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• W2520003619 abstract "•Activation of OxtrINs is anxiolytic in males and prosocial in females•OxtrINs specifically express CRHBP, an inhibitor of the stress hormone CRH•CRHBP blocks activation of layer 2/3 pyramidal cells by CRH only in males•OxtrINs in the mPFC coordinate sexually dimorphic social/emotional behaviors The frequency of human social and emotional disorders varies significantly between males and females. We have recently reported that oxytocin receptor interneurons (OxtrINs) modulate female sociosexual behavior. Here, we show that, in male mice, OxtrINs regulate anxiety-related behaviors. We demonstrate that corticotropin-releasing-hormone-binding protein (CRHBP), an antagonist of the stress hormone CRH, is specifically expressed in OxtrINs. Production of CRHBP blocks the CRH-induced potentiation of postsynaptic layer 2/3 pyramidal cell activity of male, but not female, mice, thus producing an anxiolytic effect. Our data identify OxtrINs as critical for modulation of social and emotional behaviors in both females and males and reveal a molecular mechanism that acts on local medial prefrontal cortex (mPFC) circuits to coordinate responses to OXT and CRH. They suggest that additional studies of the impact of the OXT/OXTR and CRHBP/CRH pathways in males and females will be important in development of gender-specific therapies. The frequency of human social and emotional disorders varies significantly between males and females. We have recently reported that oxytocin receptor interneurons (OxtrINs) modulate female sociosexual behavior. Here, we show that, in male mice, OxtrINs regulate anxiety-related behaviors. We demonstrate that corticotropin-releasing-hormone-binding protein (CRHBP), an antagonist of the stress hormone CRH, is specifically expressed in OxtrINs. Production of CRHBP blocks the CRH-induced potentiation of postsynaptic layer 2/3 pyramidal cell activity of male, but not female, mice, thus producing an anxiolytic effect. Our data identify OxtrINs as critical for modulation of social and emotional behaviors in both females and males and reveal a molecular mechanism that acts on local medial prefrontal cortex (mPFC) circuits to coordinate responses to OXT and CRH. They suggest that additional studies of the impact of the OXT/OXTR and CRHBP/CRH pathways in males and females will be important in development of gender-specific therapies. Gender-specific differences in the occurrence of social and emotional disorders are well documented. Autism spectrum disorder (ASD), which is characterized by impaired social communication and restrictive, repetitive behaviors, is several-fold more prevalent in males than females (Rubenstein et al., 2015Rubenstein E. Wiggins L.D. Lee L.C. A review of the differences in developmental, psychiatric, and medical endophenotypes between males and females with autism spectrum disorder.J. Dev. Phys. Disabil. 2015; 27: 119-139Crossref PubMed Scopus (48) Google Scholar). In contrast, anxiety-related disorders such as post-traumatic stress disorder, generalized anxiety disorder, panic disorder, and social anxiety disorder occur approximately twice as frequently in females than in males (Altemus et al., 2014Altemus M. Sarvaiya N. Neill Epperson C. Sex differences in anxiety and depression clinical perspectives.Front. Neuroendocrinol. 2014; 35: 320-330Crossref PubMed Scopus (627) Google Scholar). Although recent studies in rodents (Yang et al., 2013Yang C.F. Chiang M.C. Gray D.C. Prabhakaran M. Alvarado M. Juntti S.A. Unger E.K. Wells J.A. Shah N.M. Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males.Cell. 2013; 153: 896-909Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, Lee et al., 2014Lee H. Kim D.W. Remedios R. Anthony T.E. Chang A. Madisen L. Zeng H. Anderson D.J. Scalable control of mounting and attack by Esr1+ neurons in the ventromedial hypothalamus.Nature. 2014; 509: 627-632Crossref PubMed Scopus (268) Google Scholar, Kunwar et al., 2015Kunwar P.S. Zelikowsky M. Remedios R. Cai H. Yilmaz M. Meister M. Anderson D.J. Ventromedial hypothalamic neurons control a defensive emotion state.eLife. 2015; 4: e06633Crossref Scopus (121) Google Scholar, Scott et al., 2015Scott N. Prigge M. Yizhar O. Kimchi T. A sexually dimorphic hypothalamic circuit controls maternal care and oxytocin secretion.Nature. 2015; 525: 519-522Crossref PubMed Scopus (146) Google Scholar) and humans (Wigton et al., 2015Wigton R. Radua J. Allen P. Averbeck B. Meyer-Lindenberg A. McGuire P. Shergill S.S. Fusar-Poli P. Neurophysiological effects of acute oxytocin administration: systematic review and meta-analysis of placebo-controlled imaging studies.J. Psychiatry Neurosci. 2015; 40: E1-E22Crossref PubMed Scopus (131) Google Scholar, Insel, 2016Insel T.R. Translating oxytocin neuroscience to the clinic: a National Institute of Mental Health Perspective.Biol. Psychiatry. 2016; 79: 153-154Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) have provided strong evidence that differences in hormonal balance and brain circuitry between males and females can contribute to complex behaviors, the specific mechanisms that are responsible for gender biases in the occurrence and treatment of social and emotional disorders are not understood. Oxytocin (OXT) is a peptide hormone that has been shown to contribute to many aspects of social behavior (Insel, 2010Insel T.R. The challenge of translation in social neuroscience: a review of oxytocin, vasopressin, and affiliative behavior.Neuron. 2010; 65: 768-779Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar, Neumann and Slattery, 2016Neumann I.D. Slattery D.A. Oxytocin in general anxiety and social fear: a translational approach.Biol. Psychiatry. 2016; 79: 213-221Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, Aoki et al., 2015Aoki Y. Watanabe T. Abe O. Kuwabara H. Yahata N. Takano Y. Iwashiro N. Natsubori T. Takao H. Kawakubo Y. et al.Oxytocin’s neurochemical effects in the medial prefrontal cortex underlie recovery of task-specific brain activity in autism: a randomized controlled trial.Mol. Psychiatry. 2015; 20: 447-453Crossref PubMed Scopus (75) Google Scholar, Guastella and Hickie, 2016Guastella A.J. Hickie I.B. Oxytocin treatment, circuitry, and autism: a critical review of the literature placing oxytocin into the autism context.Biol. Psychiatry. 2016; 79: 234-242Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). It is released in response to a variety of social cues and acts through OXT receptors (OXTRs) that are widely dispersed in the brain and periphery (Owen et al., 2013Owen S.F. Tuncdemir S.N. Bader P.L. Tirko N.N. Fishell G. Tsien R.W. Oxytocin enhances hippocampal spike transmission by modulating fast-spiking interneurons.Nature. 2013; 500: 458-462Crossref PubMed Scopus (233) Google Scholar). Studies of behaviors altered in response to systemic or local administration of OXT and analysis of behavioral phenotypes evident in knockout mice lacking Oxt or Oxtr have demonstrated definitively that this hormone system plays essential roles in maternal care, social cognition, and affiliative behaviors. Stressful and anxiogenic stimuli result in the release of OXT, which acts as a powerful modulator of anxiety- and stress-related behaviors. These studies have led to a great deal of interest in OXT as a potential treatment for human social disorders, resulting in a large number of clinical trials to assess its therapeutic efficacy. Although the results of these trials have been encouraging in the context of ASD and anxiety disorders, our understanding of the brain circuits engaged by endogenous or exogenously supplied OXT and their role in specific behaviors remains incomplete. We have recently identified a specific class of OXTR interneurons (OxtrINs) in the mouse medial prefrontal cortex (mPFC) that express Oxtr and are activated in response to OXT (Nakajima et al., 2014Nakajima M. Görlich A. Heintz N. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons.Cell. 2014; 159: 295-305Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Genetic and pharmacological studies of these neurons revealed that they modulate female social interactions with male mice during the sexually responsive, estrus phase of their cycle. Although the number of OxtrINs in the male mPFC is equivalent to that in the female mPFC, social behavior of male mice was not altered by the silencing of OxtrINs or blockade of OXT signaling in the mPFC. While it is possible that these neurons are dedicated to modulation of social behaviors in female mice, OXT administration can impact a wide range of behaviors in both males and females. Furthermore, human imaging studies have indicated that OXT elicits activity in cortical and subcortical sites in both sexes, although gender-specific differences in these responses have been noted (Bethlehem et al., 2013Bethlehem R.A. van Honk J. Auyeung B. Baron-Cohen S. Oxytocin, brain physiology, and functional connectivity: a review of intranasal oxytocin fMRI studies.Psychoneuroendocrinology. 2013; 38: 962-974Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, MacDonald and Feifel, 2014MacDonald K. Feifel D. Oxytocin’s role in anxiety: a critical appraisal.Brain Res. 2014; 1580: 22-56Crossref PubMed Scopus (100) Google Scholar). It seems probable, therefore, that OxtrINs in the mPFC of male mice also modulate behavior in response to changing levels of OXT, but that gender-specific differences in physiology or circuitry result in sexually dimorphic behavioral outcomes. Here, we report that optogenetic activation of OxtrINs in male mice has a strong anxiolytic effect and no impact on social interaction, whereas activation of these neurons in female mice results in increased sociality and no change in anxiety-related behaviors. Postsynaptic responses to activation of OxtrINs in cortical pyramidal cells are primarily inhibitory. Layer 2/3 neurons respond more robustly in male mice, whereas postsynaptic responses in layer 5 are enhanced in females. TRAP (translational ribosome affinity purification) translational profiling (Heiman et al., 2008Heiman M. Schaefer A. Gong S. Peterson J.D. Day M. Ramsey K.E. Suarez-Farinas M. Schwarz C. Stephan D.A. Surmeier D.J. et al.A translational profiling approach for the molecular characterization of CNS cell types.Cell. 2008; 135: 738-748Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar) revealed that expression of the corticotropin-releasing-hormone (CHP)-binding protein (CRHBP) gene (Crhbp) (Van Den Eede et al., 2005Van Den Eede F. Van Broeckhoven C. Claes S.J. Corticotropin-releasing factor-binding protein, stress and major depression.Ageing Res. Rev. 2005; 4: 213-239Crossref PubMed Scopus (34) Google Scholar), an inhibitor of the stress hormone CRH (Laryea et al., 2012Laryea G. Arnett M.G. Muglia L.J. Behavioral studies and genetic alterations in corticotropin-releasing hormone (CRH) neurocircuitry: insights into human psychiatric disorders.Behav. Sci. (Basel). 2012; 2: 135-171Crossref PubMed Scopus (36) Google Scholar), is specifically enriched in OxtrINs. Upon CRH application, induced activity of male, but not female, layer 2/3 pyramidal cells in mPFC slice recordings was potentiated. This response to CRH was blocked by optogenetic stimulation of OxtrINs and enhanced by bath application of a CRHBP antagonist. Infusion of OXT into the mPFC resulted in a significant decrease of anxiety-related behavior in male mice. This anxiolytic effect was blocked by co-infusion of a CRHBP antagonist. Furthermore, short-hairpin-RNA (shRNA)-mediated knockdown of Crhbp expression in OxtrINs of the mPFC resulted in increased anxiety-related behaviors only in male mice. Finally, CRH expression in female mice is strongly elevated. Taken together, these data identify a molecular mechanism regulating male-specific anxiety-related behaviors. They demonstrate that OXT acts through OxtrINs and CRHBP to moderate the anxiogenic effects of the stress hormone CRH in the mPFC. When taken together with our previous study of the role of OxtrINs in female sociosexual behavior, these results provide an important illustration of the concept that higher brain circuits regulating complex behaviors may be identical in males and females yet retain the ability to generate gender-specific behaviors based on differential sensitivity to sexually dimorphic hormones. They suggest that coordination of the local actions of OXT and CRH in the cerebral cortex by OxtrINs and CRHBP may be involved in the modulation of a wide variety of gender-specific cognitive and behavioral functions. To investigate the function of OxtrINs in male mice, we performed two types of genetic manipulations. First, we used Oxtr-Cre mice (Nakajima et al., 2014Nakajima M. Görlich A. Heintz N. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons.Cell. 2014; 159: 295-305Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) for optogenetic activation of OxtrINs (Figures 1A–1I). For this purpose, the mPFC of male and female Oxtr-Cre mice were stereotactically injected with a Cre-dependent AAV virus expressing channelrhodopsin (ChR2-H134R), and fiber-optic cannulas were implanted in the prelimbic cortex (Figures 1A–1C; Figures S1A–S1D). Behavioral responses during blue-light (BL) on and off periods were scored in the three-chamber social interaction test, the open field (OF) test, and the elevated plus maze (EPM). Optogenetic activation of OxtrINs in the mPFC of female mice promoted social preference toward male mice (Figure 1D), consistent with the suppression of sociosexual preference observed by silencing these same neurons (Nakajima et al., 2014Nakajima M. Görlich A. Heintz N. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons.Cell. 2014; 159: 295-305Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). However, male mice showed no significant changes in social interaction upon light activation of OxtrINs (Figure 1E). Given that the oxytocin system can also affect behavioral responses to stress (Neumann and Slattery, 2016Neumann I.D. Slattery D.A. Oxytocin in general anxiety and social fear: a translational approach.Biol. Psychiatry. 2016; 79: 213-221Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), we next examined whether OxtrINs can contribute to the regulation of anxiety-related behaviors in males. Avoidance of the open arms of the EPM and decreased exploration time in the center of the OF are established behavioral tests that are used to assess anxiety-like behavior in rodents. In female mice, activation of OxtrINs by BL had no impact on the behavior of the animals in either of these assays (Figures 1F and 1H). However, optogenetic activation of OxtrINs in the mPFC of male mice resulted in more exploration of the open arms of the EPM (Figure 1G) and increased time in the center area of the OF arena (Figure 1I) compared to the light-off phase. As expected, the strength of the anxiety phenotype in male mice correlated directly with the number of OxtrINs expressing ChR2 (Figures S1A–S1C). These data demonstrate that activation of OxtrINs in the mPFC results in the induction of distinct gender-specific behaviors. They confirm our previous studies demonstrating a role for OxtrINs in female sociosexual behavior (Nakajima et al., 2014Nakajima M. Görlich A. Heintz N. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons.Cell. 2014; 159: 295-305Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) and establish a role for these same neurons in the modulation of anxiety-related behaviors in male mice.Figure S1Efficacy of Viral Injections and Locomotor Activity in Oxtr-Cre and Oxtrf/f Mice, Related to Figure 1Show full caption(A-B) Representative confocal images of Oxtr-Cre::ChR2 mice showing a low (A) or high (B) number of OtxrINs expressing ChR2-mCherry in the mPFC. Scale bars, 600 μm.(C-D) Graphs showing the percentage of OxtrINs expressing ChR2 with respect to the total number of OxtrINs in mPFC in male (C) and females (D), plotted against the strength of the anxiety-like phenotype calculated as the fold change in light on versus light off periods. (C) In male mice, the percentage of ChR2 expressing OxtrINs correlates with the anxiety level. (D) In females a high percentage of OxtrINs express ChR2 but anxiety levels are unchanged by light activation.(E) Anxiety-like behavior measured in the EPM does not change in control and Oxtr-CKO female mice during estrus and diestrus. Data are presented as mean ± SEM n = 6 to 9 mice for each condition. Two-way ANOVA, Bonferroni’s multiple comparison test. n.s. p > 0.05.(F-G) Optogenetic activation of OxtrINs does not affect locomotion measured in the open field (OF) for 30 min. (F) Oxtr-Cre::ChR2 female mice show no differences in the distance traveled in the OF during light on and light off epochs. (G) Oxtr-Cre::ChR2 male mice show no differences in the distance traveled in the OF during light on and light off epochs. Data are represented as mean ± SEM. F to G, n = 9 to 16 mice per group. Mann Whitney test. n.s. p > 0.05.(H-I) Locomotion in mice with conditional deletion of the Oxtr in mPFC. (H) Oxtr f/f:Cre female mice show a small increase in the distance traveled relative to Oxtr f/f:GFP control mice. (I) Oxtr f/f:Cre male mice show no differences in the distance traveled relative to Oxtr f/f:GFP control mice. Data are presented as mean ± SEM n = 10 to 20 mice per group. Mann Whitney test. n.s. p > 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A-B) Representative confocal images of Oxtr-Cre::ChR2 mice showing a low (A) or high (B) number of OtxrINs expressing ChR2-mCherry in the mPFC. Scale bars, 600 μm. (C-D) Graphs showing the percentage of OxtrINs expressing ChR2 with respect to the total number of OxtrINs in mPFC in male (C) and females (D), plotted against the strength of the anxiety-like phenotype calculated as the fold change in light on versus light off periods. (C) In male mice, the percentage of ChR2 expressing OxtrINs correlates with the anxiety level. (D) In females a high percentage of OxtrINs express ChR2 but anxiety levels are unchanged by light activation. (E) Anxiety-like behavior measured in the EPM does not change in control and Oxtr-CKO female mice during estrus and diestrus. Data are presented as mean ± SEM n = 6 to 9 mice for each condition. Two-way ANOVA, Bonferroni’s multiple comparison test. n.s. p > 0.05. (F-G) Optogenetic activation of OxtrINs does not affect locomotion measured in the open field (OF) for 30 min. (F) Oxtr-Cre::ChR2 female mice show no differences in the distance traveled in the OF during light on and light off epochs. (G) Oxtr-Cre::ChR2 male mice show no differences in the distance traveled in the OF during light on and light off epochs. Data are represented as mean ± SEM. F to G, n = 9 to 16 mice per group. Mann Whitney test. n.s. p > 0.05.(H-I) Locomotion in mice with conditional deletion of the Oxtr in mPFC. (H) Oxtr f/f:Cre female mice show a small increase in the distance traveled relative to Oxtr f/f:GFP control mice. (I) Oxtr f/f:Cre male mice show no differences in the distance traveled relative to Oxtr f/f:GFP control mice. Data are presented as mean ± SEM n = 10 to 20 mice per group. Mann Whitney test. n.s. p > 0.05. To determine whether the anxiolytic effect of OxtrINs in male mice requires OXT/OXTR signaling, as we have shown previously for sociosexual behavior in females (Nakajima et al., 2014Nakajima M. Görlich A. Heintz N. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons.Cell. 2014; 159: 295-305Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), we conducted a second set of experiments using Cre-expressing AAV viruses injected into mice carrying a conditional allele of the Oxtr gene (Oxtrflox/flox) (Lee et al., 2008Lee H.J. Caldwell H.K. Macbeth A.H. Tolu S.G. Young 3rd, W.S. A conditional knockout mouse line of the oxytocin receptor.Endocrinology. 2008; 149: 3256-3263Crossref PubMed Scopus (189) Google Scholar). We bilaterally injected the mPFC of adult male and female Oxtrflox/flox mice with AAV-expressing Cre to delete the receptor (Oxtrf/f:Cre). As a control, we injected control Oxtrflox/flox mice with an AAV-expressing GFP (Oxtrf/f:GFP) that cannot result in deletion of Oxtr. As shown in Figures 1J and 1L, deletion of the Oxtr in female mice has no significant effect on performance in either the OF or EPM assays. However, in male mice, deletion of Oxtr resulted in a strong anxiogenic effect. Thus, male Oxtrf/f:Cre mice spent significantly less time in open arms of the EPM and in the center of the OF arena than did the control male Oxtrf/f:GFP mice (Figures 1K and 1M). There were no significant differences in anxiety levels between control males and females (Figures 1F–1M), and between females during estrus and diestrus (Figure S1E). There were also no major differences in total locomotor behavior as a consequence of optogenetic activation or Oxtr deletion, although a slight increase in female locomotion in the OF test was observed with Oxtr deletion (Figures S1F–S1I). We conclude that OXT action on OxtrINs in the mPFC regulates distinct, gender-specific behaviors. OxtrINs are regular spiking, GABAergic interneurons that increase their rate of firing in response to OXT (Nakajima et al., 2014Nakajima M. Görlich A. Heintz N. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons.Cell. 2014; 159: 295-305Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). To investigate the mechanism by which OxtrINs activation regulates local circuit activity, we performed whole-cell recordings of layer 2/3 and layer 5 pyramidal neurons in acute brain slices of the mPFC during photostimulation of ChR2-expressing OxtrINs (Figure 2). Brief light stimulation (470 nm) efficiently evoked action potentials in ChR2-mCherry expressing OxtrINs in slice preparations (Figures 2A–2C). We observed no differences in the response of male and female OxtrINs to BL at different frequencies (Figure 2C). To measure postsynaptic responses to OxtrINs activation, light-evoked responses of layer 2/3 and layer 5 pyramidal neurons (Figure 2D) were recorded at 0-mV and −60-mV holding potentials, allowing independent detection of both GABA- and AMPA-receptor-mediated currents (Figures S2A and S2B). OxtrINs synapse extensively with layer 2/3 and layer 5 pyramidal neurons in both genders. Thus, in males, 91.3% of layer 2/3 and 97% of layer 5 neurons responded to OxtrINs activation, whereas 95.2% of layer 2/3 and 95.6% of layer 5 pyramidal neurons responded in females (Figure 2E).Figure S2EPSCs and IPSCs Recorded in Layer 2/3 and Layer 5 upon BL Stimulation, Related to Figure 2Show full caption(A, B) Representative responses of layer 2/3 pyramidal cells to blue light pulses elicited picrotoxin sensitive inhibitory (IPSC) responses at 0mV (blue) and CNQX and AP5 sensitive excitatory (EPSC) responses at −60mV (red).(C, D) Quantitation of IPSCs amplitudes in layer 2/3 and layer 5 pyramidal cells at medium and low BL intensities indicates that the differences in IPSCs response sizes between male and females are detected at all laser intensities. Data are presented as mean ± SEM n = 16 to 21 cells recorded in female and n = 23 to 29 cells recorded in male. Multiple t test, Holm-Sidak multiple comparison test. ∗p < 0.05.(E, F) The synaptic latencies of IPSCs and EPSCs in layer 2/3 (C) and layer 5(D) cells in males and females. Data are represented as mean ± SEM n = 16 cells recorded in female and n = 23 cells recorded in male in layer 2/3 and n = 21 in female, n = 29 in male layer 5 cells. Two-way ANOVA, Bonferroni’s multiple comparison test. ∗p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A, B) Representative responses of layer 2/3 pyramidal cells to blue light pulses elicited picrotoxin sensitive inhibitory (IPSC) responses at 0mV (blue) and CNQX and AP5 sensitive excitatory (EPSC) responses at −60mV (red). (C, D) Quantitation of IPSCs amplitudes in layer 2/3 and layer 5 pyramidal cells at medium and low BL intensities indicates that the differences in IPSCs response sizes between male and females are detected at all laser intensities. Data are presented as mean ± SEM n = 16 to 21 cells recorded in female and n = 23 to 29 cells recorded in male. Multiple t test, Holm-Sidak multiple comparison test. ∗p < 0.05. (E, F) The synaptic latencies of IPSCs and EPSCs in layer 2/3 (C) and layer 5(D) cells in males and females. Data are represented as mean ± SEM n = 16 cells recorded in female and n = 23 cells recorded in male in layer 2/3 and n = 21 in female, n = 29 in male layer 5 cells. Two-way ANOVA, Bonferroni’s multiple comparison test. ∗p < 0.05. Analysis of the amplitudes of postsynaptic responses elicited by optogenetic activation of OxtrINs revealed that GABAergic responses to activation of OxtrINs differed between males and females (Figures 2F–2K). Inhibitory postsynaptic currents (IPSCs) elicited in response of BL activation were significantly larger in male layer 2/3 pyramidal neurons (Figures 2F and 2H), whereas IPSCs in layer 5 postsynaptic neurons were larger in females (Figures 2I and 2K). These differences in IPSCs between males and females were observed at different BL intensities (Figures S2C and S2D). Excitatory postsynaptic current (EPSC) amplitudes were also larger in females in layer 5 (Figures 2J and 2K). No differences in EPSC amplitudes in layer 2/3 (Figures 2G and 2H) were observed between male and female neurons. Thus, the electrophysiological evidence presented here establishes that there is broad connectivity between OxtrINs and pyramidal cells in the mPFC of both sexes and that the strength of inhibition in male and female neurons in the mPFC is both laminar and gender specific. Although the differences in postsynaptic inhibitory responses we observed between male and female neurons may explain the gender-specific anxiolytic effect of OXT, the response differences between genders suggested that additional factors might be involved. To gain insight into signaling pathways that are active in OxtrINs and to elucidate candidate mechanisms that might play a role in the anxiolytic effects of OxtrINs activation, we used TRAP to identify proteins actively synthesized in these cells in the mPFC. This was accomplished using Oxtr-Cre mice (Nakajima et al., 2014Nakajima M. Görlich A. Heintz N. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons.Cell. 2014; 159: 295-305Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) crossed to EGFP-L10a reporter mice (Liu et al., 2014Liu J. Krautzberger A.M. Sui S.H. Hofmann O.M. Chen Y. Baetscher M. Grgic I. Kumar S. Humphreys B.D. Hide W.A. McMahon A.P. Cell-specific translational profiling in acute kidney injury.J. Clin. Invest. 2014; 124: 1242-1254Crossref PubMed Scopus (127) Google Scholar) (Figures S3A and S3B), followed by TRAP profiling as described previously (Heiman et al., 2008Heiman M. Schaefer A. Gong S. Peterson J.D. Day M. Ramsey K.E. Suarez-Farinas M. Schwarz C. Stephan D.A. Surmeier D.J. et al.A translational profiling approach for the molecular characterization of CNS cell types.Cell. 2008; 135: 738-748Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). Six replicate samples were collected from the mPFC of Oxtr-Cre/EGFP-L10a mice, and the resulting RNA was sequenced from each sample. Normalized values from each of the six replicates and their averages were obtained for both the INPUT and immunoprecipitation (IP) samples (Table S1). These data provided us with a more comprehensive and quantitative list of highly translated mRNAs than that available from our previous microarray analyses and allowed us to confirm the reported (Nakajima et al., 2014Nakajima M. Görlich A. Heintz N. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons.Cell. 2014; 159: 295-305Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) high expression and enrichment of somatostatin (Sst), glutamic acid decarboxylase 1 (Gad1), and Oxtr in OxtrINs. As shown in the scatterplot in Figure 3A, these markers are expressed at quite different levels, and each of them is significantly enriched in the OxtrINs IP data relative to the sample taken from the intact mPFC prior to IP (INPUT). To evaluate expressed genes as possible candidates for further analysis, we first removed genes whose mRNAs were present at low levels (base mean < 25.00) and then sorted for mRNAs that are highly enriched in the OxtrINs IP data (log2-fold change IP v INPUT). This resulted in the identification of a large number of known interneuron markers, and a variety of transcription factors, receptors, and signaling proteins that were not known previously to be enriched in OxtrINs (Table S1).Figure 3TRAP Profiling Reveals Cell-Specific Enrichment of CRHBP in OxtrINsShow full caption(A) Scatterplot of average TRAP IP samples (y axis) versus (v) INPUT samples (x axis) from the mPFC of Oxtr-EFGP-L10a mic" @default.
• W2520003619 created "2016-09-23" @default.
• W2520003619 creator A5006564093 @default.
• W2520003619 creator A5010210504 @default.
• W2520003619 creator A5036990231 @default.
• W2520003619 creator A5077354952 @default.
• W2520003619 date "2016-09-01" @default.
• W2520003619 modified "2023-10-18" @default.
• W2520003619 title "A Cortical Circuit for Sexually Dimorphic Oxytocin-Dependent Anxiety Behaviors" @default.
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