Abstract
Key in aversive emotional states are serotonergic (5-HT) projections to the basolateral amygdala (BLA) originating from the dorsal raphe nucleus (DRN). Despite being critical in aversive states, the precise anatomical location of 5-HT DRN neurons that project to the BLA is not well delineated. Vital in recent advancements in neuroscience are neurotropic adeno-associated viral (AAV) vectors, which allow for long-term expression of transgenes. However, a recent study using retrograde transport optimized AAV (rAAV2-retro) tracers has shown that transfection of axon terminals and subsequent retrograde transport may not be as ubiquitous as traditional non-transgenic tracer techniques. In this study, we wanted to not only map the precise location of neurons within the DRN that project to the BLA but also test the extent to which rAAV2-retro vectors were useful tools to study these projection neurons. In C57 mice, we iontophoretically deposited (+3-μA positive current at 7s intervals for 10 min, n=3 mice) or pressure nanoinjected (100 nl, n=3 mice) rAAV2-retro expressing the fluorescent reporter GFP (rAAV2-retro-CAG-GFP) into the BLA. After allowing two weeks for GFP expression, coronal sections were prepared from perfusion-fixated tissue and GFP expression was assessed in the DRN as well as the medial entorhinal cortex (mEC), a well validated input to the BLA. We found that iontophoretic deposition failed to robustly label the DRN and mEC. In contrast, while pressure nanoinjection failed to label many DRN neurons, the mEC was robustly labeled. These results may suggest a lack of DRN axon terminals within the viral injection sites or a failure of AAV retrograde tracer transduction in DRN projections to the BLA. Future studies comparing traditional retrograde tracer techniques to our results are warranted. In sum, these results caution the use of rAAV2-retro vectors to study DRN inputs to the BLA. MH093320 (LCD & GMT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Published Version
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