Oxytocin receptors in the Magel2 mouse model of autism: Specific region, age, sex and oxytocin treatment effects.

The neurohormone oxytocin (OXT) has been implicated in the regulation of social behavior and is intensively investigated as a potential therapeutic treatment in neurodevelopmental disorders characterized by social deficits. In the Magel2-knockout (KO) mouse, a model of Schaaf-Yang Syndrome, an early postnatal administration of OXT rescued autistic-like behavior and cognition at adulthood, making this model relevant for understanding the actions of OXT in (re)programming postnatal brain development. The oxytocin receptor (OXTR), the main brain target of OXT, was dysregulated in the hippocampus of Magel2-KO adult males, and normalized upon OXT treatment at birth. Here we have analyzed male and female Magel2-KO brains at postnatal day 8 (P8) and at postnatal day 90 (P90), investigating age, genotype and OXT treatment effects on OXTR levels in several regions of the brain. We found that, at P8, male and female Magel2-KOs displayed a widespread, substantial, down-regulation of OXTR levels compared to wild type (WT) animals. Most intriguingly, the postnatal OXT treatment did not affect Magel2-KO OXTR levels at P8 and, consistently, did not rescue the ultrasonic vocalization deficits observed at this age. On the contrary, the postnatal OXT treatment reduced OXTR levels at P90 in male Magel2-KO in a region-specific way, restoring normal OXTR levels in regions where the Magel2-KO OXTR was upregulated (central amygdala, hippocampus and piriform cortex). Interestingly, Magel2-KO females, previously shown to lack the social deficits observed in Magel2-KO males, were characterized by a different trend in receptor expression compared to males; as a result, the dimorphic expression of OXTR observed in WT animals, with higher OXTR expression observed in females, was abolished in Magel2-KO mice. In conclusion, our data indicate that in Magel2-KO mice, OXTRs undergo region-specific modifications related to age, sex and postnatal OXT treatment. These results are instrumental to design precisely-timed OXT-based therapeutic strategies that, by acting at specific brain regions, could modify the outcome of social deficits in Schaaf-Yang Syndrome patients.

Region-Specific KCC2 Rescue by rhIGF-1 and Oxytocin in a Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is characterized by dysfunction in neuronal excitation/inhibition (E/I) balance, potentially impacting seizure susceptibility via deficits in K+/Cl- cotransporter 2 (KCC2) function. Mice lacking the Methyl-CpG binding protein 2 (MeCP2) recapitulate many symptoms of RTT, and recombinant human insulin-like growth factor-1 (rhIGF-1) restores KCC2 expression and E/I balance in MeCP2 KO mice. However, clinical trial outcomes of rhIGF-1 in RTT have been variable, and increasing its therapeutic efficacy is highly desirable. To this end, the neuropeptide oxytocin (OXT) is promising, as it also critically modulates KCC2 function during early postnatal development. We measured basal KCC2 expression levels in MeCP2 KO mice and identified 3 key frontal brain regions showing KCC2 alterations in young adult mice, but not in postnatal P10 animals. We hypothesized that deficits in an IGF-1/OXT signaling crosstalk modulating KCC2 may occur in RTT during postnatal development. Consistently, we detected alterations of IGF-1 receptor and OXT receptor levels in those brain areas. rhIGF-1 and OXT treatments in KO mice rescued KCC2 expression in a region-specific and complementary manner. These results suggest that region-selective combinatorial pharmacotherapeutic strategies could be most effective at normalizing E/I balance in key brain regions subtending the RTT pathophysiology.

The ligand-bound state of a G protein-coupled receptor stabilizes the interaction of functional cholesterol molecules.

Cholesterol is a major component of mammalian plasma membranes that not only affects the physical properties of the lipid bilayer but also is the function of many membrane proteins including G protein-coupled receptors. The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined. Using the ion channel-coupled receptor technology, we developed a functional assay of cholesterol modulation of G protein-coupled receptors that is independent of intracellular signaling pathways and operational in living cells. Using this assay, we discovered a stable binding of cholesterol molecules to the receptor when it adopts an orthosteric ligand-bound state. This stable interaction preserves the cholesterol-dependent activity of the receptor in cholesterol-depleted membranes. This mechanism was confirmed using time-resolved FRET experiments on WT OXTR expressed in CHO cells. Consequently, a positive cross-regulation sequentially occurs in OXTR between cholesterol and orthosteric ligands.

Impaired approach to novelty and striatal alterations in the oxytocin receptor deficient mouse model of autism.

Long-standing studies established a role for the oxytocin system in social behavior, social reward, pair bonding and affiliation. Oxytocin receptors, implicated in pathological conditions affecting the social sphere such as autism spectrum disorders, can also modulate cognitive processes, an aspect generally overlooked. Here we examined the effect of acute (pharmacological) or genetic (Oxtr-/-) inactivation of oxytocin receptor-mediated signaling, in male mice, in several cognitive tests. In the novel object recognition test, both oxytocin receptor antagonist treated wild type animals and Oxtr-/- mice lacked the typical preference for novelty. Oxtr-/- mice even preferred the familiar object; moreover, their performance in the Morris water maze did not differ from wild types, suggesting that oxytocin receptor inactivation did not disrupt learning. Because the preference for novel objects could be rescued in Oxtr-/- mice with longer habituation periods, we propose that the loss of novelty preferences following Oxtr inactivation is due to altered processing of novel contextual information. Finally, we observed an increased expression of excitatory synaptic markers in the striatum of Oxtr-/- mice and a greater arborization and higher number of spines/neuron in the dorsolateral area of this structure, which drives habit formation. Our data also indicate a specific reshaping of dorsolateral striatal spines in Oxtr-/- mice after exposure to a novel environment, which might subtend their altered approach to novelty, and support previous work pointing at this structure as an important substrate for autistic behaviors.

Molecular Basis of Oxytocin Receptor Signalling in the Brain: What We Know and What We Need to Know.

Oxytocin (OT), a hypothalamic neuropeptide involved in regulating the social behaviour of all vertebrates, has been proposed as a treatment for a number of neuropsychiatric disorders characterised by deficits in the social domain. Over the last few decades, advances focused on understanding the social effects of OT and its role in physiological conditions and brain diseases, but much less has been done to clarify the molecular cascade of events involved in mediating such effects and in particular the cellular and molecular pharmacology of OT and its target receptor (OTR) in neuronal and glial cells.The entity and persistence of OT activity in the brain is closely related to the expression and regulation of the OTR expressed on the cell surface, which transmits the signal intracellularly and permits OT to affect cell function. Understanding the various signalling mechanisms mediating OTR-induced cell responses is crucial to determine the different responses in different cells and brain regions, and the success of OT and OT-derived analogues in the treatment of neurodevelopmental and psychiatric diseases depends on how well we can control such responses. In this review, we will consider the most important aspects of OT/OTR signalling by focusing on the molecular events involved in OT binding and coupling, on the main signalling pathways activated by the OTR in neuronal cells and on intracellular and plasma membrane OTR trafficking, all of which contribute to the quantitative and qualitative features of OT responses in the brain.

Oxytocin Receptors in the Anteromedial Bed Nucleus of the Stria Terminalis Promote Stress-Induced Social Avoidance in Female California Mice.

BACKGROUND: The neuropeptide oxytocin (OT) is a key regulator of social and emotional behaviors. The effects of OT are context dependent, and it has been proposed that OT increases the salience of both positive and negative social cues. Here we tested whether the bed nucleus of the stria terminalis (BNST) mediates anxiogenic effects of OT. METHODS: First, we studied the effects of systemic administration of an OT receptor (OTR) antagonist L-368,899 on social behavior in male and female California mice exposed to social defeat. We examined the effect of L-368,899 on G protein activation and used early growth response factor 1 immunohistochemistry to identify potential sites of OTR action. Finally, we examined the effects of L-368,899 infused in the BNST on behavior. RESULTS: A single dose of systemic L-368,899 increased social approach in stressed female mice and decreased social approach in male mice naïve to defeat. L-368,899 prevented OT activation of G proteins and did not activate G proteins in the absence of OT. Intranasal OT, which reduces social approach in female mice but not male mice, increased early growth response factor 1 immunoreactivity in the nucleus accumbens core and anteromedial BNST in female mice but not in male mice. Stressed female mice that received an infusion of L-368,899 into the anteromedial BNST but not the nucleus accumbens core increased social approach and decreased social vigilance responses. CONCLUSIONS: Our results suggest that OTR activation in anteromedial BNST induces a vigilance response in which individuals avoid, yet attend to, unfamiliar social contexts. Our results suggest that OTR antagonists may have unappreciated therapeutic potential for stress-induced psychiatric disorders.

Subtle modifications to oxytocin produce ligands that retain potency and improved selectivity across species.

Oxytocin and vasopressin mediate various physiological functions that are important for osmoregulation, reproduction, cardiovascular function, social behavior, memory, and learning through four G protein-coupled receptors that are also implicated in high-profile disorders. Targeting these receptors is challenging because of the difficulty in obtaining ligands that retain selectivity across rodents and humans for translational studies. We identified a selective and more stable oxytocin receptor (OTR) agonist by subtly modifying the pharmacophore framework of human oxytocin and vasopressin. [Se-Se]-oxytocin-OH displayed similar potency to oxytocin but improved selectivity for OTR, an effect that was retained in mice. Centrally infused [Se-Se]-oxytocin-OH potently reversed social fear in mice, confirming that this action was mediated by OTR and not by V1a or V1b vasopressin receptors. In addition, [Se-Se]-oxytocin-OH produced a more regular contraction pattern than did oxytocin in a preclinical labor induction and augmentation model using myometrial strips from cesarean sections. [Se-Se]-oxytocin-OH had no activity in human cardiomyocytes, indicating a potentially improved safety profile and therapeutic window compared to those of clinically used oxytocin. In conclusion, [Se-Se]-oxytocin-OH is a novel probe for validating OTR as a therapeutic target in various biological systems and is a promising new lead for therapeutic development. Our medicinal chemistry approach may also be applicable to other peptidergic signaling systems with similar selectivity issues.

Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo.

Thromboxane A2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPß homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations.

Design and Characterization of Superpotent Bivalent Ligands Targeting Oxytocin Receptor Dimers via a Channel-Like Structure.

Dimeric/oligomeric states of G-protein coupled receptors have been difficult to target. We report here bivalent ligands consisting of two identical oxytocin-mimetics that induce a three order magnitude boost in G-protein signaling of oxytocin receptors (OTRs) in vitro and a 100- and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish. Through receptor mutagenesis and interference experiments with synthetic peptides mimicking transmembrane helices (TMH), we show that such superpotent behavior follows from the binding of the bivalent ligands to dimeric receptors based on a TMH1-TMH2 interface. Moreover, in this arrangement, only the analogues with a well-defined spacer length (∼25 Å) precisely fit inside a channel-like passage between the two protomers of the dimer. The newly discovered oxytocin bivalent ligands represent a powerful tool for targeting dimeric OTR in neurodevelopmental and psychiatric disorders and, in general, provide a framework to untangle specific arrangements of G-protein coupled receptor dimers.

The Timing of the Excitatory-to-Inhibitory GABA Switch Is Regulated by the Oxytocin Receptor via KCC2.

Oxytocin and its receptor (Oxtr) play a crucial role in the postnatal transition of neuronal GABA neurotransmission from excitatory to inhibitory, a developmental process known as the GABA switch. Using hippocampal neurons from Oxtr-null mice, we show that (1) Oxtr is necessary for the correct timing of the GABA switch by upregulating activity of the chloride cotransporter KCC2, (2) Oxtr, in a very early and narrow time window, directly modulates the functional activity of KCC2 by promoting its phosphorylation and insertion/stabilization at the neuronal surface, and (3) in the absence of Oxtr, electrophysiological alterations are recorded in mature neurons, a finding consistent with a reduced level of KCC2 and increased susceptibility to seizures observed in adult Oxtr-null mice. These data identify KCC2 as a key target of oxytocin in postnatal events that may be linked to pathogenesis of neurodevelopmental disorders.

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.

Analysis of G Protein and ß-Arrestin Activation in Chemokine Receptors Signaling.

Chemokines are key regulators of leukocyte migration and play fundamental roles in immune responses. The chemokine system includes a set of over 40 ligands which engage in a promiscuous fashion a panel of over 25 receptors belonging to a distinct family of 7 transmembrane-domain receptors (7TM) widely expressed on a variety of cells. Although responses evoked by chemokine receptors have long been considered the result of balanced activation of the G protein- and ß-arrestin-dependent signaling modules, evidence is accumulating showing that these receptors are capable, as other 7TMs, to activate different signaling modules in a ligand- and cell/tissue-specific manner. This biased signaling, or functional selectivity, confers a hitherto largely uncharacterized level of complexity to the chemokine system and challenges our present understanding of its redundancy. At the same time, it also provides new insights of relevance for chemokine receptors targeting drug development plans. Here, we provide current methods to study biased signaling of chemokine receptors by dissecting G proteins and ß-arrestins activation upon chemokine stimulation.

Assembling the Puzzle: Pathways of Oxytocin Signaling in the Brain.

Oxytocin (OT) is a neuropeptide, which can be seen to be one of the molecules of the decade due to its profound prosocial effects in nonvertebrate and vertebrate species, including humans. Although OT can be detected in various physiological fluids (blood, saliva, urine, cerebrospinal fluid) and brain tissue, it is unclear whether peripheral and central OT releases match and synergize. Moreover, the pathways of OT delivery to brain regions involved in specific behaviors are far from clear. Here, we discuss the evolutionarily and ontogenetically determined pathways of OT delivery and OT signaling, which orchestrate activity of the mesolimbic social decision-making network. Furthermore, we speculate that both the alteration in OT delivery and OT receptor expression may cause behavioral abnormalities in patients afflicted with psychosocial diseases.

Region specific up-regulation of oxytocin receptors in the opioid oprm1 (-/-) mouse model of autism.

Autism spectrum disorders (ASDs) are characterized by impaired communication, social impairments, and restricted and repetitive behaviors and interests. Recently, altered motivation and reward processes have been suggested to participate in the physiopathology of ASDs, and µ-opioid receptors (MORs) have been investigated in relation to social reward due to their involvement in the neural circuitry of reward. Mice lacking a functional MOR gene (Oprm1 (-/-) mice) display abnormal social behavior and major autistic-like core symptoms, making them an animal model of autism. The oxytocin (OXT) system is a key regulator of social behavior and co-operates with the opioidergic system in the modulation of social behavior. To better understand the opioid-OXT interplay in the central nervous system, we first determined the expression of the oxytocin receptor (OXTR) in the brain of WT C57BL6/J mice by quantitative autoradiography; we then evaluated OXTR regional alterations in Oprm1 (-/-) mice. Moreover, we tested these mice in a paradigm of social behavior, the male-female social interaction test, and analyzed the effects of acute intranasal OXT treatment on their performance. In autoradiography, Oprm1 (-/-) mice selectively displayed increased OXTR expression in the Medial Anterior Olfactory Nucleus, the Central and Medial Amygdaloid nuclei, and the Nucleus Accumbens. Our behavioral results confirmed that Oprm1 (-/-) male mice displayed social impairments, as indicated by reduced ultrasonic calls, and that these were rescued by a single intranasal administration of OXT. Taken together, our results provide evidence of an interaction between OXT and opioids in socially relevant brain areas and in the modulation of social behavior. Moreover, they suggest that the oxytocinergic system may act as a compensative mechanism to bypass and/or restore alterations in circuits linked to impaired social behavior.

Germline prokineticin receptor 2 (PROKR2) variants associated with central hypogonadism cause differental modulation of distinct intracellular pathways.

INTRODUCTION: Defects of prokineticin pathway affect the neuroendocrine control of reproduction, but their role in the pathogenesis of central hypogonadism remains undefined, and the functional impact of the missense PROKR2 variants has been incompletely characterized. MATERIAL AND METHODS: In a series of 246 idiopathic central hypogonadism patients, we found three novel (p.V158I, p.V334M, and p.N15TfsX30) and six already known (p.L173R, p.T260M, p.R268C, p.V274D, p.V331M, and p.H20MfsX23) germline variants in the PROKR2 gene. We evaluated the effects of seven missense alterations on two different prokineticin receptor 2 (PROKR2)-dependent pathways: inositol phosphate-Ca(2+) (Gq coupling) and cAMP (Gs coupling). RESULTS: PROKR2 variants were found in 16 patients (6.5%). Expression levels of variants p.V158I and p.V331M were moderately reduced, whereas they were markedly impaired in the remaining cases, except p.V334M, which was significantly overexpressed. The variants p.T260M, p.R268C, and p.V331M showed no remarkable changes in cAMP response (EC50) whereas the IP signaling appeared more profoundly affected. In contrast, cAMP accumulation cannot be stimulated through the p.L173R and p.V274D, but IP EC50 was similar to wt inp.L173R and increased by 10-fold in p.V274D. The variant p.V334M led to a 3-fold increase of EC50 for both cAMP and IP. CONCLUSION: Our study shows that single PROKR2 missense allelic variants can either affect both signaling pathways differently or selectively. Thus, the integrity of both PROKR2-dependent cAMP and IP signals should be evaluated for a complete functional testing of novel identified allelic variants.

Chronic and acute intranasal oxytocin produce divergent social effects in mice.

Intranasal administration of oxytocin (OXT) might be a promising new adjunctive therapy for mental disorders characterized by social behavioral alterations such as autism and schizophrenia. Despite promising initial studies in humans, it is not yet clear the specificity of the behavioral effects induced by chronic intranasal OXT and if chronic intranasal OXT could have different effects compared with single administration. This is critical for the aforementioned chronic mental disorders that might potentially involve life-long treatments. As a first step to address these issues, here we report that chronic intranasal OXT treatment in wild-type C57BL/6J adult mice produced a selective reduction of social behaviors concomitant to a reduction of the OXT receptors throughout the brain. Conversely, acute intranasal OXT treatment produced partial increases in social behaviors towards opposite-sex novel-stimulus female mice, while on the other hand, it decreased social exploration of same-sex novel stimulus male mice, without affecting social behavior towards familiar stimulus male mice. Finally, prolonged exposure to intranasal OXT treatments did not alter, in wild-type animals, parameters of general health such as body weight, locomotor activity, olfactory and auditory functions, nor parameters of memory and sensorimotor gating abilities. These results indicate that a prolonged over-stimulation of a 'healthy' oxytocinergic brain system, with no inherent deficits in social interaction and normal endogenous levels of OXT, results in specific detrimental effects in social behaviors.

Selective and potent agonists and antagonists for investigating the role of mouse oxytocin receptors.

The neuropeptides oxytocin (OT) and vasopressin (AVP) have been shown to play a central role in social behaviors; as a consequence, they have been recognized as potential drugs to treat neurodevelopmental and psychiatric disorders characterized by impaired social interactions. However, despite the basic and preclinical relevance of mouse strains carrying genetic alterations in the OT/AVP systems to basic and preclinical translational neuroscience, the pharmacological profile of mouse OT/AVP receptor subtypes has not been fully characterized. To fill in this gap, we have characterized a number of OT and AVP agonists and antagonists at three murine OT/AVP receptors expressed in the nervous system as follows: the oxytocin (mOTR) and vasopressin V1a (mV1aR) and V1b (mV1bR) subtypes. These three receptors were transiently expressed in vitro for binding and intracellular signaling assays, and then a homology model of the mOTR structure was constructed to investigate how its molecular features compare with human and rat OTR orthologs. Our data indicate that the selectivity profile of the natural ligands, OT and AVP, is conserved in humans, rats, and mice. Furthermore, we found that the synthetic peptide [Thr(4)Gly(7)]OT (TGOT) is remarkably selective for the mOTR and, like the endogenous OT ligand, activates Gq and Gi and recruits ß-arrestins. Finally, we report three antagonists that exhibit remarkably high affinities and selectivities at mOTRs. These highly selective pharmacological tools will contribute to the investigation of the specific physiologic and pathologic roles of mOTR for the development of selective OT-based therapeutics.

Full and partial agonists of thromboxane prostanoid receptor unveil fine tuning of receptor superactive conformation and G protein activation.

The intrahelical salt bridge between E/D(3.49) and R(3.50) within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E(3.49/6.30) in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

Deciphering the specific role of G(αi/o) isoforms: functional selective oxytocin ligands and somatostatin SST5 receptor mutants.

Receptor coupling to different G-proteins and ß-arrestins has been described for a number of GPCRs (G-protein-coupled receptors), suggesting a multi-state model of receptor activation in which each receptor can assume a number of different active conformations, each capable of promoting the coupling to a specific effector. Consistently, functional-selective ligands and biased agonists have been described to be able to induce and/or stabilize only a subset of specific active conformations. Furthermore, GPCR mutants deficient in selective coupling have been reported. Functional selective ligands and receptor mutants thus constitute unique tools to dissect the specific roles of different effectors, in particular among the Gi/o family. In the present mini-review, we focus on (i) the identification of functional selective OXT (oxytocin)-derived peptides capable of activating single Gi/o isoforms, namely Gi1 or Gi3; and (ii) the characterization of an SS (somatostatin) receptor SST5 mutant selectively impaired in its GoA coupling. These analogues and receptor mutants represent unique tools for examining the contribution of Gi/o isoforms in complex biological responses and open the way for the development of drugs with peculiar selectivity profiles.