Jonathan P. Fadok

Neuronal circuits for fear and anxiety

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.

Disruption of NMDAR-dependent burst firing by dopamine neurons provides selective assessment of phasic dopamine-dependent behavior

Midbrain dopamine (DA) neurons fire in 2 characteristic modes, tonic and phasic, which are thought to modulate distinct aspects of behavior. However, the inability to selectively disrupt these patterns of activity has hampered the precise definition of the function of these modes of signaling. Here, we addressed the role of phasic DA in learning and other DA-dependent behaviors by attenuating DA neuron burst firing and subsequent DA release, without altering tonic neural activity. Disruption of phasic DA was achieved by selective genetic inactivation of NMDA-type, ionotropic glutamate receptors in DA neurons. Disruption of phasic DA neuron activity impaired the acquisition of numerous conditioned behavioral responses, and dramatically attenuated learning about cues that predicted rewarding and aversive events while leaving many other DA-dependent behaviors unaffected.

Activation of dopamine neurons is critical for aversive conditioning and prevention of generalized anxiety

Generalized anxiety is thought to result, in part, from impairments in contingency awareness during conditioning to cues that predict aversive or fearful outcomes. Dopamine neurons of the ventral midbrain exhibit heterogeneous responses to aversive stimuli that are thought to provide a critical modulatory signal to facilitate orientation to environmental changes and assignment of motivational value to unexpected events. Here we describe a mouse model in which activation of dopamine neurons in response to an aversive stimulus is attenuated by conditional genetic inactivation of functional NMDA receptors on dopamine neurons. We discovered that altering the magnitude of excitatory responses by dopamine neurons in response to an aversive stimulus was associated with impaired conditioning to a cue that predicts an aversive outcome. Impaired conditioning by these mice was associated with the development of a persistent, generalized anxiety-like phenotype. These data are consistent with a role for dopamine in facilitating contingency awareness that is critical for the prevention of generalized anxiety.

Dopamine Is Necessary for Cue-Dependent Fear Conditioning

Dopamine (DA) is implicated in many behaviors, including motor function, cognition, and reward processing; however, the role of DA in fear processing remains equivocal. To examine the role of DA in fear-related learning, dopamine-deficient (DD) mice were tested in a fear-potentiated startle paradigm. DA synthesis can be restored in DD mice through administration of 3, 4-dihydroxy-l-phenylalanine (l-Dopa), thereby permitting the assessment of fear processing in either a DA-depleted or -replete state. Fear-potentiated startle was absent in DD mice but could be restored by l-Dopa administration immediately after fear conditioning. Selective viral-mediated restoration of DA synthesis within the ventral tegmental area fully restored fear learning in DD mice, and restoration of DA synthesis to DA neurons projecting to the basolateral amygdala restored short-term memory but not long-term memory or shock sensitization. We also demonstrate that the DA D1 receptor (D1R) and D2-like receptors are necessary for cue-dependent fear learning. These findings indicate that DA acting on multiple receptor subtypes within multiple target regions facilitates the stabilization of fear memory.

Requirement of dopamine signaling in the amygdala and striatum for learning and maintenance of a conditioned avoidance response

Two-way active avoidance (2WAA) involves learning Pavlovian (association of a sound cue with a foot shock) and instrumental (shock avoidance) contingencies. To identify regions where dopamine (DA) is involved in mediating 2WAA, we restored DA signaling in specific brain areas of dopamine-deficient (DD) mice by local reactivation of conditionally inactivated Th genes using viral gene therapy. Among all targeted areas--prefrontal cortex (PFC), amygdala, ventral striatum, dorsal striatum, and whole striatum--only restoration of DA signaling to both the whole striatum together with the amygdala enabled DD mice to acquire 2WAA. However, after prolonged overtraining during which DD mice had DA synthesis systemically reconstituted pharmacologically with L-3,4-dihydroxyphenylalanine (L-Dopa), DA signaling in the striatum alone was sufficient to maintain 2WAA, whereas DA signaling in the PFC together with the amygdala was insufficient to maintain 2WAA. Our results indicate that learning 2WAA requires DA signaling in both the amygdala and the entire striatum; however, after sufficient training, DA signaling in the striatum alone can maintain the learned avoidance behavior.

Regulating anxiety with extrasynaptic inhibition

Aversive experiences can lead to complex behavioral adaptations including increased levels of anxiety and fear generalization. The neuronal mechanisms underlying such maladaptive behavioral changes, however, are poorly understood. Here, using a combination of behavioral, physiological and optogenetic approaches in mouse, we identify a specific subpopulation of central amygdala neurons expressing protein kinase C δ (PKCδ) as key elements of the neuronal circuitry controlling anxiety. Moreover, we show that aversive experiences induce anxiety and fear generalization by regulating the activity of PKCδ+ neurons via extrasynaptic inhibition mediated by α5 subunit-containing GABAA receptors. Our findings reveal that the neuronal circuits that mediate fear and anxiety overlap at the level of defined subpopulations of central amygdala neurons and demonstrate that persistent changes in the excitability of a single cell type can orchestrate complex behavioral changes.

Long-Term Memory for Pavlovian Fear Conditioning Requires Dopamine in the Nucleus Accumbens and Basolateral Amygdala

The neurotransmitter dopamine (DA) is essential for learning in a Pavlovian fear conditioning paradigm known as fear-potentiated startle (FPS). Mice lacking the ability to synthesize DA fail to learn the association between the conditioned stimulus and the fear-inducing footshock. Previously, we demonstrated that restoration of DA synthesis to neurons of the ventral tegmental area (VTA) was sufficient to restore FPS. Here, we used a target-selective viral restoration approach to determine which mesocorticolimbic brain regions receiving DA signaling from the VTA require DA for FPS. We demonstrate that restoration of DA synthesis to both the basolateral amygdala (BLA) and nucleus accumbens (NAc) is required for long-term memory of FPS. These data provide crucial insight into the dopamine-dependent circuitry involved in the formation of fear-related memory.

A competitive inhibitory circuit for selection of active and passive fear responses

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.

A behavioral genetics approach to understanding D1 receptor involvement in phasic dopamine signaling.

Dopamine-producing neurons fire with both basal level tonic patterns and phasic bursts. Varying affinities of the five dopamine receptors have led to a hypothesis that higher affinity receptors are primarily activated by basal level tonic dopamine, while lower affinity receptors may be tuned to be sensitive to higher levels caused by phasic bursts. Genetically modified mice provide a method to begin to probe this hypothesis. Here we discuss three mouse models. Dopamine-deficient mice were used to determine which behaviors require dopamine. These behaviors were then analyzed in mice lacking D1 receptors and in mice with reduced phasic dopamine release. Comparison of the latter two mouse models revealed a similar failure to learn about and respond normally to cues that indicate either a positive or negative outcome, giving support to the hypothesis that phasic dopamine release and the D1 receptor act in the same pathway. However, the D1 receptor likely has additional roles beyond those of phasic dopamine detection, because D1 receptor knockout mice have deficits in addition to what has been observed in mice with reduced phasic dopamine release.

New perspectives on central amygdala function

The central nucleus of the amygdala (CEA) is a striatum-like structure orchestrating a diverse set of adaptive behaviors, including defensive and appetitive responses [1-3]. Studies using anatomical, electrophysiological, imaging and optogenetic approaches revealed that the CEA network consists of recurrent inhibitory circuits comprised of precisely connected functionally and genetically defined cell types that can select and control specific behavioral outputs [3,4,5•,6•,7-9,11,12]. While bivalent functionality of the CEA in adaptive behavior has been clearly demonstrated, we are just beginning to understand to which degree individual CEA circuit elements are functionally segregated or overlapping. Importantly, recent studies seem to suggest that optogenetic manipulations of the same, or overlapping cell populations can give rise to distinct, or sometimes even opposite, behavioral phenotypes [5•,6•,9-12]. In this review, we discuss recent progress in our understanding of how defined CEA circuits can control defensive and appetitive behaviors, and how seemingly contradictory results could point to an integrated concept of CEA function.