International Journal of Molecular Sciences Article Sex Differences in the Hypothalamic Oxytocin Pathway to Locus Coeruleus and Augmented Attention with Chemogenetic Activation of Hypothalamic Oxytocin Neurons Xin Wang, Joan B. Escobar and David Mendelowitz * Department of Pharmacology and Physiology, School of Medicine and Health Sciences, The George University Washington, Washington, DC 20037, USA; [email protected] (X.W.); [email protected] (J.B.E.) * Correspondence: [email protected] Abstract: The tightly localized noradrenergic neurons (NA) in the locus coeruleus (LC) are well recognized as essential for focused arousal and novelty-oriented responses, while many children with autism spectrum disorder (ASD) exhibit diminished attention, engagement and orienting to exogenous stimuli. This has led to the hypothesis that atypical LC activity may be involved in ASD. Oxytocin (OXT) neurons and receptors are known to play an important role in social behavior, pair bonding and cognitive processes and are under investigation as a potential treatment for ASD. However, little is known about the neurotransmission from hypothalamic paraventricular (PVN) OXT neurons to LC NA neurons. In this study, we test, in male and female rats, whether PVN OXT neurons excite LC neurons, whether oxytocin is released and involved in this neurotransmission, Citation: Wang, X.; Escobar, J.B.; and whether activation of PVN OXT neurons alters novel object recognition. Using “oxytocin sniffer Mendelowitz, D. Sex Differences in cells” (CHO cells that express the human oxytocin receptor and a Ca indicator) we show that there is the Hypothalamic Oxytocin Pathway release of OXT from hypothalamic PVN OXT fibers in the LC. Optogenetic excitation of PVN OXT to Locus Coeruleus and Augmented fibers excites LC NA neurons by co-release of OXT and glutamate, and this neurotransmission is Attention with Chemogenetic Activation of Hypothalamic Oxytocin greater in males than females. In male, but not in female animals, chemogenetic activation of PVN Neurons. Int. J. Mol. Sci. 2021, 22, OXT neurons increases attention to novel objects. 8510. https://doi.org/10.3390/ ijms22168510 Keywords: hypothalamus; oxytocin; locus coeruleus; paraventricular nucleus; attention; sex differences; novel object Academic Editors: Adele Romano and Gustavo Provens Received: 22 June 2021 1. Introduction Accepted: 4 August 2021 The tightly localized noradrenergic neurons (NA) in the locus coeruleus (LC) are Published: 7 August 2021 well recognized as essential for focused attention, arousal, novelty-oriented responses and promoting wakefulness [1]. LC neurons possess slow irregular firing during quiet Publisher’s Note: MDPI stays neutral wakefulness which increases to sustained higher frequencies of firing during heightened with regard to jurisdictional claims in vigilance and/or exposure to novel stimuli [2]. Children with autism spectrum disorder published maps and institutional affil- iations. (ASD) exhibit diminished attention, engagement and orienting to exogenous stimuli, and it has been suggested that atypical LC-NA activity is involved in aberrant mental focus and responsiveness in children with ASD [3]. The oxytocin network within the CNS is known to play an important role in social behavior, pair bonding and cognitive processes in both sexes and is under investigation Copyright: © 2021 by the authors. as a potential treatment for ASD [4]. In human subjects, oxytocin (OXT) administration Licensee MDPI, Basel, Switzerland. enhances stimulus-induced pupil dilation, consistent with oxytocin augmenting attention This article is an open access article towards socially relevant stimuli [5]. OXT administration in ASD children increases activity distributed under the terms and in brain regions important for perceiving social-emotional information [6]. Pharmacological conditions of the Creative Commons Attribution (CC BY) license (https:// and genetic (Oxtr-/-) inactivation of oxytocin receptor-mediated network function in mice creativecommons.org/licenses/by/ diminishes the typical preference for novelty, not due to impairment in learning, but 4.0/). likely via decreased processing of novel contextual information [7]. Exposure to a social Int. J. Mol. Sci. 2021, 22, 8510. https://doi.org/10.3390/ijms22168510 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 10 Int. J. Mol. Sci. 2021, 22, 8510 2 of 10 information [7]. Exposure to a social stimulus activates paraventricular nucleus of the hypothalamus (PVN) OXT neurons, and chemogenetic activation of PVN OXT of male stimulus activates paraventricular nucleus of the hypothalamus (PVN) OXT neurons, and mice enhances social investigation during a social choice test, while chemogenetic chemogenetic activation of PVN OXT of male mice enhances social investigation during a inhibition diminishes social preferences [8]. social choice test, while chemogenetic inhibition diminishes social preferences [8]. OXT is only synthesized in a limited number of discrete brain regions: the PVN, OXT is only synthesized in a limited number of discrete brain regions: the PVN, supraoptic and accessory nuclei of the hypothalamus [9]. While it is well known that PVN supraoptic and accessory nuclei of the hypothalamus [9]. While it is well known that OXT neurons project to the LC in rats [10], and OXT fibers are present in the LC over its PVN OXT neurons project to the LC in rats [10], and OXT fibers are present in the LC entire rostrocaudal extension in the human brain [11], little is known about the over its entire rostrocaudal extension in the human brain [11], little is known about the neurotransmission from PVN OXT neurons to LC NA neurons. In this study, we test, in neurotransmission from PVN OXT neurons to LC NA neurons. In this study, we test, male and female animals, whether PVN OXT neurons monosynaptically excite LC in male and female animals, whether PVN OXT neurons monosynaptically excite LC neurons,neurons, whether whether oxytocin oxytocin is released is released and involvedand involved in this neurotransmission,in this neurotransmission, and whether and activationwhether activation of PVN OXT of PVN neurons OXT alters neurons novel alte objectrs novel recognition. object recognition. Here, using Here, oxytocin- using sensitiveoxytocin-sensitive sniffer cells, sniffer we cells, show we that show there that is releasethere is ofrelease OXT of from OXT hypothalamic from hypothalamic PVN OXTPVN fibers OXT infibers the LC,in the optogenetic LC, optogenetic excitation excitation of PVN OXTof PVN fibers OXT excites fibers LC excites NA neurons LC NA byneurons co-release by co-release of OXT and of OXT glutamate, and glutamate, and finally and chemogeneticfinally chemogenetic activation activation of PVN of OXT PVN neuronsOXT neurons increases increases attention attention assessed assessed by novel by object novel recognition object recognition tests. Surprisingly, tests. Surprisingly, there is significantthere is significant sex dependent sex dependent modulation modulation in these responses.in these responses. 2.2. Results Results ToTo test test whether whether activation activation of of PVN PVN OXT OXT neurons neurons alters alters the the response response to to a a novel novel object, object, wewe selectively selectively activated activated PVNPVN OXT OXT neurons neurons using using DREADDs DREADDs in in 10 10 male male and and six six female female animals.animals. ChemogeneticChemogenetic activationactivation ofof PVNPVN OXTOXT neuronsneurons inin femalesfemales diddid not not change change the the proportionproportion of of time time spent spent with with a a new new object object (see (see Figure Figure1 ,1, left), left), suggesting suggesting that that PVN PVN OXT OXT neuronsneurons do do not not play play an an important important role role in in this this behavior behavior in in females. females. InIn contrast, contrast, however,however, chemogeneticchemogenetic activationactivation ofof PVNPVN OXTOXT neurons neurons in in males males significantly significantly increased increased the the time time spentspent with with a a new, new, rather rather than than familiar familiar object object (see (see Figure Figure1 ,1, right), right), indicating indicating that that PVN PVN OXT OXT neuronneuron activation activation can can facilitate facilitate attention attention to to novel novel objects objects in in male, male, but but not not female, female, rats. rats. FigureFigure 1. 1.Male Male rats rats spent spent more more time time interacting interacting with with a a novel novel subject subject than than female female rats. rats. A A graphic graphic rep- representation of the novel object paradigm is shown (top). Animals received an injection of resentation of the novel object paradigm is shown (top). Animals received an injection of Clozapine- Clozapine-N-Oxide (CNO) or saline 1 h prior to acclimation. Two randomly selected objects were N-Oxide (CNO) or saline 1 h prior to acclimation. Two randomly selected objects were placed within placed within the cage and the animal was allowed to interact with both objects for 15 min. Both theobjects cage were and theremoved animal for was one allowed hour, and to then interact one withof the both former objects objects, for 15along min. with Both one objects novel wereobject removedwas placed for in one the hour, cage and for 15 then min. one Histogram of the former (bottom) objects, shows along the with proportion one novel of time object interacting was placed inwith the a cage