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. 2018 Sep 12;37(21):e100087. doi: 10.15252/embj.2018100087

Figure 1. Hypothalamic corticotropin‐releasing hormone (CRH)‐releasing neurons innervate ependymal cells lining the 3rd ventricle.

Figure 1

  1. 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).
  2. 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.
  3. 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.
  4. (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).
  5. 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).
  6. 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).