Fear conditioning- and extinction-induced neuronal plasticity in the mouse amygdala

Experience-dependent changes in behavior are mediated by long-term functional modifications in brain circuits. To study the underlying mechanisms, our lab is using classical auditory fear conditioning, a simple and robust form of associative learning. In classical fear conditioning, the subject is exposed to a noxious unconditioned stimulus (US), such as a foot-shock, in conjunction with a neutral conditioned stimulus (CS), such as a tone or a light. As a result of the training, the tone acquires aversive properties and when subsequently presented alone, will elicit a fear response. In rodents, such responses include freezing behavior, alterations in autonomic nervous system activity, release of stress hormones, analgesia, and facilitation of reflexes. Subsequently, conditioned fear can be suppressed when the conditioned stimulus is repeatedly presented alone, a phenomenon called fear extinction. It emerges from a large number of studies in animals and humans that the amygdala is a key brain structure mediating fear conditioning. The amygdala consists of several distinct nuclei, including the lateral (LA) and basal (BA) nuclei, and the central nucleus (CEA). In the classical circuit model of fear conditioning, the LA is thought of as the primary site where CS-US associations are formed and stored. The formation of CS-US associations in the LA is mediated by N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) at glutamatergic sensory inputs originating from auditory thalamus and cortex. In contrast to the LA, the CEA has been considered to be primarily involved in the behavioral expression of conditioned fear responses. While the mechanisms and the circuitry underlying fear conditioning in the LA have been extensively studied, much less is known about the neuronal substrates underlying fear extinction. The question of how conditioned fear can be inhibited by extinction is attracting increasing interest because of its clinical importance for the therapy of anxiety disorders. The amygdala is also a potential site of extinction-associated plasticity since intra-amygdala blockade of NMDA receptors or the MAPK signaling pathway prevents extinction. In the first part of this thesis, a combination of behavioral, pharmacological and in vivo electrophysiological approaches was used to study the role of distinct amygdala sub-nuclei in fear exinction. Single unit recordings in behaving mice revealed that the BA contains distinct types of neurons that are specifically activated upon fear conditioning or extinction, respectively. During acquisition of extinction, the activity of “fear neurons” gradually declines, while “extinction neurons” increase their activity. Conversely, when extinguished fear responses are recovered by placing the animal in an unsafe environment, “extinction neurons” switch off, while “fear neurons” switch on. Using local micro-iontophoretic injection of the GABAA receptor agonist muscimol, we found that inactivation of the BA completely prevents the acquisition of extinction or context-dependent fear recovery, depending on the injection time point. Finally, we could show that “fear neurons” and “extinction neurons” are differentially connected with the medial prefrontal cortex (mPFC) and the ventral hippocampus (vHC), two brain areas involved in context-dependent extinction. In contrast to previous models suggesting that amygdala neurons are active during states of high fear and inactive during states of low fear, our findings indicate that activity in specific neuronal circuits within the amygdala may cause opposite behavioral outcomes, thus providing a new framework for understanding context-dependent expression and extinction of fear behavior. In the second part of the thesis, I examined how inhibitory circuits in the central nucleus of the amygdala (CEA) contribute to fear conditioning. While many studies have demonstrated that neuronal plasticity in the LA is necessary for fear conditioning, the role of the CEA, which is mainly composed of GABAergic inhibitory neurons, is poorly understood. In the classical circuit model, the CEA has been thought of as a passive relay station conveying LA output to downstream targets in the hypothalamus and in the brain stem. However, recent in vivo pharmacological experiments suggest a more active role for the CEA during fear conditioning. To address the role of CEA inhibitory circuits in fear conditioning, we obtained single unit recordings from neurons located in the lateral (CEl) and medial (CEm) subdivisions of the CEA in behaving mice. We found that CEm output neurons, that control fear behavior via projections to brainstem targets, are under tight inhibitory control from a subpopulation of neurons located in CEl. Fear conditioning induced opposite changes in phasic and tonic inhibition in the CEl to CEm pathway. Targeted pharmacological inactivation of CEl and CEm revealed that whereas plasticity of phasic inhibition is necessary for gating CEm output during fear learning and expression, changes in tonic inhibitory network activity control signal-to-noise ratio and stimulus discrimination. Our results identify CEA inhibitory circuits as a major site of plasticity in fear conditioning, and suggest that regulation of tonic activity of inhibitory circuits may be an important mechanism for controlling sensitivity and specificity in associative learning. Taken together, these findings suggest that the amygdala is not a functionally homogeneous structure. Rather, our results reveal that the BA and the CEA contain specialized and discrete neuronal populations that contribute to distinct aspects of fear conditioning and extinction. Ultimately, elucidating these mechanisms is fundamental for an understanding of memory processes in the brain in general, and should also inform novel therapeutic strategies for psychiatric disorders involving excessive fear responses associated with amygdala hypersensitivity such as post-traumatic stress disorder and other anxiety disorders

Firing patterns of ventral hippocampal neurons predict the exploration of anxiogenic locations.

The ventral hippocampus (vH) plays a crucial role in anxiety-related behaviour and vH neurons increase their firing when animals explore anxiogenic environments. However, if and how such neuronal activity induces or restricts the exploration of an anxiogenic location remains unexplained. Here, we developed a novel behavioural paradigm to motivate rats to explore an anxiogenic area. Male rats ran along an elevated linear maze with protective sidewalls, which were subsequently removed in parts of the track to introduce an anxiogenic location. We recorded neuronal action potentials during task performance and found that vH neurons exhibited remapping of activity, overrepresenting anxiogenic locations. Direction-dependent firing was homogenised by the anxiogenic experience. We further showed that the activity of vH neurons predicted the extent of exploration of the anxiogenic location. Our data suggest that anxiety-related firing does not solely depend on the exploration of anxiogenic environments, but also on intentions to explore them

Fear extinction relies on ventral hippocampal safety codes shaped by the amygdala.

Extinction memory retrieval is influenced by spatial contextual information that determines responding to conditioned stimuli (CS). However, it is poorly understood whether contextual representations are imbued with emotional values to support memory selection. Here, we performed activity-dependent engram tagging and in vivo single-unit electrophysiological recordings from the ventral hippocampus (vH) while optogenetically manipulating basolateral amygdala (BLA) inputs during the formation of cued fear extinction memory. During fear extinction when CS acquire safety properties, we found that CS-related activity in the vH reactivated during sleep consolidation and was strengthened upon memory retrieval. Moreover, fear extinction memory was facilitated when the extinction context exhibited precise coding of its affective zones. Last, these activity patterns along with the retrieval of the fear extinction memory were dependent on glutamatergic transmission from the BLA during extinction learning. Thus, fear extinction memory relies on the formation of contextual and stimulus safety representations in the vH instructed by the BLA

Anxiety-related activity of ventral hippocampal interneurons.

Anxiety is an aversive mood reflecting the anticipation of potential threats. The ventral hippocampus (vH) is a key brain region involved in the genesis of anxiety responses. Recent studies have shown that anxiety is mediated by the activation of vH pyramidal neurons targeting various limbic structures. Throughout the cortex, the activity of pyramidal neurons is controlled by GABA-releasing inhibitory interneurons and the GABAergic system represents an important target of anxiolytic drugs. However, how the activity of vH inhibitory interneurons is related to different anxiety behaviours has not been investigated so far. Here, we integratedin vivoelectrophysiology with behavioural phenotyping of distinct anxiety exploration behaviours in rats. We showed that pyramidal neurons and interneurons of the vH are selectively active when animals explore specific compartments of the elevated-plus-maze (EPM), an anxiety task for rodents. Moreover, rats with prior goal-related experience exhibited low-anxiety exploratory behaviour and showed a larger trajectory-related activity of vH interneurons during EPM exploration compared to high anxiety rats. Finally, in low anxiety rats, trajectory-related vH interneurons exhibited opposite activity to pyramidal neurons specifically in the open arms (i.e. more anxiogenic) of the EPM. Our results suggest that vH inhibitory micro-circuits could act as critical elements underlying different anxiety states

Activity of ventral hippocampal parvalbumin interneurons during anxiety.

Anxiety plays a key role in guiding behavior in response to potential threats. Anxiety is mediated by the activation of pyramidal neurons in the ventral hippocampus (vH), whose activity is controlled by GABAergic inhibitory interneurons. However, how different vH interneurons might contribute to anxiety-related processes is unclear. Here, we investigate the role of vH parvalbumin (PV)-expressing interneurons while mice transition from safe to more anxiogenic compartments of the elevated plus maze (EPM). We find that vH PV interneurons increase their activity in anxiogenic EPM compartments concomitant with dynamic changes in inhibitory interactions between PV interneurons and pyramidal neurons. By optogenetically inhibiting PV interneurons, we induce an increase in the activity of vH pyramidal neurons and persistent anxiety. Collectively, our results suggest that vH inhibitory microcircuits may act as a trigger for enduring anxiety states

Fluid network dynamics in the prefrontal cortex during multiple strategy switching

Coordinated shifts of neuronal activity in the prefrontal cortex are associated with strategy adaptations in behavioural tasks, when animals switch from following one rule to another. However, network dynamics related to multiple-rule changes are scarcely known. We show how firing rates of individual neurons in the prelimbic and cingulate cortex correlate with the performance of rats trained to change their navigation multiple times according to allocentric and egocentric strategies. The concerted population activity exhibits a stable firing during the performance of one rule but shifted to another neuronal firing state when a new rule is learnt. Interestingly, when the same rule is presented a second time within the same session, neuronal firing does not revert back to the original neuronal firing state, but a new activity-state is formed. Our data indicate that neuronal firing of prefrontal cortical neurons represents changes in strategy and task-performance rather than specific strategies or rules