Abstract: Article12 September 2018Open Access Transparent process Hypothalamic CNTF volume transmission shapes cortical noradrenergic excitability upon acute stress Alán Alpár Corresponding Author Alán Alpár [email protected] SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, Budapest, Hungary Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Search for more papers by this author Péter Zahola Péter Zahola SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, Budapest, Hungary Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Search for more papers by this author János Hanics János Hanics SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, Budapest, Hungary Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Search for more papers by this author Zsófia Hevesi Zsófia Hevesi SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, Budapest, Hungary Search for more papers by this author Solomiia Korchynska Solomiia Korchynska Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Marco Benevento Marco Benevento Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Christian Pifl Christian Pifl Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Gergely Zachar Gergely Zachar Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Search for more papers by this author Jessica Perugini Jessica Perugini Section of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, Marche Polytechnic University, Ancona, Italy Search for more papers by this author Ilenia Severi Ilenia Severi Section of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, Marche Polytechnic University, Ancona, Italy Search for more papers by this author Patrick Leitgeb Patrick Leitgeb Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Joanne Bakker Joanne Bakker Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Andras G Miklosi Andras G Miklosi Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Evgenii Tretiakov Evgenii Tretiakov Immanuel Kant Baltic Federal University, Kaliningrad, Russia Search for more papers by this author Erik Keimpema Erik Keimpema Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Gloria Arque Gloria Arque Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Ramon O Tasan Ramon O Tasan Department of Pharmacology, Medical University Innsbruck, Innsbruck, Austria Search for more papers by this author Günther Sperk Günther Sperk Department of Pharmacology, Medical University Innsbruck, Innsbruck, Austria Search for more papers by this author Katarzyna Malenczyk Katarzyna Malenczyk Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Zoltán Máté Zoltán Máté Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Search for more papers by this author Ferenc Erdélyi Ferenc Erdélyi Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Search for more papers by this author Gábor Szabó Gábor Szabó Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Search for more papers by this author Gert Lubec Gert Lubec Paracelsus Medical University, Salzburg, Austria Search for more papers by this author Miklós Palkovits Miklós Palkovits Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Human Brain Tissue Bank and Laboratory, Semmelweis University, Budapest, Hungary Search for more papers by this author Antonio Giordano Antonio Giordano Section of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, Marche Polytechnic University, Ancona, Italy Search for more papers by this author Tomas GM Hökfelt Tomas GM Hökfelt Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Roman A Romanov Roman A Romanov Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Immanuel Kant Baltic Federal University, Kaliningrad, Russia Search for more papers by this author Tamas L Horvath Tamas L Horvath Program in Integrative Cell Signaling and Neurobiology of Metabolism, Departments of Comparative Medicine and Neuroscience, Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA Department of Anatomy and Histology, University of Veterinary Medicine, Budapest, Hungary Search for more papers by this author Tibor Harkany Corresponding Author Tibor Harkany [email protected] orcid.org/0000-0002-6637-5900 Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Section of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, Marche Polytechnic University, Ancona, Italy Search for more papers by this author Alán Alpár Corresponding Author Alán Alpár [email protected] SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, Budapest, Hungary Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Search for more papers by this author Péter Zahola Péter Zahola SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, Budapest, Hungary Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Search for more papers by this author János Hanics János Hanics SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, Budapest, Hungary Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Search for more papers by this author Zsófia Hevesi Zsófia Hevesi SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, Budapest, Hungary Search for more papers by this author Solomiia Korchynska Solomiia Korchynska Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Marco Benevento Marco Benevento Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Christian Pifl Christian Pifl Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Gergely Zachar Gergely Zachar Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Search for more papers by this author Jessica Perugini Jessica Perugini Section of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, Marche Polytechnic University, Ancona, Italy Search for more papers by this author Ilenia Severi Ilenia Severi Section of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, Marche Polytechnic University, Ancona, Italy Search for more papers by this author Patrick Leitgeb Patrick Leitgeb Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Joanne Bakker Joanne Bakker Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Andras G Miklosi Andras G Miklosi Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Evgenii Tretiakov Evgenii Tretiakov Immanuel Kant Baltic Federal University, Kaliningrad, Russia Search for more papers by this author Erik Keimpema Erik Keimpema Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Gloria Arque Gloria Arque Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Ramon O Tasan Ramon O Tasan Department of Pharmacology, Medical University Innsbruck, Innsbruck, Austria Search for more papers by this author Günther Sperk Günther Sperk Department of Pharmacology, Medical University Innsbruck, Innsbruck, Austria Search for more papers by this author Katarzyna Malenczyk Katarzyna Malenczyk Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Zoltán Máté Zoltán Máté Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Search for more papers by this author Ferenc Erdélyi Ferenc Erdélyi Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Search for more papers by this author Gábor Szabó Gábor Szabó Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Search for more papers by this author Gert Lubec Gert Lubec Paracelsus Medical University, Salzburg, Austria Search for more papers by this author Miklós Palkovits Miklós Palkovits Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary Human Brain Tissue Bank and Laboratory, Semmelweis University, Budapest, Hungary Search for more papers by this author Antonio Giordano Antonio Giordano Section of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, Marche Polytechnic University, Ancona, Italy Search for more papers by this author Tomas GM Hökfelt Tomas GM Hökfelt Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Roman A Romanov Roman A Romanov Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Immanuel Kant Baltic Federal University, Kaliningrad, Russia Search for more papers by this author Tamas L Horvath Tamas L Horvath Program in Integrative Cell Signaling and Neurobiology of Metabolism, Departments of Comparative Medicine and Neuroscience, Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA Department of Anatomy and Histology, University of Veterinary Medicine, Budapest, Hungary Search for more papers by this author Tibor Harkany Corresponding Author Tibor Harkany tibor.harka[email protected] orcid.org/0000-0002-6637-5900 Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria Section of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, Marche Polytechnic University, Ancona, Italy Search for more papers by this author Author Information Alán Alpár *,1,2,‡,‡, Péter Zahola1,2,‡, János Hanics1,2, Zsófia Hevesi1, Solomiia Korchynska3, Marco Benevento3, Christian Pifl3, Gergely Zachar2, Jessica Perugini4, Ilenia Severi4, Patrick Leitgeb3, Joanne Bakker5, Andras G Miklosi3, Evgenii Tretiakov6, Erik Keimpema3, Gloria Arque3, Ramon O Tasan7, Günther Sperk7, Katarzyna Malenczyk3, Zoltán Máté8, Ferenc Erdélyi8, Gábor Szabó8, Gert Lubec9, Miklós Palkovits2,10, Antonio Giordano4, Tomas GM Hökfelt5, Roman A Romanov3,6,‡, Tamas L Horvath11,12,‡ and Tibor Harkany *,3,4,‡ 1SE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, Budapest, Hungary 2Department of Anatomy, Histology, and Embryology, Semmelweis University, Budapest, Hungary 3Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria 4Section of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, Marche Polytechnic University, Ancona, Italy 5Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 6Immanuel Kant Baltic Federal University, Kaliningrad, Russia 7Department of Pharmacology, Medical University Innsbruck, Innsbruck, Austria 8Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary 9Paracelsus Medical University, Salzburg, Austria 10Human Brain Tissue Bank and Laboratory, Semmelweis University, Budapest, Hungary 11Program in Integrative Cell Signaling and Neurobiology of Metabolism, Departments of Comparative Medicine and Neuroscience, Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA 12Department of Anatomy and Histology, University of Veterinary Medicine, Budapest, Hungary ‡These authors contributed equally to this work as first authors ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +36 1 2156 920/53609; E-mail: [email protected] *Corresponding author (Lead contact). Tel: +43 1 40160 34050; E-mail: [email protected] The EMBO Journal (2018)37:e100087https://doi.org/10.15252/embj.2018100087 See also: D Pozzi & M Matteoli (November 2018) [The copyright line of this article was changed on 26 November 2018 after original online publication.] PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Stress-induced cortical alertness is maintained by a heightened excitability of noradrenergic neurons innervating, notably, the prefrontal cortex. However, neither the signaling axis linking hypothalamic activation to delayed and lasting noradrenergic excitability nor the molecular cascade gating noradrenaline synthesis is defined. Here, we show that hypothalamic corticotropin-releasing hormone-releasing neurons innervate ependymal cells of the 3rd ventricle to induce ciliary neurotrophic factor (CNTF) release for transport through the brain's aqueductal system. CNTF binding to its cognate receptors on norepinephrinergic neurons in the locus coeruleus then initiates sequential phosphorylation of extracellular signal-regulated kinase 1 and tyrosine hydroxylase with the Ca2+-sensor secretagogin ensuring activity dependence in both rodent and human brains. Both CNTF and secretagogin ablation occlude stress-induced cortical norepinephrine synthesis, ensuing neuronal excitation and behavioral stereotypes. Cumulatively, we identify a multimodal pathway that is rate-limited by CNTF volume transmission and poised to directly convert hypothalamic activation into long-lasting cortical excitability following acute stress. Synopsis A multimodal signaling pathway initiated by CRH neurons using aqueductal CNTF volume transmission explains how acute stress is communicated from the hypothalamus to the cerebral cortex. Hypothalamic CRH output gates cortical norepinephrine (NE) signaling upon stress. CRH neurons induce ciliary neurotrophic factor (CNTF) release from ventricular ependyma. CNTF primes NE neurons by volume transmission. CNTF signaling recruits secretagogin to activate tyrosine hydroxylase for NE production. CNTF infusion or driving secretagogin+ NE neuron excitability accentuates stress-induced freezing. Introduction Ensuring species' survival in perilous environments is a primary evolutionary demand. Therefore, central stress pathways, efficiently linking the brain and periphery, have evolved to form the hypothalamus–pituitary–adrenal axis (HPA) (Selye & Fortier, 1949; Bale & Vale, 2004; McEwen, 2008). By principle, corticotropin-releasing hormone (CRH)-containing(+) neurons of the paraventricular hypothalamic nucleus (PVN) gate output from the central nervous system (Swanson & Sawchenko, 1980) with CRH (Swanson et al, 1983, 1986), through pituitary amplification steps, triggering corticosteroid release from the adrenal glands for immediate metabolic mobilization (Rivier & Vale, 1983; Kovacs & Sawchenko, 1996). Nevertheless, stress, whether due to, e.g., predation or competition for reproduction, is unlikely a singular event. Therefore, secondary response pathways to enable an individual's prolonged vigilance might have evolved to confer added evolutionary benefit when responding to recurrent challenges. Seminal studies (Schulkin et al, 1994; McEwen & Sapolsky, 1995; Popoli et al, 2011) support this notion by documenting that corticosteroids released from the adrenals do not only produce feedback inhibition of hypothalamic and pituitary hormone secretion (Akana et al, 1992) but directly regulate limbic and reward circuits (Sapolsky, 2003) to gate coping and flexibility (e.g., "flight or fight" behaviors; Eriksen et al, 1999; McEwen et al, 2012), motivation (McEwen, 2005), memory (Roozendaal et al, 2009), and fear extinction (Korte, 2001; McEwen, 2005). The prefrontal cortex (PFC) has emerged as a central site to orchestrate coordinated responses to acute stress (McEwen, 2007) with glucocorticoid and mineralocorticoid receptors modulating its ability to integrate upstream emotional, sensory, cognitive, and spatial inputs (Patel et al, 2008; Gadek-Michalska et al, 2013; Caudal et al, 2014). In general terms, corticosteroids can change neuronal excitability through the cell-type-specific engagement of mineralocorticoid and glucocorticoid receptors with the former increasing and the latter suppressing neuronal activity (Joels & de Kloet, 1992). However, stress-induced alertness (defined as heightened cortical network excitability for prolonged periods) might benefit from a single neural trigger, such as CRH+ neuroendocrine cells, for the tight temporal coupling and scaling of cortical excitability for conscious execution of stereotyped behaviors associated with vigilance and HPA-induced peripheral energy mobilization. A neural link between CRH+ parvocellular cells and norepinephrinergic (NE) neurons of the locus coeruleus (LC) is of particular appeal because NE activity increases with the severity and duration of stress (Aston-Jones et al, 1996; Chowdhury et al, 2000) and NE afferents of the PFC are poised to facilitate adaptive behaviors (Uematsu et al, 2017). Recently, CRH+ innervation of NE neurons has been described (Zhang et al, 2017), including at the ultrastructural level (Van Bockstaele et al, 1996). However, a monosynaptic circuit operating through excitatory CRH [i.e., CRH acting at Gs protein-coupled Crhr1 and/or Crhr2 receptors (De Souza, 1995)] seems insufficient to functionally convert short-lived surges of excitability into long-lasting NE sensitization for cortical stress adaptation, particularly since neuropeptide release likely commences only upon intense burst firing (Overton & Clark, 1997). Here, we unmask an efficient mechanism coordinated by glutamate release from CRH neurons onto ependymal cells that line the wall of the 3rd ventricle to trigger long-range volume transmission by ciliary neurotrophic factor (CNTF) in the brain aqueductal system. Once reaching the LC, CNTF heightens NE output (Fig 1A), as opposed to fast synaptic coupling known to evoke anxiety acutely (Zhang et al, 2017). We show the maintenance of NE excitability through CNTF-induced sequential recruitment of secretagogin (Scgn) and extracellular signal-regulated kinase 1 (Erk1) to increase tyrosine hydroxylase (TH) activity by phosphorylation for cortical NE production. Despite extensive NE innervation of the entire cortical mantle (Fuxe et al, 1968; Moore & Bloom, 1979; Aston-Jones, 1995), this mechanism centers on the PFC where it is poised to efficiently reset network excitability (Fig 1A; McCormick et al, 1991). Thus, the combination of genetic manipulation of Cntf and Scgn with opto-/chemogenetics and biochemistry not only uncovers previously undescribed molecular determinants gating stress-induced behavioral phenotypes but also offers targets for stress resilience. Figure 1. Hypothalamic corticotropin-releasing hormone (CRH)-releasing neurons innervate ependymal cells lining the 3rd ventricle Cartoon depicting a multimodal signaling axis including a direct pathway between the paraventricular hypothalamic nucleus (PVN) and ventricular ependyma (1), volume transmission to the locus coeruleus (LC; 2) with norepinephrinergic projections to the prefrontal cortex (PFC; 3). Microinjection of AAV-DIO-mCherry virus particles into the PVN of Crh-Ires-Cre mice reveals mCherry-containing processes oriented toward the 3rd ventricle (3V; arrowheads). Scale bar = 60 μm. Single-cell RNA-seq reveals infrequent expression of Crhr1, Crhr2, and Crhbp, as opposed to glutamate and GABA receptor subunits (in red), by ependymal cells. Ependymal cells were classified by Enkur and Foxj1 expression (Romanov et al, 2017b), and also contained Cntf mRNAs. (C1) Reconstruction of GRIA1+ ependymal cells receiving VGLUT2+ synapses (arrowheads). Asterisks denote nuclei. Scale bar = 12 μm. (1) Electron micrograph showing gap junction coupling (arrowheads) between ependymal cells. Scale bar = 250 nm. (2) Dye transfer among ependymal cells. "1st" indicates the cell probed directly. Arrowheads indicate secondary labeling in adjacent cells. Scale bar = 15 μm. (3) Postsynaptic currents (arrowheads) recorded in ependymal cells in control and upon exposure to AMPA (10 μM). Upper panel: Tonic inward current produced by bath-applied AMPA (10 μM). Lower panel: Quantitative data from ependymal cells from n > 3 mice. Data in box plots represent medians and 10th, 25th, 75th, and 90th percentiles. **P < 0.01 vs. baseline and wash-out (ANOVA). Activating DREADD (hM3Dq) was microinjected into the PVN of Crh-Ires-Cre mice 14–17 days prior to ex vivo recordings. (1) Reconstruction of mCherry-labeled terminals (arrowheads) in apposition to nestin+ ependyma. Scale bar = 7 μm. (2) DREADD activation by CNO in CRH terminals innervating ependymal cells induces inward currents (arrowheads), which are sensitive to NBQX, an AMPA receptor antagonist (20 μM). Download figure Download PowerPoint Results Ependymal cells are an intrahypothalamic target of CRH neurons Paraventricular CRH neurons of the hypothalamus release CRH into the median eminence to control the HPA stress axis by facilitating adrenocorticotropic hormone (ACTH) release from the anterior pituitary (Bale & Vale, 2004). However, whether paraventricular CRH neurons project to other targets within the hypothalamus, as proposed for other types of parvocellular neurons (Ter Horst & Luiten, 1987; Dai et al, 1998), remains undefined. First, we addressed alternative synaptic sites for CRH neurons by microinjecting adeno-associated virus (AAV8) particles encapsulating an mCherry reporter into adult Crh-Ires-Cre mice that are commonly used to test stress-related behaviors (Fuzesi et al, 2016). By postoperative days 5–7, mCherry-labeled processes emanating from CRH neurons that reside in the PVN coursed toward the 3rd ventricle (Fig 1B) with mCherry+ bouton-like varicosities lining the outermost ependymal layer of the 3rd ventricle. Next, we tested whether ependymal cells could directly respond to synaptic signals of CRH+ neurons by using single-cell RNA-seq to survey their CRH, glutamate, and GABA receptor contents (Romanov et al, 2017b). Ependymal cells, clustered by their expression of Enkur and Foxj1 protogenes (Romanov et al, 2017b), predominantly expressed mRNA transcripts for glutamate and select GABAA receptor subunits (Fig 1C) with unexpectedly sparse mRNA content for Crhr1 and Crhr2 receptors. These data suggest that ependymal cells could respond to glutamate (co-)released from "stress-on" CRH+ neuroendocrine cells (Romanov et al, 2015, 2017b). We have developed Crh-Ires-Cre::egfp mice to demonstrate that EGFP+ nerve endings contained vesicular glutamate transporter 2 (VGLUT2; Fig EV1A and A1) and less so VGLUT1 (Fig EV1A) along the 3rd ventricle wall, suggesting the likelihood of glutamate release from CRH+ terminals. We then confirmed that VGLUT2+ nerve endings apposed ependymal cells that expressed GRIA1 (Fig 1C1), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit most abundantly expressed by ependymal cells at the mRNA level (Fig 1C). Notably, our three-dimensional tissue reconstructions revealed that only a subset of ependymal cells received VGLUT2+ innervation (Fig 1C1), which could preclude their widespread and synchronous synaptic activation. However, ultrastructural analysis demonstrated that ependymal cells in the dorsolateral segment of the 3rd ventricle wall are connected by gap junctions (Fig 1D1) with their plasmalemma often convoluted (Fig EV1B) to increase surface contact (Vanslembrouck et al, 2018). These data were substantiated by dye transfer from biocytin-loaded ependymal cells to their closest neighbors (Fig 1D2). Thus, gap junctions are the structural basis to convert the synaptic activation of "first-responder" ependymal cells to synchronous cell-state changes in a larger ependymal network. Click here to expand this figure. Figure EV1. Glutamatergic inputs to ependymal cells lining the 3rd ventricle (related to Fig 1) EGFP+ nerve endings from Crh-Ires-Cre::egfp mice along the wall of the 3rd ventricle (bottom edge of each image) contained either VGLUT2 (1) or VGLUT1 (2) immunoreactivities (open arrowheads). Orthogonal projections. (A1) Three-dimensional rendering of VGLUT2+ nerve endings (open arrowheads) along nestin+ ependymal cells. Scale bars = 3 μm (1, 2) and 10 μm (A1). Electron micrographs showing gap junctions (arrowheads) between the convoluting plasmalemma of ependymal cells. Scale bars = 250 nm. Biophysical parameters of ependymal cell membranes, including resting membrane potential (C), and current-clamp (C1) and voltage-clamp (C2) profiles in response to 20 pA and 10 mV depolarization steps, respectively. I-V relationship is shown in (C3). Data from n = 10 cells are shown. AMPA superfusion significantly increases the frequency of spontaneous postsynaptic currents in ventricular ependyma. **P < 0.01 (paired Student's t-test), n = 10 cells/group. In the meantime, the amplitude (D1), rise time (D2), and decay time (D3) of such currents remained unchanged (for all parameters, P > 0.1). Data information: Data are expressed as means ± s.e.m. Download figure Download PowerPoint We used patch-clamp electrophysiology ex vivo to monitor whether ependymal cells receive synaptic inputs. Firstly, ependymal cells (for basic membrane properties, see Fig EV1C–C3) produced spontaneous postsynaptic currents, which increased in frequency when bath-applying AMPA (10 μM; Figs 1D3 and EV1D–D3). Secondly, they invariably responded to AMPA superfusion by generating long-lasting inward currents when held at −70 mV (Fig 1E). We then addressed whether glutamatergic innervation of ependymal cells originates from CRH neurons by microinjecting adeno-associated virus (AAV) particles carrying Cre-dependent activating DREADD (hM3Dq) in tandem with an mCherry reporter (Alexander et al, 2009) into the PVN (Fig 1F). Histochemical localization of mCherry recapitulated the distribution of VGLUT2+ synaptic puncta along ventricular ependyma (Fig 1F1), supporting the existence of a direct projection from parvocellular CRH neurons. Thereafter, we applied the DREADD agonist clozapine N-oxide (CNO, 10 μM) to acute brain slices to show the emergence of inward currents in ependymal cells, which were completely abolished by superfusion of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]chinoxalin-2,3-dion (NBQX, 20 μM), an AMPA receptor antagonist (Fig 1F2). Overall, these data suggest that ependymal cells along the anterior-dorsal segment of the 3rd ventricle are intrahypothalamic targets for CRH neurons and are tonically excited by glutamate. Glutamatergic neurotransmission facilitates CNTF release into the cerebrospinal fluid upon acute stress The existence of a monosynaptic pathway originating from CRH+ neurons and innervating ventricular ependyma points to the stress-induced release of bioactive substances into the cerebrospinal fluid. Crh-Ires-Cre::egfp mice were informative to reveal the genuine extent of EGFP+ innervation within the proximity (< 15 μm) of the wall of the 3rd ventricle through lifetime synapse labeling (Fig 2A). In turn, quantitative histochemistry for CRH showed that acute formalin stress significantly increases the density of CRH+ boutons targeting the wall of the 3rd ventricle (in rats: 6.93 ± 0.67 in control vs. 13.41 ± 0.93 20 min after stress, P < 0.05; Fig 2A1; Appendix Fig S1A). Subsequently, we used cfos-CreERT2::ROSA26-stop-ZsGreen1f/f mice in an activity "TRAP" approach (Guenthner et al, 2013; Fig 2B), as well as histochemical detection of c-Fos itself (Fig 2C; Appendix Fig S1B) to show that formalin stress significantly increases the density of both ZsGreen1+ (at 48 h; Fig 2B1) and c-Fos+ (at 2 h; Fig 2C) ependymal cells that co-express glial