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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:277–282Date published: (11 November 2010)

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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)

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

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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.

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TECHNOLOGY

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TECHNOLOGY

Opto genetics

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TECHNOLOGY

For more information, go to www.stanford.edu/group/dlab/optogenetics/

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TECHNOLOGY

Microiontophoresis is the techniquewhereby ions and charged moleculescan be ejected in very small amounts from solutions contained in micropipettes

OFTEN USED FOR:(1) deposition of dyes and neural transport tracers for histologicalexamination (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 www.kationscientific.com/basics.html

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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

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RESULTS

• Bilateral activation of CEm induced strong and reversible freezing responses

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RESULTSMICROIONTOPHORESIS (Inhibition)Fluorescently labelled GABAA 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.

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RESULTS

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

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RESULTS3. 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

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CONCLUSION 1

Conditioned and unconditioned freezing behaviouris driven by CEm output neurons which are under tonic inhibitory control originating in CEl.

CEl as an essential component of the neuronal circuitryunderlying the acquisition of conditioned fear.

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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)

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RESULTS

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RESULTS

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

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RESULTS

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

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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

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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

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RESULTS

2.2Whether CElon and CEloff neurons functionally inhibit CEm neurons

Two distinct subclasses of CEl neurons inhibit CEm neurons in vivo

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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-evoke

d inhibition or disinhibition.

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RESULTS

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RESULTS

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

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CONCLUSION 2

CEl contains two functionally distinct subpopulationsof neurons forming highly organized local inhibitory circuits which inhibit CEm output neurons.

Conditioned fear responses are driven by CS-evokeddisinhibition of CEm output neurons

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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 ?

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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

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RESULTS

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RESULTS

3.What might be the behavioural relevance of plasticity of tonicactivity in CEA inhibitory circuits?

After fear conditioning, absolute and z-scored levels of tonic activity were only poorly correlated with freezing

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RESULTS

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

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RESULTS

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

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SUMMARY

SECTION 3SECTION 1、 2

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Thank you !