1 Encoding of conditioned fear in central amygdala inhibitory circuits Stephane Ciocchi1*, Cyril Herry1*{, François Grenier1, Steffen B. E.Wolff1, Johannes J. Letzkus1, Ioannis Vlachos2, Ingrid Ehrlich1{, Rolf Sprengel3, Karl Deisseroth4, Michael B. Stadler1, Christian Mu¨ller1 & Andreas Lu¨thi1 Nature Volume:468, Pages:277–282 Date published: (11 November 2010)

2 BACKGROUND The central amygdala (CEA)has been considered to be primarily involved in the behavioural expression of conditioned fear responses CEAoutput neurons(CEm) project to downstream targets in the brainstem and in the hypothalamus where they orchestrate conditioned autonomic and motor responses CEm output neurons are under tight inhibitory control from the lateral and capsular subdivisions (together referred to as CEl)

3 How the acquisition and expression of conditioned fear are encoded within CEA inhibitory circuits ?

4 WHAT WE WILL KNOW IN THIS ARTICLE Neuronal activity in the lateral subdivision of the central amygdala (CEl) is required for fear acquisition. Conditioned fear responses are driven by output neurons in the medial subdivision (CEm). Inhibitory CEA microcircuits are highly organized Cell-type-specific plasticity of phasic and tonic activity in the CEl to CEm pathway may gate fear expression and regulate fear generalization.

5 TECHNOLOGY

6 Opto genetics

7 TECHNOLOGY For more information, go to

8 TECHNOLOGY Microiontophoresis is the technique whereby ions and charged molecules can be ejected in very small amounts from solutions contained in micropipettes OFTEN USED FOR: (1) deposition of dyes and neural transport tracers for histological examination (2) for administration of neuroactive compounds (e.g. transmitters, modulators, drugs or hormones) by microiontophoresis to examine their effects on firing parameters of single neurons in vivo. For more information, go to

9 RESULTS Differential role of CEl and CEm 1.The impact of neuronal activity in CEm on freezing behavior OPTOGENETICS (Activation) Virus expressing ChR2 in neurons

10 RESULTS Bilateral activation of CEm induced strong and reversible freezing responses

11 RESULTS MICROIONTOPHORESIS (Inhibition) Fluorescently labelled GABA A receptor agonist (muscimol-bodipy (BPY)) Bilateral inactivation of CEm, or of the entire CEA (CEm and CEl), did not elicit freezing behaviour. Inactivation of CEl alone induced unconditioned freezing.

12 RESULTS 2. The contribution of distinct CEA subnuclei to the acquisition of conditioned freezing

13 RESULTS 3. The role of CEl and CEm for memory retrieval or expression Local application of muscimol-BPY 24 h after conditioning Inactivation of the entire CEA or CEm resulted in a retrieval/ expression deficit. Inactivation of CEl did not reduce conditioned freezing levels

14 CONCLUSION 1 Conditioned and unconditioned freezing behaviour is driven by CEm output neurons which are under tonic inhibitory control originating in CEl. CEl as an essential component of the neuronal circuitry underlying the acquisition of conditioned fear.

15 RESULTS Organization of CEA inhibitory networks 1. Fear conditioning-induced changes in CS evoked neuronal firing in the CEl 30% of units acquired an excitatory response (CElon neurons) (Fig.a) 25% of CEl neurons displayed a strong inhibitory response to the CS1 after fear conditioning (CEloff neurons) (Fig.b)

16 RESULTS

17 RESULTS The inverse direction of fear-conditioning-induced plasticity in CEIon and CEIoff neurons indicated that...

18 RESULTS CElon to CEloff, 9 of 35 pairs; CEloff to CElon, 3 of 35 pairs;

19 RESULTS 2.Anatomical and functional connectivity between CEl and CEm. Injections into CEm resulted in intense retrograde labelling of neurons in CEl CEm remained largely devoid of GFP after injections into CEl

20 RESULTS 2.1 Whether identified CElon or CEloff neurons project to CEm The axons of both subtypes arborize locally within CEl, and send collaterals to CEm

21 RESULTS 2.2Whether CElon and CEloff neurons functionally inhibit CEm neurons Two distinct subclasses of CEl neurons inhibit CEm neurons in vivo

22 3.Considering that CElon and CEloff neurons exhibited opposite changes in CS-evoked firing during fear conditioning, this raises the question of whether at the level of CEm output neurons fear conditioning results in CS-evoked inhibition or disinhibition.

23 RESULTS

24 RESULTS Conditioned CS responses of CEm output neurons reflect the integration of both excitatory and disinhibitory inputs.

25 CONCLUSION 2 CEl contains two functionally distinct subpopulations of neurons forming highly organized local inhibitory circuits which inhibit CEm output neurons. Conditioned fear responses are driven by CS-evoked disinhibition of CEm output neurons

26 RESULTS Tonic inhibition and fear generalization 1.Whether spontaneous activity in CEl and CEm is subject to regulation ? 2.How plasticity of spontaneous activity might contribute to the encoding of conditioned fear responses ?

27 RESULTS Spontaneous activity of CEm output neurons was markedly decreased after fear conditioning. CEloff neurons exhibited increased spontaneous activity after fear conditioning On average CElon neurons showed a slight decrease

28 RESULTS

29 RESULTS 3.What might be the behavioural relevance of plasticity of tonic activity in CEA inhibitory circuits? After fear conditioning, absolute and z-scored levels of tonic activity were only poorly correlated with freezing

30 RESULTS Fear-conditioning induced changes in tonic activity were not limited to periods of CS + exposure, but were also manifest during CS - stimulation

31 RESULTS A decrease in tonic activity of CEm output neurons was associated with generalization, CEloff neuronsand CElon neurons exhibited the inverse correlation.

32 SUMMARY SECTION 3 SECTION 1 、 2

33 Thank you !