Himmelfarb Health Sciences Library, The George Washington University Health Sciences Research Commons Pediatrics Faculty Publications Pediatrics 5-2018 Amygdala Corticofugal Input Shapes Mitral Cell Responses in the Accessory Olfactory Bulb Livio Oboti George Washington University Eleonora Russo Tuyen Tran Daniel Durstewitz Joshua G. Corbin George Washington University Follow this and additional works at: https://hsrc.himmelfarb.gwu.edu/smhs_peds_facpubs Part of the Nervous System Diseases Commons, Neurology Commons, and the Pediatrics Commons APA Citation Oboti, L., Russo, E., Tran, T., Durstewitz, D., & Corbin, J. G. (2018). Amygdala Corticofugal Input Shapes Mitral Cell Responses in the Accessory Olfactory Bulb. eNeuro, 5 (3). http://dx.doi.org/10.1523/ENEURO.0175-18.2018 This Journal Article is brought to you for free and open access by the Pediatrics at Health Sciences Research Commons. It has been accepted for inclusion in Pediatrics Faculty Publications by an authorized administrator of Health Sciences Research Commons. For more information, please contact [email protected]. New Research Sensory and Motor Systems Amygdala Corticofugal Input Shapes Mitral Cell Responses in the Accessory Olfactory Bulb Livio Oboti,1 Eleonora Russo,2 Tuyen Tran,1 Daniel Durstewitz,2 and Joshua G. Corbin1 DOI:http://dx.doi.org/10.1523/ENEURO.0175-18.2018 1Center for Neuroscience Research, Children’s National Health System, Washington, DC 20010 and 2Department of Theoretical Neuroscience, Bernstein Center for Computational Neuroscience, Central Institute of Mental Health, Medical Faculty Mannheim of Heidelberg University, 68159 Mannheim, Germany Abstract Interconnections between the olfactory bulb and the amygdala are a major pathway for triggering strong behavioral responses to a variety of odorants. However, while this broad mapping has been established, the patterns of amygdala feedback connectivity and the influence on olfactory circuitry remain unknown. Here, using a combination of neuronal tracing approaches, we dissect the connectivity of a cortical amygdala [posteromedial cortical nucleus (PmCo)] feedback circuit innervating the mouse accessory olfactory bulb. Optogenetic activation of PmCo feedback mainly results in feedforward mitral cell (MC) inhibition through direct excitation of GABAergic granule cells. In addition, LED-driven activity of corticofugal afferents increases the gain of MC responses to olfactory nerve stimulation. Thus, through corticofugal pathways, the PmCo likely regulates primary olfactory and social odor processing. Key words: accessory olfactory bulb; amygdala; circuitry; connectivity; mitral cells Significance Statement Olfactory inputs are relayed directly through the amygdala to hypothalamic and limbic system nuclei, regulating essential responses in the context of social behavior. However, it is not clear whether and how amygdala circuits participate in the earlier steps of olfactory processing at the level of the olfactory bulb. Unraveling the organization of this circuitry is critical to understand the function of amygdala circuits. Combining cre-dependent viral tracing with optogenetic-assisted patch-clamp electrophysiology, the present work maps the synaptic connectivity and physiology of a cortical amygdala pathway innervating primary olfactory circuits. Introduction Accessory olfactory bulb (AOB) neurons processing these The accessory olfactory system (AOS) plays a crucial chemical signals relay their output directly to the amyg- role in the detection of sensory signals used for individual dala, which in turn provides feedback projections to AOB recognition in the context of social, reproductive and parental relationships (Winans and Powers, 1977; Halp- Acknowledgments: We would like to thank K. Briggman (Department of Com- ern, 1987; Meredith, 1991; Dulac and Wagner, 2006). putational Neuroethology-Ceasar Institut-Bonn, Germnay) for Pcdh21cre mice; J. Elmquist (Department of Internal Medicine-UT Southwestern Medical Center, USA) for Sim1cre mice; R. Araneda, K. Sokolowski, P. Li, S. Mahapatra, E. Jacobi, Received May 3, 2018; accepted May 10, 2018; First published May 22, 2018. W. Kelsch, F. Albeanu and his lab, E. Demir, A. Sheikh and members of the Triplett The authors declare no competing financial interests or conflict of interest. and Corbin labs for helpful discussions and input. Author contributions: L.O. designed research; L.O. and T.T. performed Correspondence should be addressed to either Livio Oboti or Joshua G. research; L.O., E.R., and D.D. analyzed data; L.O., E.R., D.D., and J.G.C. wrote Corbin, Center for Neuroscience Research, Children’s National Health the paper. System, Washington, DC 20010, E-mail: [email protected] or This work was supported by National Institutes of Health (NIH) Grants [email protected]. R01-NIDA020140 (J.G.C.) and R01-DC-012050 (J.G.C.). Core support was DOI:http://dx.doi.org/10.1523/ENEURO.0175-18.2018 provided by the Children’s National Health Center Intellectual and Develop- Copyright © 2018 Oboti et al. mental Disabilities Research Center Imaging and Microscopy Core (NIH ID- This is an open-access article distributed under the terms of the Creative DRC Grant U54-HD-090257). E.R. and D.D. were supported by grants from the Commons Attribution 4.0 International license, which permits unrestricted use, German Science (Department of Computational Neuroethology - Caesar Insti- distribution and reproduction in any medium provided that the original work is tut - Bonn, Germany) Foundation (Du 354/8-2, CRC-1134). properly attributed. May/June 2018, 5(3) e0175-18.2018 1–16 New Research 2 of 16 circuits (Raisman, 1972). Although the precise cell-to-cell Arenk/J; stock #024708), GADcre mice (Gad2 Ͻ tm2(cre) connectivity of these circuits is largely unknown, the lack ZjhϾ/J; RRID:MGI:4418723), and Dlx5/6cre mice (Tg(dlx6a- of thalamic relays implies that any refinement of the in- cre)1Mekk/J; RRID:IMSR_JAX:008199) were all obtained coming sensory information must be conducted by pri- from The Jackson Laboratory. Sim1cre mice were provided mary AOS circuits, amygdala feedback projections, or by Joel Elmquist (Tg(Sim1-cre)1Lowl/J; RRID:IMSR_JAX: both. 006395), and Pcdh21cre animals were provided by The AOS detects olfactory information through sensory Dr. Kevin Briggman [Tg(Cdhr1-cre) KG76Gsat; RRID: neurons localized in the vomeronasal organ (VNO). Each MMRRC_036074-UCD]. sensory neuron innervates multiple glomeruli in the AOB, the most posterior-dorsal bulbar region (Belluscio et al., Viral vectors and stereotaxic injections 1999). Here, mitral cells (MCs) integrate inputs from multiple The following procedures were followed for each tracer glomeruli (Wagner et al., 2006) before relaying this informa- or viral vector injected: postpubertal mice (postnatal day tion directly to the medial amygdala (MeA) and cortical [pos- 30–50) were anesthetized with an intraperitoneal injection teromedial cortical nucleus (PmCo)] amygdala subnuclei ofa10␮l/g of anesthetic cocktail (8.5 ml sterile saline, 1 (Winans and Scalia, 1970). Importantly, this connectivity dif- ml 100 mg/ml ketamine, 0.5 ml 20 mg/ml xylazine). Injec- fers dramatically from the main olfactory bulb (MOB), where tion sites targeting the PmCo were determined based on each MC contacts a single glomerulus composed of input coordinates that referred to bregma: X, Ϫ2.5; Y, 2.6; Z, from sensory neurons expressing the same receptor sub- Ϫ5.3. Injections (50–100 nl) were made bilaterally using class. Therefore, whereas in the MOB each MC primarily beveled glass pipettes (Kingston Glass) at depths of 5.1– encodes inputs from single odorants, AOB MCs convey to 5.3 mm from the pial surface. Viral injections were man- the amygdala related blends of chemical ligands, which ually assisted by the use of a Pico Injector (catalog #pli- can be as complex as the number of afferent receptor 100, Harvard Apparatus), each pressure step delivering neurons on a given MC. Surprisingly, AOB MCs are ca- 10–20 nl, 1 per minute. Ten minutes after the final injec- pable of highly selective responses to complex individual tion, the glass pipette was withdrawn and the wound odor signatures (Luo et al., 2003; Ben-Shaul et al., 2010), sutured. Pseudotyped rabies virus (PRV) tracing from the yet how such narrow tuning is achieved is unclear. Among AOB was preferably performed using the RABV mouse the possible mechanisms, lateral inhibition through local line due to problems encountered with tissue damage and GABAergic granule interneurons [granule cells (GCs)] has starter cell viability, especially in AOB GCs. been proposed for both the MOB and AOB (Hendrickson Cholera toxin subunit-B (Ct-b; Alexa Fluor 555 Conju- et al., 2008; Geramita et al., 2016). In the MOB, in addition gate, C34776; Alexa Fluor 488 Conjugate, C22841; to these horizontal interactions, GC activity is also Thermo Fisher Scientific) was diluted 10 ␮g/␮l in sterile strongly modulated by top-down feedback from the piri- PBS, aliquoted, and stored at 4°C until use. The following form cortex (Balu et al., 2007; Matsutani, 2010; Boyd viral vectors were obtained as follows: University of North et al., 2012). Not only has it become increasingly evident Carolina Vector Core: double-floxed reporter, rAAV5/ that this modulatory feedback represents a critical com- EF1a-DIO-eYFP; University of Pennsylvania Vector Core: ponent of olfactory perception (Boyd et al., 2012; Marko- double-floxed channelrhodopsin 2 (ChR2), AAV9.EF1.dflox. poulos et al., 2012; Otazu et al., 2015; Oettl