Effects of College Athlete Life Stressors on Baseline Concussion Measures.

CONTEXT: Concussion baseline testing helps injury evaluation by allowing postinjury comparisons to preinjury measures. To facilitate best practice, common neurocognitive, balance, and symptom report metrics used in concussion baseline testing merit examination relative to participant life stressors. OBJECTIVE: The purpose of this study was to determine if life stressors are associated with college athlete neurocognitive function, postural control, and symptom scores at preseason baseline assessment. DESIGN: All study variables were collected in a single laboratory session where athletes completed valid and reliable psychometrics as well as a computerized neurocognitive and balance assessments. SETTING: Sports medicine research center on an American university campus. PARTICIPANTS: A convenience sample of 123 college student-athletes: 47 females (age = 18.9 [4.3] y) and 76 males (age = 19.4 [1.6] y). MAIN OUTCOME MEASURES: Participants were categorized into low, moderate, or high life stressors groups using scores from the Social Readjustment Rating Scale-Revised. Dependent variables included outcomes from the CNS Vitals Signs test, the Sensory Organization Test, and the graded symptom checklist indexing neurocognition, balance, and symptom severity, respectfully. RESULTS: One-way analysis of variance revealed that the moderate life stressors group performed significantly worse than the low life stressors group on the baseline verbal memory domain of the CNS Vital Signs (F2,119 = 3.28; P = .04) only. CONCLUSION: In the current college athlete sample, few baseline concussion assessment variables were found to be significantly associated with life stressors. Considering the clinical significance of these variables, psychological life stressors may not be a confounding factor in concussion evaluation.

Sex differences in main olfactory system pathways involved in psychosexual function.

We summarize literature from animal and human studies assessing sex differences in the ability of the main olfactory system to detect and process sex-specific olfactory signals ("pheromones") that control the expression of psychosexual functions in males and females. A case is made in non primate mammals for an obligatory role of pheromonal signaling via the main olfactory system (in addition to the vomeronasal-accessory olfactory system) in mate recognition and sexual arousal, with male-specific as well as female-specific pheromones subserving these functions in the opposite sex. Although the case for an obligatory role of pheromones in mate recognition and mating among old world primates, including humans, is weaker, we review the current literature assessing the role of putative human pheromones (eg, AND, EST, "copulin"), detected by the main olfactory system, in promoting mate choice and mating in men and women. Based on animal studies, we hypothesize that sexually dimorphic effects of putative human pheromones are mediated via main olfactory inputs to the medial amygdala which, in turn, transmits olfactory information to sites in the hypothalamus that regulate reproduction.

Effect of Ovarian Hormones and Mating Experience on the Preference of Female Mice to Investigate Male Urinary Pheromones.

In female mice, the expression of receptive lordosis behavior requires estradiol and progesterone actions in the nervous system; however, the contribution of these hormones to females' motivation to seek out male pheromones is less clear. In an initial experiment, sexually naïve ovary-intact female mice preferred to investigate (make nasal contact with) testes-intact male as opposed to estrous female urine, provided they were in vaginal estrus. In a second experiment, groups of sexually naïve and mating-experienced, ovariectomized females were tested for urinary pheromone preference first without and then with ovarian hormone replacement. Without hormone replacement, sexually naïve ovariectomized females showed no preference for male over female urinary pheromones whereas mating-experienced females preferred to investigate male pheromones. Ovariectomized females in both groups preferred male over female urine after sequential s.c. injections with estradiol benzoate followed 2 days later with progesterone and after prolonged (7 days) exposure to estradiol alone. Our results indicate that in sexually naïve female mice estradiol, perhaps aided by progesterone, is required to motivate a preference to seek out male pheromones whereas after mating experience females' preference to investigate male pheromones persists even in the absence of ovarian hormone action.

Hormone-dependent medial preoptic/lumbar spinal cord/autonomic coordination supporting male sexual behaviors.

Testosterone (T) can act directly through neural androgen receptors (AR) to facilitate male sexual behavior; however, T's metabolites also can play complicated and interesting roles in the control of mating. One metabolite, dihydrotestosterone (DHT) binds to AR with significantly greater affinity than that of T. Is that important behaviorally? Another metabolite, estradiol (E), offers a potential alternative route of facilitating male mating behavior by acting through estradiol receptors (ER). In this review we explore the roles and relative importance of T as well as E and DHT at various levels of the neuroaxis for the activation of male sex behavior in common laboratory animals and, when relevant research findings are available, in man.

DREADD-induced silencing of the medial amygdala reduces the preference for male pheromones and the expression of lordosis in estrous female mice.

Sexually naïve estrous female mice seek out male urinary pheromones; however, they initially display little receptive (lordosis) behavior in response to male mounts. Vomeronasal-accessory olfactory bulb inputs to the medial amygdala (Me) regulate courtship in female rodents. We used a reversible inhibitory chemogenetic technique (Designer Receptors Exclusively Activated by Designer Drugs; DREADDs) to assess the contribution of Me signaling to females' preference for male pheromones and improvement in receptivity normally seen with repeated testing. Sexually naïve females received bilateral Me injections of an adeno-associated virus carrying an inhibitory DREADD. Females were later ovariectomized, treated with ovarian hormones, and given behavioral tests following intraperitoneal injections of saline or clozapine-N-oxide (CNO; which hyperpolarizes infected Me neurons). CNO attenuated females' preference to investigate male vs. female urinary odors. Repeated CNO treatment also slowed the increase in lordosis otherwise seen in females given saline. However, when saline was given to females previously treated with CNO, their lordosis quotients were as high as other females repeatedly given saline. No disruptive behavioral effects of CNO were seen in estrous females lacking DREADD infections of the Me. Finally, CNO attenuated the ability of male pheromones to stimulate Fos expression in the Me of DREADD-infected mice but not in non-infected females. Our results affirm the importance of Me signaling in females' chemosensory preferences and in the acute expression of lordosis. However, they provide no indication that Me signaling is required for the increase in receptivity normally seen after repeated hormone priming and testing with a male.

Optogenetic Activation of Accessory Olfactory Bulb Input to the Forebrain Differentially Modulates Investigation of Opposite versus Same-Sex Urinary Chemosignals and Stimulates Mating in Male Mice.

Surgical or genetic disruption of vomeronasal organ (VNO)-accessory olfactory bulb (AOB) function previously eliminated the ability of male mice to processes pheromones that elicit territorial behavior and aggression. By contrast, neither disruption significantly affected mating behaviors, although VNO lesions reduced males' investigation of nonvolatile female pheromones. We explored the contribution of VNO-AOB pheromonal processing to male courtship using optogenetic activation of AOB projections to the forebrain. Protocadherin-Cre male transgenic mice received bilateral AOB infections with channelrhodopsin2 (ChR2) viral vectors, and an optical fiber was implanted above the AOB. In olfactory choice tests, males preferred estrous female urine (EFU) over water; however, this preference was eliminated when diluted (5%) EFU was substituted for 100% EFU. Optogenetic AOB activation concurrent with nasal contact significantly augmented males' investigation compared to 5% EFU alone. Conversely, concurrent optogenetic AOB activation significantly reduced males' nasal investigation of diluted urine from gonadally intact males (5% IMU) compared to 5% IMU alone. These divergent effects of AOB optogenetic activation were lost when males were prevented from making direct nasal contact. Optogenetic AOB stimulation also failed to augment males' nasal investigation of deionized water or of food odors. Finally, during mating tests, optogenetic AOB stimulation delivered for 30 s when the male was in physical contact with an estrous female significantly facilitated the occurrence of penile intromission. Our results suggest that VNO-AOB signaling differentially modifies males' motivation to seek out female vs male urinary pheromones while augmenting males' sexual arousal leading to intromission and improved reproductive performance.

A comparison of the effects of male pheromone priming and optogenetic inhibition of accessory olfactory bulb forebrain inputs on the sexual behavior of estrous female mice.

Previous research has shown that repeated testing with a stimulus male is required for ovariectomized, hormone-primed female mice to become sexually receptive (show maximal lordosis quotients; LQs) and that drug-induced, epigenetic enhancement of estradiol receptor function accelerated the improvement in LQs otherwise shown by estrous females with repeated testing. We asked whether pre-exposure to male pheromones ('pheromone priming') would also accelerate the improvement in LQs with repeated tests and whether optogenetic inhibition of accessory olfactory bulb (AOB) projection neurons could inhibit lordosis in sexually experienced estrous female mice. In Experiment 1, repeated priming with soiled male bedding failed to accelerate the progressive improvement in LQs shown by estrous female mice across 5 tests, although the duration of each lordosis response and females' investigation of male body parts during the first test was augmented by such priming. In Experiment 2, acute optogenetic inhibition of AOB inputs to the forebrain during freely moving behavioral tests significantly reduced LQs, suggesting that continued AOB signaling to the forebrain during mating is required for maximal lordotic responsiveness even in sexually experienced females. Our results also suggest that pheromonal stimulation, by itself, cannot substitute for the full complement of sensory stimulation received by estrous females from mounting males that normally leads to the progressive improvement in their LQs with repeated testing.

DREADD-Induced Silencing of the Medial Olfactory Tubercle Disrupts the Preference of Female Mice for Opposite-Sex Chemosignals(1,2,3).

Attraction to opposite-sex pheromones during rodent courtship involves a pathway that includes inputs to the medial amygdala (Me) from the main and accessory olfactory bulbs, and projections from the Me to nuclei in the medial hypothalamus that control reproduction. However, the consideration of circuitry that attributes hedonic properties to opposite-sex odors has been lacking. The medial olfactory tubercle (mOT) has been implicated in the reinforcing effects of natural stimuli and drugs of abuse. We performed a tract-tracing study wherein estrous female mice that had received injections of the retrograde tracer, cholera toxin B, into the mOT were exposed to volatile odors from soiled bedding. Both the anterior Me and ventral tegmental area sent direct projections to the mOT, of which a significant subset was selectively activated (expressed Fos protein) by testes-intact male (but not female) volatile odors from soiled bedding. Next, the inhibitory DREADD (designer receptors exclusively activated by designer drugs) receptor hM4Di was bilaterally expressed in the mOT of female mice. Urinary preferences were then assessed after intraperitoneal injection of either saline or clozapine-N-oxide (CNO), which binds to the hM4Di receptor to hyperpolarize infected neurons. After receiving CNO, estrous females lost their preference for male over female urinary odors, whereas the ability to discriminate these odors remained intact. Male odor preference returned after vehicle treatment in counterbalanced tests. There were no deficits in locomotor activity or preference for food odors when subject mice received CNO injections prior to testing. The mOT appears to be a critical segment in the pheromone-reward pathway of female mice.

Processing by the main olfactory system of chemosignals that facilitate mammalian reproduction.

This article is part of a Special Issue "Chemosignals and Reproduction". Most mammalian species possess two parallel circuits that process olfactory information. One of these circuits, the accessory system, originates with sensory neurons in the vomeronasal organ (VNO). This system has long been known to detect non-volatile pheromonal odorants from conspecifics that influence numerous aspects of social communication, including sexual attraction and mating as well as the release of luteinizing hormone from the pituitary gland. A second circuit, the main olfactory system, originates with sensory neurons in the main olfactory epithelium (MOE). This system detects a wide range of non-pheromonal odors relevant to survival (e.g., food and predator odors). Over the past decade evidence has accrued showing that the main olfactory system also detects a range of volatile odorants that function as pheromones to facilitate mate recognition and activate the hypothalamic-pituitary-gonadal neuroendocrine axis. We review early studies as well as the new literature supporting the view that the main olfactory system processes a variety of different pheromonal cues that facilitate mammalian reproduction.

6-Hydroxydopamine lesions of the anteromedial ventral striatum impair opposite-sex urinary odor preference in female mice.

Rodents rely upon their olfactory modality to perceive opposite-sex pheromonal odors needed to motivate courtship behaviors. Volatile and nonvolatile components of pheromonal odors are processed by the main (MOS) and accessory olfactory system (AOS), respectively, with inputs converging in the medial amygdala (Me). The Me in turn targets the mesolimbic dopamine system, including the nucleus accumbens core (AcbC) and shell (AcbSh), the ventral pallidum (VP), medial olfactory tubercle (mOT) and ventral tegmental area (VTA). We hypothesized that pheromone-induced dopamine (DA) release in the ventral striatum (particularly in the mAcb and mOT) may mediate the normal preference of female mice to investigate male pheromones. We made bilateral 6-OHDA lesions of DA fibers innervating either the mAcb alone or the mAcb+mOT in female mice and tested estrous females' preference for opposite-sex urinary odors. We found that 6-OHDA lesions of either the mAcb alone or the mAcb+mOT significantly reduced the preference of sexually naïve female mice to investigate breeding male urinary odors (volatiles as well as volatiles+nonvolatiles) vs. estrous female urinary odors. These same neurotoxic lesions had no effect on subjects' ability to discriminate between these two urinary odors, on their locomotor activity, or on their preference for consuming sucrose. The integrity of the dopaminergic innervation of the mAcb and mOT is required for female mice to prefer investigating male pheromones.

A quantitative comparison of the efferent projections of the anterior and posterior subdivisions of the medial amygdala in female mice.

In rodents, many aspects of sociosexual behavior are mediated by chemosignals released by opposite-sex conspecifics. These chemosignals are relayed via the main (MOS) and accessory olfactory systems (AOS) to the medial amygdala (Me). The Me is subdivided into anterior (MeA) and posterior (MeP) subnuclei, and lesions targeting these regions have different effects on proceptive courtship behaviors in female mice. Differential behavioral effects of MeA vs. MeP lesions could reflect a difference in the projections of neurons located in these Me subnuclei. To examine this question, we injected female mice with the anterograde tracer, Fluoro-Ruby into either the MeA or MeP and quantified labeled puncta in 11 forebrain target sites implicated in courtship behaviors using confocal fluorescence microscopy. We found that the MeP more densely innervates the medial and intermediate regions of the posterior bed nucleus of the stria terminalis (pBNST) and the posteromedial cortical amygdala (PMCo), while the MeA more densely innervates the horizontal diagonal band of Broca (HDB) and the medial olfactory tubercle (mOT), a region that may be a component of the circuitry responsible for olfactory-mediated motivated behaviors.

Assessment of urinary pheromone discrimination, partner preference, and mating behaviors in female mice.

Behavioral testing methods are described for determining whether female mice can discriminate between volatile urinary pheromones of conspecifics of the same vs. opposite sex and/or in different endocrine conditions, for determining sexual partner preference, for quantifying receptive (lordosis) behavior, and for monitoring the expression of male-typical mounting behavior in female mice.

Either main or accessory olfactory system signaling can mediate the rewarding effects of estrous female chemosignals in sexually naive male mice.

A long-held view has been that interest of male mice in female body odors reflects an activation of reward circuits in the male brain following their detection by the vomeronasal organ (VNO) and processing via the accessory olfactory system. We found that adult, sexually naive male mice acquired a conditioned place preference (CPP) after repeatedly receiving estrous female urine on the nose and being placed in an initially nonpreferred chamber with soiled estrous bedding on the floor. CPP was not acquired in control mice that received saline on the nose before being placed in a nonpreferred chamber with clean bedding. Robust acquisition of a CPP using estrous female odors as the reward persisted in separate groups of mice in which VNO-accessory olfactory function was disrupted by bilateral lesioning of the accessory olfactory bulb (AOB) or in which main olfactory function was disrupted by zinc sulfate lesions of the main olfactory epithelium (MOE). By contrast, no CPP was acquired for estrous odors in males that received combined AOB and MOE lesions. Either the main or the accessory olfactory system suffices to mediate the rewarding effects of estrous female odors in the male mouse, even in the absence of prior mating experience. The main olfactory system is part of the circuitry that responds to chemosignals involved in motivated behavior, a role that may be particularly important for humans who lack a functional accessory olfactory system.

Roles of sex and gonadal steroids in mammalian pheromonal communication.

A brain circuit (the accessory olfactory system) that originates in the vomeronasal organ (VNO) and includes the accessory olfactory bulb (AOB) plus additional forebrain regions mediates many of the effects of pheromones, typically comprised of a variety of non-volatile and volatile compounds, on aspects of social behavior. A second, parallel circuit (the main olfactory system) that originates in the main olfactory epithelium (MOE) and includes the main olfactory bulb (MOB) has also been shown to detect volatile pheromones from conspecifics. Studies are reviewed that point to specific roles of several different steroids and their water-soluble metabolites as putative pheromones. Other studies are reviewed that establish an adult, 'activational' role of circulating sex hormones along with sex differences in the detection and/or processing of non-steroidal pheromones by these two olfactory circuits. Persisting questions about the role of sex steroids in pheromonal processing are posed for future investigation.

Contribution of pheromones processed by the main olfactory system to mate recognition in female mammals.

Until recently it was widely believed that the ability of female mammals (with the likely exception of women) to identify and seek out a male breeding partner relied on the detection of non-volatile male pheromones by the female's vomeronasal organ (VNO) and their subsequent processing by a neural circuit that includes the accessory olfactory bulb (AOB), vomeronasal amygdala, and hypothalamus. Emperical data are reviewed in this paper that demonstrate the detection of volatile pheromones by the main olfactory epithelium (MOE) of female mice which, in turn, leads to the activation of a population of glomeruli and abutting mitral cells in the main olfactory bulb (MOB). Anatomical results along with functional neuroanatomical data demonstrate that some of these MOB mitral cells project to the vomeronasal amygdala. These particular MOB mitral cells were selectively activated (i.e., expressed Fos protein) by exposure to male as opposed to female urinary volatiles. A similar selectivity to opposite sex urinary volatiles was also seen in mitral cells of the AOB of female mice. Behavioral data from female mouse, ferret, and human are reviewed that implicate the main olfactory system, in some cases interacting with the accessory olfactory system, in mate recognition.

Disruption of urinary odor preference and lordosis behavior in female mice given lesions of the medial amygdala.

Previous research showed that axonal inputs to both anterior and posterior subdivisions of the medial amygdala from the main and accessory olfactory bulbs of female mice, respectively, process volatile and non-volatile pheromonal signals from male conspecifics. In the present study we found that bilateral electrolytic lesions that included posterior portions, but not the anterior subdivision alone of the medial amygdala (Me) blocked the preference of estrous female mice to investigate volatile urinary odors from testes-intact vs. castrated males. Similar results were obtained in separate tests in which nasal contact with urinary stimuli was permitted. In addition, total time investigating volatile urinary stimuli was reduced in subjects with posterior Me lesions. Subjects were able to discriminate volatile urinary odors from testes-intact vs. castrated male mice, suggesting that this disruption of odor preference did not result from the inability of females given amygdaloid lesions to discriminate these male urinary odors. Bilateral lesions of the Me that were either restricted to the anterior or posterior subdivisions, or included areas of both regions, caused significant reductions in the display of lordosis behavior in estrous female mice. Our results suggest that the Me is a critical segment of the olfactory circuit that controls both mate recognition and mating behavior in the female mouse.

The main and accessory olfactory systems of female mice are activated differentially by dominant versus subordinate male urinary odors.

Previous studies have shown that female preferences for male pheromones depend on the female's reproductive condition and the dominance status of the male. However, it is unknown which olfactory system detects the odors that result in a preference for a dominant male. Therefore, in the present study, we asked whether dominant versus subordinate male urinary odors differentially activate the main and accessory olfactory systems in female (C57Bl/6j) mice by monitoring the induction of the immediate early gene, c-fos. A more robust induction of Fos was observed in female mice which had direct nasal contact with dominant male urinary odors in four specific segments of the accessory olfactory system, i.e., the posteroventral part of the medial amygdala, the bed nucleus of the stria terminalis, the medial part of the preoptic nucleus and the ventrolateral part of the ventromedial hypothalamus, compared to females that were exposed to subordinate male urine. This greater activation of the accessory olfactory pathway by dominant male urine suggests that there are differences in the nonvolatile components of dominant versus subordinate male urine that are detected by the vomeronasal organ. By contrast, subordinate male urinary odors induced a greater activation in the piriform cortex which is part of the main olfactory system, suggesting that female mice discriminate between dominant and subordinate male urine using their main olfactory system as well.

The development of female sexual behavior requires prepubertal estradiol.

The classic view of brain and behavioral sexual differentiation holds that the neural mechanisms controlling sexual behavior in female rodents develop in the absence of ovarian sex hormone actions. However, in a previous study, female aromatase knock-out (ArKO) mice, which cannot convert testosterone to estradiol, showed deficient male-oriented partner preference and lordosis behaviors in response to adult ovarian hormones, raising the possibility that estradiol may contribute to the development of these female sexual behaviors. In the present experiments, administering estradiol prepubertally [between postnatal day 15 (P15) and P25] significantly enhanced the ability of ArKO female mice to display lordosis behavior in response to ovarian hormones administered later in adulthood, whereas treatment with estradiol over an earlier postnatal period (P5-P15) had no such effect. Treatment of ArKO females with estradiol between P15 and P25 also rescued their later preference to approach distal cues from an intact male over an estrous female. ArKO females also displayed significantly less female-directed (male-typical) mounting behavior than wild-type control females when treated with testosterone in adulthood. Prepubertal estradiol treatment failed to reverse this deficit in ArKO females, whereas earlier postnatal estradiol augmented later mounting in both genotypes. Our results provide new evidence for an organizing role of prepubertal estradiol in the development of neural mechanisms that control female-typical sexual behavior.