; 16: 5256-5265
        • Burns L.H.
        • Robbins T.W.
        • Everitt B.J.
        Differential effects of excitotoxic lesions of the basolateral amygdala, ventral subiculum and medial prefrontal cortex on responding with conditioned reinforcement and locomotor activity potentiated by intra-accumbens infusions ofd-amphetamine.
        Behavioural Brain Research. 1993; 55: 167-183
        • Setlow B.
        • Gallagher M.
        • Holland P.C.
        The basolateral complex of the amygdala is necessary for acquisition but not expression of CS motivational value in appetitive Pavlovian second-order conditioning.
        Eur J Neurosci. 2002; 15: 1841-1853
      3. Chesworth R, Corbit LJTAWESP, Learning, Memories (2017): The contribution of the amygdala to reward-related learning and extinction.305-325.

        • Toyomitsu Y.
        • Nishijo H.
        • Uwano T.
        • Kuratsu J.
        • Ono T.
        Neuronal responses of the rat amygdala during extinction and reassociation learning in elementary and configural associative tasks.
        Eur J Neurosci. 2002; 15: 753-768
        • Haney R.Z.
        • Calu D.J.
        • Takahashi Y.K.
        • Hughes B.W.
        • Schoenbaum G.
        Inactivation of the central but not the basolateral nucleus of the amygdala disrupts learning in response to overexpectation of reward.
        Journal of Neuroscience. 2010; 30: 2911-2917
        • Cedarbaum J.M.
        • Aghajanian G.K.
        Afferent projections to the rat locus coeruleus as determined by a retrograde tracing technique.
        J Comp Neurol. 1978; 178: 1-16
        • Van Bockstaele E.J.
        • Colago E.E.
        • Valentino R.J.
        Amygdaloid corticotropin-releasing factor targets locus coeruleus dendrites: substrate for the co-ordination of emotional and cognitive limbs of the stress response.
        J Neuroendocrinol. 1998; 10: 743-757
        • Valentino R.J.
        • Van Bockstaele E.
        Convergent regulation of locus coeruleus activity as an adaptive response to stress.
        Eur J Pharmacol. 2008; 583: 194-203
        • Giustino T.F.
        • Maren S.
        Noradrenergic Modulation of Fear Conditioning and Extinction.
        Front Behav Neurosci. 2018; 12: 43
      4. McCall JG, Al-Hasani R, Siuda ER, Hong DY, Norris AJ, Ford CP, et al. (2015): CRH engagement of the locus coeruleus noradrenergic system mediates stress-induced anxiety. 87:605-620.

        • Prouty E.W.
        • Waterhouse B.D.
        • Chandler D.J.
        Corticotropin releasing factor dose-dependently modulates excitatory synaptic transmission in the noradrenergic nucleus locus coeruleus.
        Eur J Neurosci. 2017; 45: 712-722
        • Schwarz L.A.
        • Miyamichi K.
        • Gao X.J.
        • Beier K.T.
        • Weissbourd B.
        • DeLoach K.E.
        • et al.
        Viral-genetic tracing of the input–output organization of a central noradrenaline circuit.
        Nature. 2015; 524: 88-92
        • Iordanova M.D.
        • Yau J.O.
        • McDannald M.A.
        • Corbit L.H.
        Neural substrates of appetitive and aversive prediction error.
        Neurosci Biobehav Rev. 2021; 123: 337-351
        • Sanford C.A.
        • Soden M.E.
        • Baird M.A.
        • Miller S.M.
        • Schulkin J.
        • Palmiter R.D.
        • et al.
        A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats.
        Neuron. 2017; 93: 164-178
        • Giustino T.F.
        • Ramanathan K.R.
        • Totty M.S.
        • Miles O.W.
        • Maren S.
        Locus Coeruleus Norepinephrine Drives Stress-Induced Increases in Basolateral Amygdala Firing and Impairs Extinction Learning.
        J Neurosci. 2020; 40: 907-916
        • Uematsu A.
        • Tan B.Z.
        • Ycu E.A.
        • Cuevas J.S.
        • Koivumaa J.
        • Junyent F.
        • et al.
        Modular organization of the brainstem noradrenaline system coordinates opposing learning states.
        Nat Neurosci. 2017; 20: 1602-1611
        • Abiri D.
        • Douglas C.E.
        • Calakos K.C.
        • Barbayannis G.
        • Roberts A.
        • Bauer E.P.
        Fear extinction learning can be impaired or enhanced by modulation of the CRF system in the basolateral nucleus of the amygdala.
        Behav Brain Res. 2014; 271: 234-239
        • Ciocchi S.
        • Herry C.
        • Grenier F.
        • Wolff S.B.
        • Letzkus J.J.
        • Vlachos I.
        • et al.
        Encoding of conditioned fear in central amygdala inhibitory circuits.
        Nature. 2010; 468: 277-282
        • Cai H.
        • Haubensak W.
        • Anthony T.E.
        • Anderson D.J.
        Central amygdala PKC-δ(+) neurons mediate the influence of multiple anorexigenic signals.
        Nat Neurosci. 2014; 17: 1240-1248
        • Nader K.
        • Schafe G.E.
        • Le Doux J.E.
        Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval.
        Nature. 2000; 406: 722-726
        • Duvarci S.
        • Nader K.
        • LeDoux J.E.
        De novo mRNA synthesis is required for both consolidation and reconsolidation of fear memories in the amygdala.
        Learn Mem. 2008; 15: 747-755