Social buffering in rats reduces fear by oxytocin triggering sustained changes in central amygdala neuronal activity.

The presence of a companion can reduce fear, but the neural mechanisms underlying this social buffering of fear are incompletely known. We studied social buffering of fear in male and female, and its encoding in the amygdala of male, auditory fear-conditioned rats. Pharmacological, opto,- and/or chemogenetic interventions showed that oxytocin signaling from hypothalamus-to-central amygdala projections underlied fear reduction acutely with a companion and social buffering retention 24 h later without a companion. Single-unit recordings with optetrodes in the central amygdala revealed fear-encoding neurons (showing increased conditioned stimulus-responses after fear conditioning) inhibited by social buffering and blue light-stimulated oxytocinergic hypothalamic projections. Other central amygdala neurons showed baseline activity enhanced by blue light and companion exposure, with increased conditioned stimulus responses that persisted without the companion. Social buffering of fear thus switches the conditioned stimulus from encoding "fear" to "safety" by oxytocin-mediated recruitment of a distinct group of central amygdala "buffer neurons".

Movement-Related Modulation in Mouse Auditory Cortex Is Widespread Yet Locally Diverse.

Neurons in the mouse auditory cortex are strongly influenced by behavior, including both suppression and enhancement of sound-evoked responses during movement. The mouse auditory cortex comprises multiple fields with different roles in sound processing and distinct connectivity to movement-related centers of the brain. Here, we asked whether movement-related modulation in male mice might differ across auditory cortical fields, thereby contributing to the heterogeneity of movement-related modulation at the single-cell level. We used wide-field calcium imaging to identify distinct cortical fields and cellular-resolution two-photon calcium imaging to visualize the activity of layer 2/3 excitatory neurons within each field. We measured each neuron's responses to three sound categories (pure tones, chirps, and amplitude-modulated white noise) as mice rested and ran on a non-motorized treadmill. We found that individual neurons in each cortical field typically respond to just one sound category. Some neurons are only active during rest and others during locomotion, and those that are responsive across conditions retain their sound-category tuning. The effects of locomotion on sound-evoked responses vary at the single-cell level, with both suppression and enhancement of neural responses, and the net modulatory effect of locomotion is largely conserved across cortical fields. Movement-related modulation in auditory cortex also reflects more complex behavioral patterns, including instantaneous running speed and nonlocomotor movements such as grooming and postural adjustments, with similar patterns seen across all auditory cortical fields. Our findings underscore the complexity of movement-related modulation throughout the mouse auditory cortex and indicate that movement-related modulation is a widespread phenomenon.

An AAV-CRISPR/Cas9 strategy for gene editing across divergent rodent species: Targeting neural oxytocin receptors as a proof of concept.

A major issue in neuroscience is the poor translatability of research results from preclinical studies in animals to clinical outcomes. Comparative neuroscience can overcome this barrier by studying multiple species to differentiate between species-specific and general mechanisms of neural circuit functioning. Targeted manipulation of neural circuits often depends on genetic dissection, and use of this technique has been restricted to only a few model species, limiting its application in comparative research. However, ongoing advances in genomics make genetic dissection attainable in a growing number of species. To demonstrate the potential of comparative gene editing approaches, we developed a viral-mediated CRISPR/Cas9 strategy that is predicted to target the oxytocin receptor (Oxtr) gene in >80 rodent species. This strategy specifically reduced OXTR levels in all evaluated species (n = 6) without causing gross neuronal toxicity. Thus, we show that CRISPR/Cas9-based tools can function in multiple species simultaneously. Thereby, we hope to encourage comparative gene editing and improve the translatability of neuroscientific research.

The modulation of emotional and social behaviors by oxytocin signaling in limbic network.

Neuropeptides can exert volume modulation in neuronal networks, which account for a well-calibrated and fine-tuned regulation that depends on the sensory and behavioral contexts. For example, oxytocin (OT) and oxytocin receptor (OTR) trigger a signaling pattern encompassing intracellular cascades, synaptic plasticity, gene expression, and network regulation, that together function to increase the signal-to-noise ratio for sensory-dependent stress/threat and social responses. Activation of OTRs in emotional circuits within the limbic forebrain is necessary to acquire stress/threat responses. When emotional memories are retrieved, OTR-expressing cells act as gatekeepers of the threat response choice/discrimination. OT signaling has also been implicated in modulating social-exposure elicited responses in the neural circuits within the limbic forebrain. In this review, we describe the cellular and molecular mechanisms that underlie the neuromodulation by OT, and how OT signaling in specific neural circuits and cell populations mediate stress/threat and social behaviors. OT and downstream signaling cascades are heavily implicated in neuropsychiatric disorders characterized by emotional and social dysregulation. Thus, a mechanistic understanding of downstream cellular effects of OT in relevant cell types and neural circuits can help design effective intervention techniques for a variety of neuropsychiatric disorders.

Acute and long-lasting effects of oxytocin in cortico-limbic circuits: consequences for fear recall and extinction.

The extinction of conditioned fear responses entrains the formation of safe new memories to decrease those behavioral responses. The knowledge in neuronal mechanisms of extinction is fundamental in the treatment of anxiety and fear disorders. Interestingly, the use of pharmacological compounds that reduce anxiety and fear has been shown as a potent co-adjuvant in extinction therapy. However, the efficiency and mechanisms by which pharmacological compounds promote extinction of fear memories remains still largely unknown and would benefit from a validation based on functional neuronal circuits, and the neurotransmitters that modulate them. From this perspective, oxytocin receptor signaling, which has been shown in cortical and limbic areas to modulate numerous functions (Eliava et al. Neuron 89(6):1291-1304, 2016), among them fear and anxiety circuits, and to enhance the salience of social stimuli (Stoop Neuron 76(1):142-59, 2012), may offer an interesting perspective. Experiments in animals and humans suggest that oxytocin could be a promising pharmacological agent at adjusting memory consolidation to boost fear extinction. Additionally, it is possible that long-term changes in endogenous oxytocin signaling can also play a role in reducing expression of fear at different brain targets. In this review, we summarize the effects reported for oxytocin in cortico-limbic circuits and on fear behavior that are of relevance for the modulation and potential extinction of fear memories.

The Basolateral Amygdala Is Essential for Rapid Escape: A Human and Rodent Study.

Rodent research delineates how the basolateral amygdala (BLA) and central amygdala (CeA) control defensive behaviors, but translation of these findings to humans is needed. Here, we compare humans with natural-selective bilateral BLA lesions to rats with a chemogenetically silenced BLA. We find, across species, an essential role for the BLA in the selection of active escape over passive freezing during exposure to imminent yet escapable threat (Timm). In response to Timm, BLA-damaged humans showed increased startle potentiation and BLA-silenced rats demonstrated increased startle potentiation, freezing, and reduced escape behavior as compared to controls. Neuroimaging in humans suggested that the BLA reduces passive defensive responses by inhibiting the brainstem via the CeA. Indeed, Timm conditioning potentiated BLA projections onto an inhibitory CeA pathway, and pharmacological activation of this pathway rescued deficient Timm responses in BLA-silenced rats. Our data reveal how the BLA, via the CeA, adaptively regulates escape behavior from imminent threat and that this mechanism is evolutionary conserved across rodents and humans.

A New Population of Parvocellular Oxytocin Neurons Controlling Magnocellular Neuron Activity and Inflammatory Pain Processing.

Oxytocin (OT) is a neuropeptide elaborated by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Magnocellular OT neurons of these nuclei innervate numerous forebrain regions and release OT into the blood from the posterior pituitary. The PVN also harbors parvocellular OT cells that project to the brainstem and spinal cord, but their function has not been directly assessed. Here, we identified a subset of approximately 30 parvocellular OT neurons, with collateral projections onto magnocellular OT neurons and neurons of deep layers of the spinal cord. Evoked OT release from these OT neurons suppresses nociception and promotes analgesia in an animal model of inflammatory pain. Our findings identify a new population of OT neurons that modulates nociception in a two tier process: (1) directly by release of OT from axons onto sensory spinal cord neurons and inhibiting their activity and (2) indirectly by stimulating OT release from SON neurons into the periphery.

Neuroanatomical distribution of oxytocin receptor binding in the female rabbit forebrain: Variations across the reproductive cycle.

Oxytocin receptors (OTR) have been characterized in the brains of several mammals, including rodents, carnivores, and primates. Their species-specific distribution in the brain has been associated with species differences in social organization, including mating strategy and parenting behavior. In several species, the density of OTR binding in specific brain regions varies according to reproductive condition, including ovarian cycle, pregnancy and lactation. Rabbits are induced ovulators, polygamous, and monoparental but their distribution and regulation of brain OTR has not been described. Here we used receptor autoradiography to quantitatively characterize OTR binding in the brains of estrous, ovariectomized, late pregnant, and lactating does. Intense binding occurred in the prefrontal cortex (PFC), preoptic area (POA), lateral septum (LS; dorsal and ventral), hippocampus, and medial amygdala. Variations among the experimental groups were seen only in PFC, POA, LS. Ovariectomy increased OTR density in PFC but had the opposite effect in POA. Lactating does had significantly reduced OTR density, relative to late pregnancy, in PFC and POA. Our results are consistent with a possible role of OT in modulating social and maternal behavior in rabbits since the brain regions sensitive to OT have been implicated in social interaction, learning and memory, olfactory processing and maternal behavior.

Conditioned same-sex partner preference in male rats is facilitated by oxytocin and dopamine: effect on sexually dimorphic brain nuclei.

Conditioned same-sex partner preference can develop in male rats that undergo cohabitation under the effects of quinpirole (QNP, D2 agonist). Herein, we assessed the development of conditioned same-sex social/sexual preference in males that received either nothing, saline, QNP, oxytocin (OT), or QNP+OT during cohabitation with another male (+) or single-caged (-). This resulted in the following groups: (1) Intact-, (2) Saline+, (3) QNP-, (4) OT-, (5) QNP+, (6) OT+ and (7) QNP/OT+. Cohabitation occurred during 24h in a clean cage with a male partner that bore almond scent on the back as conditioned stimulus. This was repeated every 4 days for a total of three trials. Social and sexual preference were assessed four days after the last conditioning trial in a drug-free test in which experimental males chose between the scented familiar male and a novel sexually receptive female. Results showed that males from groups Intact-, Saline+, QNP- and OT- displayed a clear preference for the female (opposite-sex), whereas groups QNP+, OT+ and QNP/OT+ displayed socio/sexual preference for the male partner (same-sex). In Experiment 2, the brains were processed for Nissl dye and the area size of two sexually dimorphic nuclei (SDN-POA and SON) was compared between groups. Males from groups OT-, OT+ and QNP/OT+ expressed a smaller SDN-POA and groups QNP+ and QNP/OT+ expressed a larger SON. Accordingly, conditioned same-sex social/sexual partner preference can develop during cohabitation under enhanced D2 or OT activity but such preference does not depend on the area size of those sexually dimorphic nuclei.

Cohabitation between male rats after ejaculation: effects on conditioned partner preference.

Male rats display a conditioned ejaculatory preference for females that bear olfactory cues associated with ejaculation+the postejaculatory interval (PEI), or with the PEI alone. This indicates that exposure to a partner during the PEI is necessary and sufficient 'for the development of conditioned sexual partner preference. In the present study we examined the effect of cohabitation between two males during the PEI on the possible development of same-sex partner preference. Males first copulated with an ovariectomized, E+P primed female to one ejaculation and were immediately removed from the female's chamber and placed in another chamber with a conspecific male scented with almond odor as a conditioned stimulus (CS+). Cohabitation lasted for 1 h and started immediately after ejaculation in the PEI group and 7h later in the control group. Conditioning occurred daily for a total of ten trials with different females, but cohabitation during the PEI occurred always with the same stimulus male partner. On trial 11, males were tested for social partner preference with two stimulus male partners. One was the familiar scented male and the other an unfamiliar unscented male. Results indicated that males did not develop any social or sexual preference for the male associated with the PEI. In fact, rats from the PEI group interacted significantly less with the scented male as compared to the unscented male, and displayed more agonistic behaviors towards the scented male than towards the unscented male. These data show that conditioned same-sex preference does not develop as a result of cohabitation during the PEI. We discuss the implications for conditioned hostility in intrasexual competition.

Enhaced D2-type receptor activity facilitates the development of conditioned same-sex partner preference in male rats.

Animal models have shown that the neural bases of social attachment, sexual preference and pair bonds, depend on dopamine D2-type receptor and oxytocin activity. In addition, studies have demonstrated that cohabitation can shape partner preference via conditioning. Herein, we used rats to explore the development of learned same-sex partner preferences in adulthood as a result of cohabitation during enhanced D2 activity. Experimental Wistar males (N=20), received saline or the D2 agonist (quinpirole) and were allowed to cohabitate during 24 h, with a stimulus male partner that bore almond scent on the back as conditioned stimulus. This was repeated every 4 days, for a total of three trials. Four days later they were drug-free tested for partner preference between the scented male partner and a sexually receptive female. Sexual partner preference was analyzed by measuring frequency and latency for appetitive and consummatory sexual behaviors, as well as non-contact erections. Social preference was also analyzed by measuring the frequency and latency of visits, body contacts and time spent together. Results indicated that only quinpirole-treated males displayed sexual and social preference for the scented male over the sexually receptive female. They spent more time together, displayed more body contacts, more female-like proceptive behaviors, and more non-contact erections. Accordingly, conditioned males appeared to be more sexually aroused and motivated by the known male than by a receptive female. We discuss the implications of this animal model on the formation of learned homosexual partner preferences.

Same-sex cohabitation under the effects of quinpirole induces a conditioned socio-sexual partner preference in males, but not in female rats.

The effects of the dopamine D2-type receptor agonist quinpirole (QNP) were examined on the development of conditioned same-sex partner preference induced by cohabitation in rats. In Experiment 1, males received either saline or QNP (1.25mg/kg) and cohabited during three trials with almond-scented stimulus males that were sexually naïve. In Experiment 2, males received six trials, and in Experiment 3 received three trials with sexually expert stimulus males. During a final drug-free preference test, males chose between the familiar or a novel male partner. In Experiments 1, 2 and 3 only QNP-treated males displayed a social preference for the familiar male, observed with more time spent together. In Experiment 3 males also displayed a sexual preference observed with more non-contact erections when were exposed to their male partner. In Experiment 4 we tested the effects on OVX, E+P primed females that received 1 systemic injection of either saline or QNP during three conditioning trials. In Experiment 5, females received 2 injections 12-h apart during each trial. Results indicated that both saline and QNP-treated females failed to develop partner preference. These data demonstrate that enhanced D2-type receptor activity during cohabitation facilitates the development of conditioned same-sex partner preference in males, but not in female rats. We discuss the implications for same-sex partner preferences.

Clitoral stimulation modulates appetitive sexual behavior and facilitates reproduction in rats.

In rats, sexual reward, appetitive sexual behaviors and reproduction are modulated by the amount and rate of vaginocervical stimulation. Here the effect of clitoral stimulation (CLS) on proceptivity was assessed. In Exp 1, ovariectomized, hormone-primed Wistar females formed three groups: G1 (1 CLS every second), G2 (1 CLS every 5s) and G3 (no CLS). Precopulatory CLS consisted of 5cycles of 1min of stimulation with the tip of a cotton swab connected to a vibrator device, followed by 1-2min of rest. CLS increased proceptive behavior in G1 compared to G2, but not compared to G3. In Exp 2, gonadally-intact rats in late proestrous received CLS prior to copulation. No differences in sexual behavior were detected between the groups, but CLS enhanced reproduction in females that received >9 intromissions. 28, 66 and 10% of females became pregnant in G1, G2, and G3, respectively. These data indicate that precopulatory CLS affects proceptive behaviors depending on the pattern and rhythm of stimulation in hormone-primed females. In virgin rats that have received sufficient vagino cervical stimulation CLS also increases fertility.