Decades of research has identified the brain areas that are involved in fear, fear extinction, anxiety and related defensive behaviours. Newly developed genetic and viral tools, optogenetics and advanced in vivo imaging techniques have now made it possible to characterize the activity, connectivity and function of specific cell types within complex neuronal circuits. Recent findings that have been made using these tools and techniques have provided mechanistic insights into the exquisite organization of the circuitry underlying internal defensive states. This Review focuses on studies that have used circuit-based approaches to gain a more detailed, and also more comprehensive and integrated, view on how the brain governs fear and anxiety and how it orchestrates adaptive defensive behaviours.

Learning causes a change in how information is processed by neuronal circuits. Whereas synaptic plasticity, an important cellular mechanism, has been studied in great detail, we know much less about how learning is implemented at the level of neuronal circuits and, in particular, how interactions between distinct types of neurons within local networks contribute to the process of learning. Here we show that acquisition of associative fear memories depends on the recruitment of a disinhibitory microcircuit in the mouse auditory cortex. Fear-conditioning-associated disinhibition in auditory cortex is driven by foot-shock-mediated cholinergic activation of layer 1 interneurons, in turn generating inhibition of layer 2/3 parvalbumin-positive interneurons. Importantly, pharmacological or optogenetic block of pyramidal neuron disinhibition abolishes fear learning. Together, these data demonstrate that stimulus convergence in the auditory cortex is necessary for associative fear learning to complex tones, define the circuit elements mediating this convergence and suggest that layer-1-mediated disinhibition is an important mechanism underlying learning and information processing in neocortical circuits.

Survival in threatening situations depends on the selection and rapid execution of an appropriate active or passive defensive response, yet the underlying brain circuitry is not understood. Here we use circuit-based optogenetic, in vivo and in vitro electrophysiological, and neuroanatomical tracing methods to define midbrain periaqueductal grey circuits for specific defensive behaviours. We identify an inhibitory pathway from the central nucleus of the amygdala to the ventrolateral periaqueductal grey that produces freezing by disinhibition of ventrolateral periaqueductal grey excitatory outputs to pre-motor targets in the magnocellular nucleus of the medulla. In addition, we provide evidence for anatomical and functional interaction of this freezing pathway with long-range and local circuits mediating flight. Our data define the neuronal circuitry underlying the execution of freezing, an evolutionarily conserved defensive behaviour, which is expressed by many species including fish, rodents and primates. In humans, dysregulation of this 'survival circuit' has been implicated in anxiety-related disorders.

Learning is mediated by experience-dependent plasticity in neuronal circuits. Activity in neuronal circuits is tightly regulated by different subtypes of inhibitory interneurons, yet their role in learning is poorly understood. Using a combination of in vivo single-unit recordings and optogenetic manipulations, we show that in the mouse basolateral amygdala, interneurons expressing parvalbumin (PV) and somatostatin (SOM) bidirectionally control the acquisition of fear conditioning--a simple form of associative learning--through two distinct disinhibitory mechanisms. During an auditory cue, PV(+) interneurons are excited and indirectly disinhibit the dendrites of basolateral amygdala principal neurons via SOM(+) interneurons, thereby enhancing auditory responses and promoting cue-shock associations. During an aversive footshock, however, both PV(+) and SOM(+) interneurons are inhibited, which boosts postsynaptic footshock responses and gates learning. These results demonstrate that associative learning is dynamically regulated by the stimulus-specific activation of distinct disinhibitory microcircuits through precise interactions between different subtypes of local interneurons.

When faced with threat, the survival of an organism is contingent upon the selection of appropriate active or passive behavioural responses. Freezing is an evolutionarily conserved passive fear response that has been used extensively to study the neuronal mechanisms of fear and fear conditioning in rodents. However, rodents also exhibit active responses such as flight under natural conditions. The central amygdala (CEA) is a forebrain structure vital for the acquisition and expression of conditioned fear responses, and the role of specific neuronal sub-populations of the CEA in freezing behaviour is well-established. Whether the CEA is also involved in flight behaviour, and how neuronal circuits for active and passive fear behaviour interact within the CEA, are not yet understood. Here, using in vivo optogenetics and extracellular recordings of identified cell types in a behavioural model in which mice switch between conditioned freezing and flight, we show that active and passive fear responses are mediated by distinct and mutually inhibitory CEA neurons. Cells expressing corticotropin-releasing factor (CRF) mediate conditioned flight, and activation of somatostatin-positive (SOM) neurons initiates passive freezing behaviour. Moreover, we find that the balance between conditioned flight and freezing behaviour is regulated by means of local inhibitory connections between CRF and SOM neurons, indicating that the selection of appropriate behavioural responses to threat is based on competitive interactions between two defined populations of inhibitory neurons, a circuit motif allowing for rapid and flexible action selection.

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Hippocampal and amygdaloid neuroplasticity are important substrates for Pavlovian fear conditioning. The hippocampus has been implicated in trace fear conditioning. However, a systematic investigation of the significance of the trace interval has not yet been performed. Therefore, this study analyzed the time-dependent involvement of N-methyl-D-aspartate (NMDA) receptors in the dorsal hippocampus in one-trial auditory trace fear conditioning in C57BL/6J mice. The NMDA receptor antagonist APV was injected bilaterally into the dorsal hippocampus 15 min before training. Mice were exposed to tone (conditioned stimulus [CS]) and footshock (unconditioned stimulus [US]) in the conditioning context without delay (0 s) or with CS-US (trace) intervals of 1-45 s. Conditioned auditory fear was determined 24 h after training by the assessment of freezing and computerized evaluation of inactivity in a new context; 2 h later, context-dependent memory was tested in the conditioning context. NMDA receptor blockade by APV markedly impaired conditioned auditory fear at trace intervals of 15 s and 30 s, but not at shorter trace intervals. A 45-s trace interval prevented the formation of conditioned tone-dependent fear. Context-dependent memory was always impaired by APV treatment independent of the trace interval. The results indicate that the dorsal hippocampus and its NMDA receptors play an important role in auditory trace fear conditioning at trace intervals of 15-30-s length. In contrast, NMDA receptors in the dorsal hippocampus are unequivocally involved in contextual fear conditioning independent of the trace interval. The results point at a time-dependent role of the dorsal hippocampus in encoding of noncontingent explicit stimuli. Preprocessing of long CS-US contingencies in the hippocampus appears to be important for the final information processing and execution of fear memories through amygdala circuits.

Highlights d Auditory cortex is selectively required for complex stimulus threat memory d Adjacent temporal association cortex controls all forms of auditory threat memory d Cortico-amygdala information transmission governs complex stimulus memory d Amygdala-projecting neurons show population plasticity for stimulus discrimination