AMYGDALA IMAGING · Functional in vivo two-photon imaging of fear memory traces in identified neuronal networks of the amygdala

Functional in vivo two-photon imaging of fear memory traces in identified neuronal networks of the amygdala

Project objectives Human anxiety disorders, such as phobias and post-traumatic stress disorders, are widespread, debilitating conditions that pose a major clinical challenge. In order to develop rational therapeutic approaches, a detailed understanding of the underlying brain areas and neuronal circuits is required. Auditory fear learning in mice shares major attributes with human anxiety disorders, making it a robust model system in which the neuronal mechanisms can be investigated with great precision, thus capitalising on a wide repertoire of established tools. While we have a reasonable understanding of the cellular and molecular mechanisms that underlie fear learning, there is a considerable lack of information regarding the interactions of neurons in local networks during memory acquisition. Neuronal circuits are composed of a large variety of neuron types, each of which is likely to have distinct computational roles, and it is largely their dynamic interactions that give rise to higher brain functions such as learning and memory formation. Therefore, the aim of this project was to characterise how fear learning is accomplished by identified neurons interacting in local circuits. Work performed The amygdala, a group of nuclei in the temporal lobe, has long been identified as a critical locus for fear learning (see below). In contrast, when we started this project, the role of the auditory cortex in fear conditioning was far less understood, with published evidence both for and against an essential involvement. Our research showed that the auditory cortex is indeed indispensable for fear conditioning with complex, naturalistic tones (Letzkus et al., 2011). Surprisingly, we found that the auditory cortex responds not only to tones, but also to mild electrical stimuli which drive fear memory formation when coinciding with tones. A detailed analysis revealed that electrical stimuli cause release of acetylcholine in the auditory cortex, which in turn activates a novel microcircuit leading to disinhibition of projection neurons. Activation of this microcircuit greatly enhanced the auditory responses of projection neurons, likely triggering induction of synaptic plasticity that may underlie fear memory formation. A selective optogenetic block of the auditory cortex's disinhibition during the electrical stimulus strongly reduced fear learning, indicating that the convergence of auditory and aversive stimuli in the auditory cortex is required for fear learning. This work clearly identifies the auditory cortex as a critical site for active association during fear conditioning. Moreover, these experiments reveal that the acetylcholine release in the neocortex acutely fires a specific class of interneuron with unanticipated temporal precision. This finding has potential implications for disorders characterised by cholinergic dysfunction, such as Alzheimer's disease and tobacco abuse. Finally, our results identify disinhibition as an important mechanism for learning in neuronal circuits (see also Ciocchi et al., 2010). This article has been highlighted in 'Nature Reviews Neuroscience' (Flight, 2011). In addition, I gave an interview on our work to 'Nature Neuropod' (see http://origin.network.nature.com/neurosci/neuropod/index-2011-12-23.html for more detail). This detailed analysis of the circuit events leading to memory formation in the auditory cortex was made possible largely by high resolution in vivo imaging techniques. However, conventional imaging approaches are limited to superficial brain structures. To overcome this fundamental limitation, we have developed a fibre-optic-based system that can be used for functional imaging of deep brain structures like the amygdala in freely-behaving mice. This approach, which represents the first successful attempt to physiological in vivo imaging in the amygdala, was spearheaded by Milica Markovic in the lab. We now employ a combination of viral and transgenic strategies to express genetically encoded calcium indicators in a cell-type specific fashion in the amygdala. Subsequently, an optical fibre is positioned just above the lateral amygdala through which excitation and emission are delivered. This novel approach will make it possible to address many open questions regarding the contribution of identified types of amygdala neurons to memory formation. Main results In summary, this project has considerably advanced our understanding of the brain areas and circuit mechanisms that are involved in associative fear learning, and has established a novel methodology for functional investigation of deep brain areas like the amygdala. This research has implications for future development of therapeutic approaches to several human disorders, including anxiety disorders, Alzheimer's disease and tobacco abuse. In addition, at a much broader level, learning and memory formation are at the heart of what makes us human, so we believe that our results will also be of intellectual interest to the wider community. References Letzkus, J. J., Wolff, S. B. E., Meyer, E. M. M., Tovote, P., Courtin, J., Herry, C., Lüthi, A. (2011). 'A disinhibitory microcircuit for associative fear learning in auditory cortex'. Nature; 480; 331 - 335. Ciocchi, S., Herry, C., Grenier, F., Wolff, S. B. E., Letzkus, J. J., Vlachos, I., Ehrlich, I., Sprengel, R., Deisseroth, K., Stadler, M. B., Müller, C., Lüthi, A. (2010). 'Encoding of conditioned fear in central amygdala inhibitory circuits'. Nature; 468; 277 - 282.

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