Impaired oxytocin signaling in the central amygdala in rats with chronic heart failure.

Aims: Heart failure (HF) patients often suffer from cognitive decline, depression, and mood impairments, but the molecular signals and brain circuits underlying these effects remain elusive. The hypothalamic neuropeptide oxytocin (OT) is critically involved in the regulation of mood, and OTergic signaling in the central amygdala (CeA) is a key mechanism controlling emotional responses including anxiety-like behaviors. Based on this, we used in this study a well-established ischemic rat HF model and aimed to study alterations in the hypothalamus-to-CeA OTergic circuit. Methods and Results: To study potential HF-induced changes in the hypothalamus-to-CeA OTertic circuit, we combined patch-clamp electrophysiology, immunohistochemical analysis, RNAScope assessment of OTR mRNA, brain region-specific stereotaxic injections of viral vectors and retrograde tracing, optogenetic stimulation and OT biosensors in the ischemic HF model. We found that most of OTergic innervation of the central amygdala (CeA) originated from the hypothalamic supraoptic nucleus (SON). While no differences in the numbers of SON→CeA OTertic neurons (or their OT content) was observed between sham and HF rats, we did observe a blunted content and release of OT from axonal terminals within the CeA. Moreover, we report downregulation of neuronal and astrocytic OT receptors, and impaired OTR-driven GABAergic synaptic activity within the CeA microcircuit of rats with HF. Conclusions: Our study provides first evidence that HF rats display various perturbations in the hypothalamus-to-amygdala OTergic circuit, and lays the foundation for future translational studies targeting either the OT system or GABAergic amygdala GABA microcircuit to ameliorate depression or mood impairments in rats or patients with chronic HF.

Astrocytes as Drivers and Disruptors of Behavior: New Advances in Basic Mechanisms and Therapeutic Targeting.

Astrocytes are emerging as key regulators of cognitive function and behavior. This review highlights some of the latest advances in the understanding of astrocyte roles in different behavioral domains across lifespan and in disease. We address specific molecular and circuit mechanisms by which astrocytes modulate behavior, discuss their functional diversity and versatility, and highlight emerging astrocyte-targeted treatment strategies that might alleviate behavioral and cognitive dysfunction in pathologic conditions. Converging evidence across different model systems and manipulations is revealing that astrocytes regulate behavioral processes in a precise and context-dependent manner. Improved understanding of these astrocytic functions may generate new therapeutic strategies for various conditions with cognitive and behavioral impairments.

CaMK1D signalling in AgRP neurons promotes ghrelin-mediated food intake.

Hypothalamic AgRP/NPY neurons are key players in the control of feeding behaviour. Ghrelin, a major orexigenic hormone, activates AgRP/NPY neurons to stimulate food intake and adiposity. However, cell-autonomous ghrelin-dependent signalling mechanisms in AgRP/NPY neurons remain poorly defined. Here we show that calcium/calmodulin-dependent protein kinase ID (CaMK1D), a genetic hot spot in type 2 diabetes, is activated upon ghrelin stimulation and acts in AgRP/NPY neurons to mediate ghrelin-dependent food intake. Global Camk1d-knockout male mice are resistant to ghrelin, gain less body weight and are protected against high-fat-diet-induced obesity. Deletion of Camk1d in AgRP/NPY, but not in POMC, neurons is sufficient to recapitulate above phenotypes. In response to ghrelin, lack of CaMK1D attenuates phosphorylation of CREB and CREB-dependent expression of the orexigenic neuropeptides AgRP/NPY in fibre projections to the paraventricular nucleus (PVN). Hence, CaMK1D links ghrelin action to transcriptional control of orexigenic neuropeptide availability in AgRP neurons.

An analgesic pathway from parvocellular oxytocin neurons to the periaqueductal gray in rats.

The hypothalamic neuropeptide oxytocin (OT) exerts prominent analgesic effects via central and peripheral action. However, the precise analgesic pathways recruited by OT are largely elusive. Here we discovered a subset of OT neurons whose projections preferentially terminate on OT receptor (OTR)-expressing neurons in the ventrolateral periaqueductal gray (vlPAG). Using a newly generated line of transgenic rats (OTR-IRES-Cre), we determined that most of the vlPAG OTR expressing cells targeted by OT projections are GABAergic. Ex vivo stimulation of parvocellular OT axons in the vlPAG induced local OT release, as measured with OT sensor GRAB. In vivo, optogenetically-evoked axonal OT release in the vlPAG of as well as chemogenetic activation of OTR vlPAG neurons resulted in a long-lasting increase of vlPAG neuronal activity. This lead to an indirect suppression of sensory neuron activity in the spinal cord and strong analgesia in both female and male rats. Altogether, we describe an OT-vlPAG-spinal cord circuit that is critical for analgesia in both inflammatory and neuropathic pain models.

Analysis of the hypothalamic oxytocin system and oxytocin receptor-expressing astrocytes in a mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hyperphagia, obesity, developmental delay and intellectual disability. Studies suggest dysfunctional signaling of the neuropeptide oxytocin as one of the key mechanisms in PWS, and administration of oxytocin via intranasal or systemic routes yielded promising results in both humans and mouse models. However, a detailed assessment of the oxytocin system in mouse models of PWS such as the Magel2-deficient Magel2tm1.Stw mouse, is lacking. In the present study, we performed an automated counting of oxytocin cells in the entire paraventricular nucleus of the hypothalamus of Magel2tm1.Stw and wild-type control mice and found a significant reduction in the caudal part, which represents the parvocellular subdivision. In addition, based on the recent discovery that some astrocytes express the oxytocin receptor (OTR), we performed detailed analysis of astrocyte numbers and morphology in various brain regions, and assessed expression levels of the astrocyte marker glial fibrillary acidic protein, which was significantly decreased in the hypothalamus, but not other brain regions in Magel2tm1.Stw mice. Finally, we analyzed the number of OTR-expressing astrocytes in various brain regions and found a significant reduction in the nucleus accumbens of Magel2tm1.Stw mice, as well as a sex-specific difference in the lateral septum. This study suggests a role for caudal paraventricular nucleus oxytocin neurons as well as OTR-expressing astrocytes in a mouse model of PWS, provides novel information about sex-specific expression of astrocytic OTRs, and presents several new brain regions containing OTR-expressing astrocytes in the mouse brain.

The Role of Oxytocin in Abnormal Brain Development: Effect on Glial Cells and Neuroinflammation.

The neonatal period is critical for brain development and determinant for long-term brain trajectory. Yet, this time concurs with a sensitivity and risk for numerous brain injuries following perinatal complications such as preterm birth. Brain injury in premature infants leads to a complex amalgam of primary destructive diseases and secondary maturational and trophic disturbances and, as a consequence, to long-term neurocognitive and behavioral problems. Neuroinflammation is an important common factor in these complications, which contributes to the adverse effects on brain development. Mediating this inflammatory response forms a key therapeutic target in protecting the vulnerable developing brain when complications arise. The neuropeptide oxytocin (OT) plays an important role in the perinatal period, and its importance for lactation and social bonding in early life are well-recognized. Yet, novel functions of OT for the developing brain are increasingly emerging. In particular, OT seems able to modulate glial activity in neuroinflammatory states, but the exact mechanisms underlying this connection are largely unknown. The current review provides an overview of the oxytocinergic system and its early life development across rodent and human. Moreover, we cover the most up-to-date understanding of the role of OT in neonatal brain development and the potential neuroprotective effects it holds when adverse neural events arise in association with neuroinflammation. A detailed assessment of the underlying mechanisms between OT treatment and astrocyte and microglia reactivity is given, as well as a focus on the amygdala, a brain region of crucial importance for socio-emotional behavior, particularly in infants born preterm.

TRP channels and monoterpenes: Past and current leads on analgesic properties.

The activation of the transient receptor potential (TRP) channels expressed by sensory neurons is essential to the transduction of thermal and mechanical sensory information. In the setting of chronic inflammatory conditions, the activation of the melastatin family member 8 (TRPM8), the TRP vanilloid 1 (TRPV1), and the TRP ankyrin 1 (TRPA1) is correlated with pain hypersensitivity reactions. Monoterpenes, among which pulegone and menthol, a major class of phytocompounds present in essential oils of medicinal plants, are known modulators of those TRP channels activity. In the present review, we correlate the monoterpene content of plants with their historical therapeutic properties. We then describe how monoterpenes exert their anti-inflammatory and antihyperalgesia effects through modulation of TRP channels activity. Finally, we discuss the importance and the potential of characterizing new plant extracts and reassessing studied plant extracts for the development of ethnopharmacology-based innovative treatments for chronic pain. This review suggests that monoterpene solutions, based on composition from traditional healing herbs, offer an interesting avenue for the development of new phytotherapeutic treatments to alleviate chronic inflammatory pain conditions.

Emerging role of astrocytes in oxytocin-mediated control of neural circuits and brain functions.

The neuropeptide oxytocin has been in the focus of scientists for decades due to its profound and pleiotropic effects on physiology, activity of neuronal circuits and behaviors, among which sociality. Until recently, it was believed that oxytocinergic action exclusively occurs through direct activation of neuronal oxytocin receptors. However, several studies demonstrated the existence and functional relevance of astroglial oxytocin receptors in various brain regions in the mouse and rat brain. Astrocytic signaling and activity is critical for many important physiological processes including metabolism, neurotransmitter clearance from the synaptic cleft and integrated brain functions. While it can be speculated that oxytocinergic action on astrocytes predominantly facilitates neuromodulation via the release of specific gliotransmitters, the precise role of astrocytic oxytocin receptors remains elusive. In this review, we discuss the latest studies on the interaction between the oxytocinergic system and astrocytes, including detailed information about intracellular cascades, and speculate about future research directions on astrocytic oxytocin signaling.

Altered PVN-to-CA2 hippocampal oxytocin pathway and reduced number of oxytocin-receptor expressing astrocytes in heart failure rats.

Oxytocinergic actions within the hippocampal CA2 are important for neuromodulation, memory processing and social recognition. However, the source of the OTergic innervation, the cellular targets expressing the OT receptors (OTRs) and whether the PVN-to-CA2 OTergic system is altered during heart failure (HF), a condition recently associated with cognitive and mood decline, remains unknown. Using immunohistochemistry along with retrograde monosynaptic tracing, RNAscope and a novel OTR-Cre rat line, we show that the PVN (but not the supraoptic nucleus) is an important source of OTergic innervation to the CA2. These OTergic fibers were found in many instances in close apposition to OTR expressing cells within the CA2. Interestingly, while only a small proportion of neurons were found to express OTRs (~15%), this expression was much more abundant in CA2 astrocytes (~40%), an even higher proportion that was recently reported for astrocytes in the central amygdala. Using an established ischemic rat heart failure (HF) model, we found that HF resulted in robust changes in the PVN-to-CA2 OTergic system, both at the source and target levels. Within the PVN, we found an increased OT immunoreactivity, along with a diminished OTR expression in PVN neurons. Within the CA2 of HF rats, we observed a blunted OTergic innervation, along with a diminished OTR expression, which appeared to be restricted to CA2 astrocytes. Taken together, our studies highlight astrocytes as key cellular targets mediating OTergic PVN inputs to the CA2 hippocampal region. Moreover, they provide the first evidence for an altered PVN-to-CA2 OTergic system in HF rats, which could potentially contribute to previously reported cognitive and mood impairments in this animal model.

Calcium imaging and BAPTA loading of amygdala astrocytes in mouse brain slices.

Astrocytes are glial cells that exhibit calcium signaling-mediated activity. Here, we present a protocol to monitor and manipulate astrocyte calcium activity from mouse amygdala slices. In the first part of this protocol, we describe the procedure of astrocyte calcium imaging. In the second part, we detail how to disrupt astrocyte calcium activity by patch-clamp-mediated loading of BAPTA. These two approaches are presented separately but they can also be used simultaneously to monitor the effects of disruption on an astrocyte network. For complete details on the use and execution of this protocol, please refer to Wahis et al. (2021).

Identification and three-dimensional reconstruction of oxytocin receptor expressing astrocytes in the rat and mouse brain.

Here, we present a step-by-step protocol for three-dimensional reconstruction of astrocyte morphology, applied to the central amygdala oxytocin receptor-expressing astrocytes. This includes RNAse-free perfusion, combination of RNAscope and immunohistochemistry, and confocal imaging. This protocol provides detailed information about tissue handling and a comprehensive description of the RNAScope technique to label rat and mouse oxytocin receptor mRNA. We also describe three-dimensional reconstruction that allows the assessment of more than 70 different cellular parameters, powerful for studying astrocyte morphology and astrocyte-astrocyte interactions. For complete details on the use and execution of this protocol, please refer to Wahis et al. (2021) and Althammer et al. (2020).

Viral vectors for opto-electrode recording and photometry-based imaging of oxytocin neurons in anesthetized and socially interacting rats.

Here, we present a step-by-step protocol to target, record, and manipulate the activity of oxytocin neurons in awake rats. The protocol includes a procedure to record the activity of oxytocin neurons from awake and socially interacting rats using opto-electrodes for simultaneous electrophysiological recording and virally based cell-type-specific opto-tagging with Channelrhodopsin 2. Furthermore, we illustrate a procedure for optically guided implantation of optic fiber and imaging of oxytocin neuron population activity expressing calcium indicator GCaMP6s with the fiber photometry technique. For complete details on the use and execution of this protocol, please refer to Tang et al., 2020.

[Emergent role of astrocytes in oxytocin-mediated modulatory control of neuronal circuits and brain functions]. / Rôle émergent des astrocytes dans le contrôle des circuits neuronaux et des fonctions cérébrales modulés par l'ocytocine.

The neuropeptide oxytocin has been in the focus of scientists for decades due to its profound and pleiotropic effects on physiology, activity of neuronal circuits and behaviors. Until recently, it was believed that oxytocinergic action exclusively occurs through direct activation of neuronal oxytocin receptors. However, several studies demonstrated the existence and functional relevance of astroglial oxytocin receptors in various brain regions in the mouse and rat brain. Astrocytic signaling and activity are critical for many important physiological processes including metabolism, neurotransmitter clearance from the synaptic cleft and integrated brain functions. While it can be speculated that oxytocinergic action on astrocytes predominantly facilitates neuromodulation via the release of gliotransmitters, the precise role of astrocytic oxytocin receptors remains elusive. In this review, we discuss the latest studies on the interaction between the oxytocinergic system and astrocytes, and give details of underlying intracellular cascades.

Title: Rôle émergent des astrocytes dans le contrôle des circuits neuronaux et des fonctions cérébrales modulés par l'ocytocine. Abstract: L'ocytocine est un neuropeptide au centre de l'attention des scientifiques depuis des décennies, en raison de ses effets puissants et pléiotropes tant sur le plan physiologique que sur l'activité des circuits neuronaux, modulant ainsi nos comportements. Jusqu'à une date récente, on pensait que l'action de l'ocytocine était induite exclusivement par l'activation directe de ses récepteurs neuronaux. Cependant, plusieurs études ont démontré l'existence et la pertinence fonctionnelle des récepteurs astrogliaux de l'ocytocine dans diverses régions du cerveau de la souris et du rat. La signalisation et l'activité astrocytaires sont essentielles à de nombreux processus physiologiques importants, notamment le métabolisme, l'élimination des neurotransmetteurs de la fente synaptique et les fonctions cérébrales intégrées. Bien que l'on puisse supposer que l'action de l'ocytocine sur les astrocytes facilite principalement la neuromodulation via la libération de gliotransmetteurs, le rôle précis des récepteurs astrocytaires de l'ocytocine reste difficile à cerner. Dans cette revue, nous discutons des dernières études sur l'interaction entre le système ocytocinergique et les astrocytes, et décrivons les cascades intracellulaires mises en jeu.

Anti-Hyperalgesic Properties of Menthol and Pulegone.

Context: Menthol, the main monoterpene found in Mentha piperita L. (M. piperita) is known to modulate nociceptive threshold and is present in different curative preparations that reduce sensory hypersensitivities in pain conditions. While for pulegone, a menthol-like monoterpene, only a limited number of studies focus on its putative analgesic effects, pulegone is the most abundant monoterpene present in Calamintha nepeta (L.) Savi (C. nepeta), a plant of the Lamiaceae family used in traditional medicine to alleviate rheumatic disorders, which counts amongst chronic inflammatory diseases. Objectives: Here, we analyzed the monoterpenes composition of C. nepeta and M. piperita. We then compared the putative anti-hyperalgesic effects of the main monoterpenes found, menthol and pulegone, in acute inflammatory pain conditions. Methods: C. nepeta and M. piperita extracts were obtained through pressurized liquid extraction and analyzed by gas chromatography-mass spectrometry. The in vitro anti-inflammatory activity of menthol or pulegone was evaluated by measuring the secretion of the tumour necrosis factor alpha (TNF α) from LPS-stimulated THP-1 cells. The in vivo anti-hyperalgesic effects of menthol and pulegone were tested on a rat inflammatory pain model. Results: Pulegone and menthol are the most abundant monoterpene found in C. nepeta (49.41%) and M. piperita (42.85%) extracts, respectively. In vitro, both pulegone and menthol act as strong anti-inflammatory molecules, with EC50 values of 1.2 ± 0.2 and 1.5 ± 0.1 mM, respectively, and exert cytotoxicity with EC50 values of 6.6 ± 0.3 and 3.5 ± 0.2 mM, respectively. In vivo, 100 mg/kg pulegone exerts a transient anti-hyperalgesic effect on both mechanical (pulegone: 274.25 ± 68.89 g, n = 8; vehicle: 160.88 ± 35.17 g, n = 8, p < 0.0001), thermal heat (pulegone: 4.09 ± 0.62 s, n = 8; vehicle: 2.25 ± 0.34 s, n = 8, p < 0.0001), and cold (pulegone: 2.25 ± 1.28 score, n = 8; vehicle: 4.75 ± 1.04 score, n = 8, p = 0.0003). In a similar way, 100 mg/kg menthol exerts a transient anti-hyperalgesic effect on both mechanical (mechanical: menthol: 281.63 ± 45.52 g, n = 8; vehicle: 166.25 ± 35.4 g, n = 8, p < 0.0001) and thermal heat (menthol: 3.65 ± 0.88 s, n = 8; vehicle: 2.19 ± 0.26 s, n = 8, <0.0001). Conclusion: Here, we show that both pulegone and menthol are anti-inflammatory and anti-hyperalgesic monoterpenes. These results might open the path towards new compound mixes to alleviate the pain sensation.

Oxytocinergic Feedback Circuitries: An Anatomical Basis for Neuromodulation of Social Behaviors.

Oxytocin (OT) is a neuropeptide produced by hypothalamic neurons and is known to modulate social behavior among other functions. Several experiments have shown that OT modulates neuronal activity in many brain areas, including sensory cortices. OT neurons thus project axons to various cortical and subcortical structures and activate neuronal subpopulations to increase the signal-to-noise ratio, and in turn, increases the saliency of social stimuli. Less is known about the origin of inputs to OT neurons, but recent studies show that cells projecting to OT neurons are often located in regions where the OT receptor (OTR) is expressed. Thus, we propose the existence of reciprocal connectivity between OT neurons and extrahypothalamic OTR neurons to tune OT neuron activity depending on the behavioral context. Furthermore, the latest studies have shown that OTR-expressing neurons located in social brain regions also project to other social brain regions containing OTR-expressing neurons. We hypothesize that OTR-expressing neurons across the brain constitute a common network coordinated by OT.

Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

Social touch promotes interfemale communication via activation of parvocellular oxytocin neurons.

Oxytocin (OT) is a great facilitator of social life but, although its effects on socially relevant brain regions have been extensively studied, OT neuron activity during actual social interactions remains unexplored. Most OT neurons are magnocellular neurons, which simultaneously project to the pituitary and forebrain regions involved in social behaviors. In the present study, we show that a much smaller population of OT neurons, parvocellular neurons that do not project to the pituitary but synapse onto magnocellular neurons, is preferentially activated by somatosensory stimuli. This activation is transmitted to the larger population of magnocellular neurons, which consequently show coordinated increases in their activity during social interactions between virgin female rats. Selectively activating these parvocellular neurons promotes social motivation, whereas inhibiting them reduces social interactions. Thus, parvocellular OT neurons receive particular inputs to control social behavior by coordinating the responses of the much larger population of magnocellular OT neurons.