This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Systems/Circuits Cortical connections position primate area 25 as a keystone for interoception, emotion, and memory Mary Kate P. Joyce1,2 and Helen Barbas1,2 1Graduate Program in Neuroscience, Boston University and School of Medicine, Boston, Massachusetts 02215 2Neural Systems Laboratory, Department of Health Sciences, Boston University, Boston, Massachusetts 02215 DOI: 10.1523/JNEUROSCI.2363-17.2017 Received: 21 August 2017 Revised: 10 November 2017 Accepted: 8 December 2017 Published: 22 January 2018 Author contributions: M.K.P.J. and H.B. designed research; M.K.P.J. and H.B. performed research; M.K.P.J. and H.B. analyzed data; M.K.P.J. and H.B. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was supported by NIH grants from NINDS and NIMH. We thank Drs. M. Á. García-Cabezas, B. Zikopoulos, and Y. John for helpful discussions. Corresponding author with complete address, including an email address and postal code: Helen Barbas, Boston University, 635 Commonwealth Avenue, Room 431, Boston, MA 02215; email: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.2363-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors ϭ Title: Cortical connections position primate area 25 as a keystone for interoception, emotion, and Ϯ memory ϯ Abbreviated title: Cortical connections of subgenual cortex ϰ Author names and affiliation, including postal codes: Mary Kate P. Joyce1,2 and Helen ϱ Barbas1,2 ϲ 1 - Graduate Program in Neuroscience, Boston University and School of Medicine, Boston, ϳ Massachusetts 02215 ϴ 2 ± Neural Systems Laboratory, Department of Health Sciences, Boston University, Boston, ϵ Massachusetts 02215 ϭϬ Corresponding author with complete address, including an email address and postal code: ϭϭ Helen Barbas, Boston University, 635 Commonwealth Avenue, Room 431, Boston, MA 02215; ϭϮ email: [email protected] ϭϯ Number of pages: 53 ϭϰ Number of figures, tables, multimedia and 3D models (separately): 12 figures; 1 table ϭϱ Number of words for Abstract, Introduction, and Discussion (separately): ϭϲ Abstract: 246 ϭϳ Introduction: 417 ϭϴ Discussion: 1583 ϭϵ Conflict of Interest: The authors declare no competing financial interests. ϮϬ Acknowledgements: This work was supported by NIH grants from NINDS and NIMH. We Ϯϭ thank Drs. M. Á. García-Cabezas, B. Zikopoulos, and Y. John for helpful discussions. ϮϮ ϭ Ϯϯ Abstract Ϯϰ The structural and functional integrity of subgenual cingulate area 25 (A25) is crucial for Ϯϱ emotional expression and equilibrium. A25 has a key role in affective networks, and its Ϯϲ disruption has been linked to mood disorders, but its cortical connections have yet to be Ϯϳ systematically or fully studied. Using neural tracers in rhesus monkeys, we found that A25 was Ϯϴ densely connected with other ventromedial and posterior orbitofrontal areas associated with Ϯϵ emotions and homeostasis. A moderate pathway linked A25 with frontopolar area 10, an area ϯϬ associated with complex cognition, which may regulate emotions and dampen negative affect. ϯϭ Beyond the frontal lobe, A25 was connected with auditory association areas and memory-related ϯϮ medial temporal cortices, and with the interoceptive-related anterior insula. A25 mostly targeted ϯϯ the superficial cortical layers of other areas, where broadly dispersed terminations comingled ϯϰ with modulatory inhibitory or disinhibitory microsystems, suggesting a dominant excitatory ϯϱ effect. The architecture and connections suggest that A25 is the consummate feedback system in ϯϲ the prefrontal cortex. Conversely, in the entorhinal cortex, A25 pathways terminated in the ϯϳ middle-deep layers amidst a strong local inhibitory microenvironment, suggesting gating of ϯϴ hippocampal output to other cortices and memory storage. The graded cortical architecture and ϯϵ associated laminar patterns of connections suggest how areas, layers, and functionally distinct ϰϬ classes of inhibitory neurons can be recruited dynamically to meet task demands. The ϰϭ complement of cortical connections of A25 with areas associated with memory, emotion, and ϰϮ somatic homeostasis provide the circuit basis to understand its vulnerability in psychiatric and ϰϯ neurologic disorders. ϰϰ ϰϱ Ϯ ϰϲ ϰϳ Significance Statement ϰϴ Integrity of the prefrontal subgenual cingulate cortex is crucial for healthy emotional ϰϵ function. Subgenual area 25 (A25) is mostly linked with other prefrontal areas associated with ϱϬ emotion in a dense network positioned to recruit large fields of cortex. In healthy states, A25 is ϱϭ associated with internal states, autonomic function, and transient negative affect. Constant ϱϮ hyperactivity in A25 is a biomarker for depression in humans, and may trigger extensive ϱϯ activation in its dominant connections with areas associated with emotions and internal balance. ϱϰ A pathway between A25 and frontopolar area 10 may provide a critical link to regulate emotions ϱϱ and dampen persistent negative affect, which may be explored for therapeutic intervention in ϱϲ depression. ϱϳ ϱϴ Introduction ϱϵ Area 25, found deep in the ventral and posterior aspect of the subgenual cingulate region ϲϬ of the ventromedial prefrontal cortex (VMPFC), has a significant role in emotion and ϲϭ visceromotor function (Drevets et al., 2008b; Drevets et al., 2008a; Hamani et al., 2011; Myers- ϲϮ Schulz and Koenigs, 2012; Gold, 2015; Palomero-Gallagher et al., 2015). Classical studies have ϲϯ shown that stimulation of A25 elicits changes in autonomic function (Kaada et al., 1949; ϲϰ Showers and Crosby, 1958), consistent with its dense projections to the hypothalamus (Nauta, ϲϱ 1971; Ongur et al., 1998; Rempel-Clower and Barbas, 1998) and brainstem monoaminergic ϲϲ systems (Freedman et al., 2000; Chiba et al., 2001). These structures have global effects on the ϲϳ brain and somatic states, like the stress response. ϯ ϲϴ Unlike most of the prefrontal cortex (PFC), A25 has both a very strong sender and ϲϵ receiver relationship with the amygdala (Ghashghaei et al., 2007), which is associated with ϳϬ affective states and projects to autonomic structures as well (Sah et al., 2003; Salzman and Fusi, ϳϭ 2010; Pessoa, 2017). Further, A25 receives direct input from the hippocampus (Rosene and Van ϳϮ Hoesen, 1977; Barbas and Blatt, 1995; Insausti and Munoz, 2001), a region known for its ϳϯ essential role in long-term and contextual memory [reviewed in Murray and Wise (2010); ϳϰ Schiller et al. (2015)], and heavily innervates the ventral striatum, associated with motivation and ϳϱ reward-driven behavior (Heimer and Van Hoesen, 2006; Heilbronner et al., 2016). Dysfunction ϳϲ of A25 is associated with psychiatric disorders characterized by abnormal affect and emotional ϳϳ expression. Accordingly, accumulating evidence has highlighted a role of A25 in major ϳϴ depression, accompanied by functional and morphological pathology (Mayberg, 1997; Drevets, ϳϵ 2001; Mayberg et al., 2005; Drevets et al., 2008a). ϴϬ In spite of the involvement of A25 in vital autonomic systems and its relevance for ϴϭ affective processes, its cortical connections have yet to be investigated systematically. Limited ϴϮ information on the cortical connections of A25 has been obtained incidentally from studies with ϴϯ a focus elsewhere [reviewed in Hamani et al. (2011); Yeterian et al. (2012)], leaving several ϴϰ questions unaddressed. Which cortical areas are connected with A25 and what are their relative ϴϱ strengths? Do these pathways interact with specific cortical layers in a systematic way and within ϴϲ functionally distinct local environments of inhibitory neurons? We addressed these questions at ϴϳ the areal and laminar level in rhesus monkeys. What emerges is a comprehensive and ϴϴ quantitatively-based cortical map at high resolution that positions A25 within the substrate ϴϵ circuitry for interoception, emotion, and memory, and provides insight into the circuitry that ϵϬ becomes disrupted in psychiatric and neurologic diseases. ϰ ϵϭ ϵϮ Materials and Methods ϵϯ Experimental design and statistical analysis overview ϵϰ Figure 1 depicts the experimental design. The goal was to map the cortical pathways of ϵϱ A25 in rhesus monkeys and investigate their laminar pattern of connections. To map incoming ϵϲ and outgoing pathways of A25, we injected four distinct neural tracers in medial or orbital A25 ϵϳ (Table 1). The tracers labeled neurons (incoming) and boutons (outgoing) in cortical areas ϵϴ connected with the injection site (Fig. 1A). The extent of each injection site was reconstructed ϵϵ from serial coronal sections, as described in Results. ϭϬϬ The rhesus monkeys were young adult (aged 3-4 years old, n=4, 1 female). Cortical ϭϬϭ connections in this species are already in place within the first postnatal year in visual as well as ϭϬϮ prefrontal cortices (Goldman and Nauta, 1977; Schwartz and Goldman-Rakic, 1991; Rodman, ϭϬϯ 1994). White matter changes continue until about 4 years (Malkova et al., 2006), and may be ϭϬϰ associated with the protracted myelination in primates (Yakovlev and Lecours, 1967). After ϭϬϱ perfusion and coronal sectioning of each brain, we used systematic sampling of coronal series