Date of Award

8-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Neuroscience

Research Advisor

Max L. Fletcher, Ph.D.

Committee

John D. Boughter, Matthew Ennis, Detlef H. Heck, Robert J. Ogg

Keywords

calcium imaging, fear learning, generalization, neuromodulation, olfactory bulb

Abstract

The exploration of how learning alters neural coding to guide behaviors remains fundamental to neuroscience. At the most basic level, the ability for organisms to flexibly adapt to changing environments and situations is paramount to biological success and often manifests in behavioral responses controlled by neural activity. For example, organisms must modify their behavior to defensive responses in the face of biological threat. Neural circuitry is involved in coordinating an initial defensive behavioral response but must undergo reorganization in order to reliably employ defensive responses in subsequent encounters based on a cue that signals imminent danger. One such form of this learning is associative fear learning, in which an organism learns to associate an initially neutral stimulus, which by itself has no biological relevance, with an innately fear-inducing stimulus. After temporally pairing the two stimuli, organisms learn that the initially neutral stimulus predicts the fear-inducing stimulus such that encountering the former induces similar behavioral responses as the latter." "Interestingly, associative fear learning is often not specific and organisms display fear responses to completely neutral stimuli that have never been associated with the innately fear-inducing stimulus. This aberrant expression of fear is referred to as generalization and is a hallmark of many psychological disorders such as anxiety and post-traumatic stress disorder. The underlying cause of fear generalization remains unknown; however, there are two predominant theories. One theory is fear generalization arises as a better-safe-than-sorry strategy, wherein sensory information remains perceptually segregated between fear predictive and neutral stimuli but the organism responds to both the same way despite the perceptual information. The second theory argues that learning alters sensory processing to make the stimuli more difficult to perceptually distinguish, which cascades into behaviorally treating them as the same. The olfactory bulb (OB) represents an ideal model system for studying the extent to which fear learning alters sensory coding in ways that support failure of perceptual discrimination due to its odotopic organization that creates unique “maps” of odor representation for each experience odor. Therefore, we used both wide-field and 2-photon (2P) calcium imaging of the OBs of awake mice in combination with classical olfactory fear conditioning to investigate how learning changes sensory representation of the conditioned odor, the initially neutral odor specifically paired with fear-inducing foot shock, as well as neutral odors never paired with shock. This allowed direct testing of the extent to which fear learning reorganizes olfactory processing in a manner that supports the failure to discriminate hypothesis of fear generalization. Both wide-field and 2P imaging revealed enhanced odor-evoked responses following fear learning that likely signal increased salience of incoming sensory information. Furthermore, the responses evoked by neutral odors became more similar to those evoked by the conditioned odor at both the population level (wide-field) and in a specific subset of OB output cells (2P), indicating neutral odors were more difficult to distinguish from the conditioned odor. Importantly, the enhanced odor-evoked responses were not attributable to behavioral state change nor top-down influence from the amygdala, the area widely believed to be involved in aberrant fear. Together, this evidence supported the failure of perceptual discrimination at early stages of sensory process as the underlying mechanism of fear generalization. We additionally investigated the role of neuromodulators in basic olfactory fear learning through in vivo pharmacology, optogenetics, and relative gene expression analysis. Manipulation of acetylcholine in the OB during olfactory fear learning established cholinergic neurotransmission can enhance the strength of learned fear and that signaling through muscarinic receptors is required for the formation of olfactory fear. Gene expression analysis revealed that several neurotransmitter receptors are downregulated 4 hours following odor-shock pairing. The majority of the downregulated genes were associated with OB inhibition suggesting that fear learning, behavioral fear generalization, and altered OB coding may arise from decreased inhibition in the OB. Altogether these results characterize neural correlates of fear generalization in the olfactory bulb at both the population and single cell level and demonstrate the importance of neuromodulation in fear learning, supporting the idea that fear generalization may initially arise from altered processing of incoming information in sensory areas. These results also highlight the importance of investigating the mechanisms of learning-induced sensory processing alterations as they relate to behavior in understanding fear generalization. This will bring insights into basic processes of learning and has the immense potential to translate to the treatment of disorders of fear generalization.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0003-4345-0135

DOI

10.21007/etd.cghs.2019.0484

Additional Files
2019-014-Ross-DOA.pdf (441 kB)
Declaration of Authorship
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