From Reductionism Toward Integration: Understanding How Social Behavior Emerges From Integrated Circuits.

Complex social behaviors are emergent properties of the brain's interconnected and overlapping neural networks. Questions aimed at understanding how brain circuits produce specific and appropriate behaviors have changed over the past half century, shifting from studies of gross anatomical and behavioral associations, to manipulating and monitoring precisely targeted cell types. This technical progression has enabled increasingly deep insights into the regulation of perception and behavior with remarkable precision. The capacity of reductionist approaches to identify the function of isolated circuits is undeniable but many behaviors require rapid integration of diverse inputs. This review examines progress toward understanding integrative social circuits and focuses on specific nodes of the social behavior network including the medial amygdala, ventromedial hypothalamus (VMH) and medial preoptic area of the hypothalamus (MPOA) as examples of broad integration between multiple interwoven brain circuits. Our understanding of mechanisms for producing social behavior has deepened in conjunction with advances in technologies for visualizing and manipulating specific neurons and, here, we consider emerging strategies to address brain circuit function in the context of integrative anatomy.

Brain-Wide Synaptic Inputs to Aromatase-Expressing Neurons in the Medial Amygdala Suggest Complex Circuitry for Modulating Social Behavior.

Here, we reveal an unbiased view of the brain regions that provide specific inputs to aromatase-expressing cells in the medial amygdala, neurons that play an outsized role in the production of sex-specific social behaviors, using rabies tracing and light sheet microscopy. While the downstream projections from these cells are known, the specific inputs to the aromatase-expressing cells in the medial amygdala remained unknown. We observed established connections to the medial amygdala (e.g., bed nucleus of the stria terminalis and accessory olfactory bulb) indicating that aromatase neurons are a major target cell type for efferent input including from regions associated with parenting and aggression. We also identified novel and unexpected inputs from areas involved in metabolism, fear and anxiety, and memory and cognition. These results confirm the central role of the medial amygdala in sex-specific social recognition and social behavior, and point to an expanded role for its aromatase-expressing neurons in the integration of multiple sensory and homeostatic factors, which are likely used to modulate many other social behaviors.

Genital anatomy and copulatory interactions in the broad snouted Caiman (Caiman latirostris).

The broad snouted caiman is a crocodylian native to South America that is subject to extensive conservation management in both wild and farming environments. Although reproductive behaviors like egg laying and clutch care have been examined in this species, little else is known about their copulatory system. We examined the anatomy of male and female cloacal and genital tissues ex vivo to build hypotheses of their interactions during copulation and the effects of that interaction on insemination. Male phallic glans tissues were artificially inflated to expand into their copulatory state, allowing the examination and quantification of structural changes at the gross and tissue levels. Digital reconstruction of MRI stacks yielded three-dimensional tissue compartment specific glans models of the inflated state. Silicone molds of female cloacae and oviducts in conjunction with dissection and diceCT analysis allowed us to assess internal geometry and infer how male and female features interact in copulo. We observed glans expansion within the female proctodeum would result in a copulatory lock limiting deeper intromission or retraction. Intromission and subsequent creation of the copulatory lock produces extensive clitoral compression, providing a possible mechanism for female assessment of male copulatory performance. Further, glans expansion forms a distal lumen that positions the glans tip in or near the vaginal openings. A coiled, muscular vagina provides a possible mechanism for postcopulatory sexual selection by excluding semen. Together, the complex male-female interaction supports evidence for cryptic selection by female choice, which can act as a driver of genital coevolution.

A cloacal opening in a non-avian dinosaur.

The Frankfurt specimen of Psittacosaurus sp. (SMF R 4970) from the Early Cretaceous Jehol deposits of Liaoning (Figure S1) exhibits exceptional preservation of scale-clad integument1. Preservation of colour patterns and countershading allowed a detailed reconstruction of this individual's physical appearance. It was previously noted that the cloacal region was preserved2, but its detailed anatomy was incorrectly reconstructed. We show here that the fine anatomy of the vent is remarkably well preserved and can be retrodeformed to illustrate its three-dimensional nature. The vent's scale anatomy and pigmentation are distinct from adjacent body regions, and although its anatomy does not reveal much information about the ecology, or sex, of this dinosaur, it suggests possible roles for visual and olfactory signalling.

Glans inflation morphology and female cloaca copulatory interactions of the male American alligator phallus†.

The phallic glans of the American alligator (Alligator mississippiensis) is the distal termination of the semen-conducting sulcus spermaticus and during copulation has the closest, most intimate mechanical interactions with the female urodeum, the middle cloacal chamber that contains the opening to the vaginal passages and oviducts. However, the details of this interface leading to insemination and gamete uptake are unclear. Here, we: (1) histologically characterize the underlying tissue types and morphologically quantify the shape changes associated with glans inflation into the copulatory conformation, (2) digitally reconstruct from MRI the 3D shape of functional tissue compartments, and (3) diffusible iodine-based contrast-enhanced computed tomography image the copulatory fit between male phallus and female cloaca. We discuss these results in relation to tissue type material properties, the transfer on intromittent forces, establishing potential copulatory lock, inflated glans volume scaling with body mass/length, the mechanics of semen targeting and insemination, and potential female cryptic choice impacting multiple clutch paternity. In part, this study further clarifies the phallic morphological variation observed among crocodylians and begins to investigate the role(s) these divergent male forms play during copulation interacting with female cloacal forms to increase reproductive success.

Synaptic Connections of Aromatase Circuits in the Medial Amygdala Are Sex Specific.

The brains of male and female mice are shaped by genetics and hormones during development. The enzyme aromatase helps establish sex differences in social behaviors and in the neural circuits that produce these behaviors. The medial amygdala of mice contains a large population of aromatase neurons and is a critical hub in the social behavior network. Moreover, the neural representation of social stimuli in the medial amygdala displays clear sex differences that track developmental changes in social behaviors. Here, we identify a potential anatomic basis for those sex differences. We found that sensory input from the accessory olfactory bulb (AOB) to aromatase neurons is derived nearly exclusively from the anterior AOB, which selectively responds to chemosensory cues from conspecific animals. Through the coordinated use of mouse transgenics and viral-based circuit-tracing strategies, we demonstrate a clear sex difference in the volume of synapses connecting the accessory olfactory bulb to aromatase-expressing neurons in the medial amygdala of male versus female mice. This difference in anatomy likely mediates, at least in part, sex differences in medial amygdala-mediated social behaviors.

Morphological changes associated with Nile crocodile (Crocodylus niloticus) phallic glans inflation.

The crocodylian phallic glans is the distal inflatable structure that makes the most direct contact with the female cloacal and associated reproductive tract openings during copulation. Therefore, its form and function directly impact female tissue sensory interactions and insemination mechanics. Compared to mammals, less is known about glans functional anatomy among other amniotes, including crocodylians. Therefore, we paired an ex vivo inflation technique with magnetic resonance imaging 3D-reconstructions and corresponding histological analyses to better characterize the morphological glans restructuring occurring in the Nile crocodile (Crocodylus niloticus) at copulation. The expansion of contiguous inflatable spongiform glans tissues is variably constrained by adjacent regions of dense irregular collagen-rich tissues. Therefore, expansion shows regional differences with greater lateral inflation than dorsal and ventral. Furthermore, this enlargement elaborates the cup-like glans lumen, dorsally reorients the glans ridge, stiffens the blunt and bifid glans tip, and putatively works to seal the ventral sulcus spermaticus semen conduit groove. We suggest how these dynamic male structures may interact with structures of the female cloacal urodeum and how these morphological changes, in concert with the varying material properties of the structural tissue compartments visualized in this study, aid copulatory gamete transfer and resulting fecundity. RESEARCH HIGHLIGHTS: Nile crocodile glans inflation produces a reproductively relevant copulatory structure directing insemination and female tissue interactions. Pairing magnetic resonance imaging 3D reconstruction with corresponding histology effectively studies functional anatomy.

Genital interactions during simulated copulation among marine mammals.

Genitalia are morphologically variable across many taxa and in physical contact during intromission, but little is known about how variation in form correlates with function during copulation. Marine mammals offer important insights into the evolutionary forces that act on genital morphology because they have diverse genitalia and are adapted to aquatic living and mating. Cetaceans have a fibroelastic penis and muscular vaginal folds, while pinnipeds have a baculum and lack vaginal folds. We examined copulatory fit in naturally deceased marine mammals to identify anatomical landmarks in contact during copulation and the potential depth of penile penetration into the vagina. Excised penises were artificially inflated to erection with pressurized saline and compared with silicone vaginal endocasts and within excised vaginas in simulated copulation using high-resolution, diffusible iodine-based, contrast-enhanced computed tomography. We found evidence suggestive of both congruent and antagonistic genital coevolution, depending on the species. We suggest that sexual selection influences morphological shape. This study improves our understanding of how mechanical interactions during copulation influence the shape of genitalia and affect fertility, and has broad applications to other taxa and species conservation.

The Morphological Diversity of Intromittent Organs: An Introduction to the Symposium.

Intromittent organs are structures that enter the female genital tract and deposit sperm; these organs are found in many animal taxa that use internal fertilization. Despite their shared function, they are fantastically diverse morphologically. Many of their species-specific shape differences are likely the result of sexual selection and coevolution between male and female reproductive tracts, but a growing number of studies have identified other factors that can also affect their functional anatomy. In this symposium, we united scientists who combine morphological methods with contemporary molecular, phylogenetic, and imaging techniques to study the reproductive performance of intromittent organs in invertebrate and vertebrate model systems. The result was a collection of studies discussing competing selective pressures that act on these structures, including the effects of life history, genes and development, sexual conflict, ecological interactions, the biomechanics of copulation, and phenotypic plasticity. There was also an overwhelming consensus that a full understanding of intromittent organ evolution will not be possible without complimentary studies of morphology and function in female reproductive tracts.

Morphological characteristics regulating phallic glans engorgement in the American alligator.

The distal part of the crocodilian phallus consists of a bulbous glans containing well-developed vascular tissues that can inflate before or during sexual activity, enlarging and elaborating the glans into a complex, though still functionally undefined, copulatory structure. An enlarged glans putatively interacts with the female cloaca and may change the shape of her reproductive tract to facilitate insemination and increase the probability of fertilization. Here, we investigated the cellular-level properties of the glans and other inflatable phallic tissues associated with the sperm-conducting sulcus spermaticus in the American alligator (Alligator mississippiensis). Using histochemical staining, we visualized and defined collagen and elastin fiber densities and orientations in these tissues. Extracellular matrix architectures provided insights about phallic glans material properties and how they may affect tissue strength and flexibility during inflation and in response to copulatory forces. We also investigated the potential sources of fluids that induce inflation in alligator phalli. Combining serial sectioning and three-dimensional reconstruction, we identified a pair of supracrucal plexus vascular bodies at the proximal end of the alligator phallus that extend distally adjacent to ventro-medial sulcus tissues. Together, our gross and histological examination of the American alligator phallic glans suggests that its tissues are arranged in a manner that would allow vascular inflation to expand the glans to a specific and repeatable shape, and potentially release secretory products into the female reproductive tract. Both elements could play roles in postcopulatory sexual selection, by mechanically and/or chemically affecting female reproductive physiology.

Intromittent Organ Morphology and Biomechanics: Defining the Physical Challenges of Copulation.

Intromittent organs-structures that place gametes into a mate for internal fertilization-evolved many times within the animal kingdom, and are remarkable for their extravagant morphological diversity. Some taxa build intromittent organs from tissues with reproductive system antecedents, but others copulate with modified fins, tentacles, or legs: anatomically, these structures can include combinations of stiff tissues, extensible tissues, and muscle. Their mechanical behavior during copulation is also diverse: males in some taxa reorient or protrude genital tissues, others inflate them and change their shape, while still other taxa combine these strategies. For these animals, the ability to ready an intromittent organ for copulation and physically interact with a mate's genital tissues is critical to reproductive success, and may be tied to aspects of postcopulatory selection such as sperm competition and sexual conflict. But we know little about their mechanical behavior during copulation. This review surveys mechanical strategies that animals may use for intromittent organ function during intromission and copulation, and discusses how they may perform when their tissues experience stresses in tension, compression, bending, torsion, or shear.

Testing hypotheses of bat baculum function with 3D models derived from microCT.

The baculum (os penis) has been extensively studied as a taxon-specific character in bats and other mammals but its mechanical function is still unclear. There is a wide consensus in the literature that the baculum is probably a sexually selected character. Using a novel approach combining postmortem manipulation and three-dimensional (3D) imaging, we tested two functional hypotheses in the common noctule bat Nyctalus noctula, the common pipistrelle Pipistrellus pipistrellus, and Nathusius' pipistrelle Pipistrellus nathusii: (i) whether the baculum can protect the distal urethra and urethral opening from compression during erection and copulation; and (ii) whether the baculum and corpora cavernosa form a functional unit to support both the penile shaft and the more distal glans tip. In freshly dead or frozen and thawed bats, we compared flaccid penises with artificially 'erect' penises that were inflated with 10% formalin. Penises were stained with alcoholic iodine and imaged with a lab-based high-resolution x-ray microtomography system. Analysis of the 3D images enabled us to compare the changes in relative positions of the baculum, corpora cavernosa, urethra, and corpus spongiosum with one another between flaccid and 'erect' penises. Our results support both functional hypotheses, indicating that the baculum probably performs two different roles during erection. Our approach should prove valuable for comparing and testing the functions of different baculum morphologies in bats and other mammals. Moreover, we have validated an essential component of the groundwork necessary to extend this approach with finite element analysis for quantitative 3D biomechanical modeling of penis function.

Social structure predicts genital morphology in African mole-rats.

BACKGROUND: African mole-rats (Bathyergidae, Rodentia) exhibit a wide range of social structures, from solitary to eusocial. We previously found a lack of sex differences in the external genitalia and morphology of the perineal muscles associated with the phallus in the eusocial naked mole-rat. This was quite surprising, as the external genitalia and perineal muscles are sexually dimorphic in all other mammals examined. We hypothesized that the lack of sex differences in naked mole-rats might be related to their unusual social structure. METHODOLOGY/PRINCIPAL FINDINGS: We compared the genitalia and perineal muscles in three African mole-rat species: the naked mole-rat, the solitary silvery mole-rat, and the Damaraland mole-rat, a species considered to be eusocial, but with less reproductive skew than naked mole-rats. Our findings support a relationship between social structure, mating system, and sexual differentiation. Naked mole-rats lack sex differences in genitalia and perineal morphology, silvery mole-rats exhibit sex differences, and Damaraland mole-rats are intermediate. CONCLUSIONS/SIGNIFICANCE: The lack of sex differences in naked mole-rats is not an attribute of all African mole-rats, but appears to have evolved in relation to their unusual social structure and reproductive biology.

Penises as variable-volume hydrostatic skeletons.

Penises are inflatable intromittent organs that transfer sperm to a female during copulation. Most of the time, males store their penises in a flexible detumesced state, but they can rapidly inflate them with blood when an opportunity for reproductive behavior arises. In mammals, the primary erectile tissue is called the corpus cavernosum; its anatomy is a close match to a model hydroskeleton reinforced by an axial orthogonal fiber array. The wall of the corpus cavernosum contains layers of highly organized collagen fibers arranged at 0 degrees and 90 degrees to the penile long axis. Flaccid wall tissue is folded. Collagen fiber straightening during erection expands the tunica albuginea and increases both its stiffness and its second moment of area. These changes make the entire penis larger and harder to bend. Axial orthogonal fiber reinforcement affects the mechanical behavior of the erect corpus cavernosum, making it resistant to tensile, compressive, and bending forces.