Serotonin (5-HT) has been linked to a large array of behavioral and affective states in both healthy and disordered brains, yet technological limitations have historically constrained interrogation of this circuitry. 5-HT is both anatomically ubiquitous and functionally heterogeneous, and consequently demonstrates a diverse scope of behavioral involvement—experimental and theoretical controversies in the field have hindered efforts to describe a unified role of 5-HT.
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Originating from the raphe nuclei, the 5-HT system exhibits distinct projections throughout the brain, engaging in widespread communication with downstream targets. Notably, the ventral tegmental area (VTA) is a midbrain structure involved in both reward and non-reward circuitry that receives modulatory input from the dorsal raphe nucleus (DRN). DRN also projects to orbitofrontal cortex (OFC), which assumes a pivotal role in top-down modulation of learning, while a separate subcortical DRN pathway showcases prominent 5-HT projections to the bed nucleus of the stria terminalis (BNST), influencing anxiety circuitry. I explored serotonergic input at each of these regions of interest during Pavlovian aversive learning, using next-generation fluorescent indicators for 5-HT developed in the lab.
Genetically-encoded neuromodulator indicators (GENIs) are powerful optical tools that can be employed to directly report neuromodulator dynamics with subsecond resolution. The indicators described in my dissertation are composed of a single fluorescent protein (FP) domain inserted into a ligand-binding scaffold—either a G-protein coupled receptor (GPCR) or a modified bacterial periplasmic binding protein (PBP). These distinct design approaches inform the properties of the indicator, such as binding affinity, dynamic range, and temporal kinetics. In turn, the in vivo performance of an indicator will be shaped by these parameters in addition to the biological context in which it is applied.
I described in vitro and in vivo characterization of fluorescent indicators for 5-HT release in OFC, BNST, and VTA during Pavlovian aversive learning. First, I compared the GPCR-based psychLight with PBP-based iSeroSnFR, demonstrating that the affinity and dynamic range of the latter render it better suited to in vivo application.
I then showed the dissociability between in vitro characterization of the iSeroSnFR suite (versions 1.0, 1.4, and 2.0) and in vivo functionality. I benchmarked iSeroSnFRs1.0,1.4, and 2.0 in each of the three regions of interest and found that biological context plays a significant role in indicator performance. I also noted conserved motifs and region-specific nuances in endogenous 5-HT release in a Pavlovian aversive learning paradigm.
Next, I examined the effects of acute (1 day) and chronic (28 days) oral administration of the selective serotonin reuptake inhibitor (SSRI) fluoxetine on 5-HT release in OFC and BNST, alongside behavioral measures of aversive learning. The results illustrate distinct behavioral and functional effects of these treatments. Acute SSRI mice exhibited heightened cue-evoked 5-HT release and freezing, while the cue-evoked 5-HT response was nearly ablated in chronic SSRI mice, although their freezing was not significantly different from no drug controls.
I then investigated the behavioral and functional effects of increasing shock intensity during Pavlovian aversive learning, specifically during conditioning and extinction. The behavioral effect of this manipulation was clear—high shock animals exhibited heightened freezing to both cue and context. The functional effect on 5-HT response, however, was less predictable. I implemented logistic regression to examine within-trial predictability of freezing by 5-HT signal, and found region- and context-specific relationships that underscore the functional heterogeneity of the serotonergic system.
Finally, I discussed the broader implications of this work, focusing on future technological directions in the field of systems neuroscience and holistic investigation of the 5-HT neuromodulatory network. I outlined important considerations for tool development and application, and presented a multi-component schema linking emotional state, behavior, and serotonergic release. By combining rigorous tool validation with behavioral interrogation, this work lays a foundation for future studies to elucidate the 5-HT dynamics underlying healthy cognition and psychiatric disease.
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