Article Figures & Data
Figures
- Figure 1.
Ciliated CSF-c neurons in the hypothalamus express GABA and somatostatin. A, Location of somatostatin-expressing CSF-c neurons (red dots) indicated in schematic drawings of transverse sections through the hypothalamic area. B, Photomicrograph of somatostatin-immunopositive CSF-c neurons in the hypothalamus (arrows). Scale bar, 200 μm. C, High-magnification photomicrograph of the boxed area in C. CSF-c neurons expressing somatostatin have short, thick apical processes with a bulb-like ending protruding into the ventricle (arrows). Scale bar, 20 μm. D, Confocal image of a somatostatin-expressing hypothalamic CSF-c neuron (green) with an α-tubulin-immunoreactive cilium (magenta, arrow). Scale bar, 5 μm. E, Somatostatin (magenta) and GABA (green) are colocalized in hypothalamic CSF-c neurons (arrows). Some of the CSF-c neurons only expressed GABA (arrowhead). Scale bar, 20 μm. F, Illustration of a reconstructed intracellularly labeled hypothalamic CSF-c neuron with a bulb-like ending protruding into the ventricle and axonal branches extending dorsally, laterally, and ventrally. G, Somatostatin-immunoreactive fibers/terminals at the level of the optic tectum. Note the dense innervation of the deep layer (DL) and absence of labeling in the retinorecipient superficial layer (SL). Scale bar, 400 μm. H, Rich somatostatin labeling in the ventral brainstem at the level of MRRN and the trigeminal nucleus (nV). Scale bar, 200 μm. Th, Thalamus; DL, deep layer; cpo, postoptic commissure; Hb, habenula; Hyp, hypothalamus; LPal, lateral pallium; MPal, medial pallium; MRRN, middle rhombencephalic reticular nucleus; ncpo, nucleus of the postoptic commissure; nh, neurohypophysis; ot, optic tract; SL, superficial layer.
- Figure 2.
Hypothalamic CSF-c neurons express connexin. A, Intracellular injection of Neurobiotin in a hypothalamic CSF-c neuron (patched cell) resulted in two filled neurons revealed by DAB staining. Scale bar, 20 μm. B, Somatostatin-expressing hypothalamic CSF-c neurons. Scale bar, 20 μm. C, Connexin 35/36 immunoreactivity in the same hypothalamic area as in B. Scale bar, 20 μm. D, Merged image showing connexin 35/36-immunoreactivity (arrows) coexpressed with somatostatin in hypothalamic CSF-c neurons. Scale bar, 20 μm. E, Western blot of a lamprey brain extract detected with a monoclonal mouse anti-connexin 35/36 antibody showing a distinct band around 35 kDa.
- Figure 3.
Electrophysiological properties of hypothalamic CSF-c neurons. A, Whole-cell current-clamp recording of a hypothalamic CSF-c neuron showing its firing pattern. A brief current injection (20 pA, 20 ms; left) elicited a single action potential, whereas longer current injections (20 pA, 500 ms; right) generated repetitive firing. B, CSF-c neuron showing spontaneous GABA- and glutamate-mediated postsynaptic potentials (top trace) that were successively blocked by gabazine (20 μm), CNQX (40 μm), and AP5 (50 μm; bottom traces), respectively. C, Voltage responses to 12 consecutive hyperpolarizing and depolarizing current injections. The red and green traces illustrate a single action potential and spike frequency adaptation evoked by depolarizing steps, respectively. D, I–V curve showing a linear current–voltage relationship.
- Figure 4.
Hypothalamic CSF-c neurons are activated by both acidic and alkaline pH. A, Change to acidic (pH 6.5) as well as to alkaline (pH 8.0) extracellular pH (gray area) depolarized the membrane potential (10–12 mV) and triggered action potentials. Upon return to pH 7.4, firing ceased and the membrane potential repolarized back to the control value (−63 mV). In this and all subsequent recordings, gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm) were applied to exclude any indirect, synaptically mediated effects. B, In the presence of TTX (1.5 μm), both acidic and alkaline pH resulted in depolarization of the membrane potential that recovered after return to pH 7.4. C, Photomicrographs of the CSF-c neuron recorded in A after intracellular labeling with Neurobiotin (green, arrow), showing that the cell expressed somatostatin (magenta). Scale bars, 20 μm. D, Mean membrane potential changes in hypothalamic CSF-c neurons for each pH condition [mean ± SD; n = 15; Student's paired t test: ***p < 0.001 significant difference vs pH 7.4 at 6.5 (p = 1.43 × 10−19, t14 = 22.7), at 6.8 (p = 5.56 × 10−18, t14 = 19.76), at 7.1 (p = 1.97 × 10−14, t14 = 14.35), at 7.7 (p = 2.20 × 10−13, t14 = 13.0), at 8.0 (p = 2.24 × 10−20, t14 = 24.33), and at 8.3 (p = 1.38 × 10−21, t14 = 26.99)]. E, In voltage-clamp mode, frequent inward current deflections appeared at pH 6.5 and 8.0 with a maximal amplitude of ∼10 pA. F, Mean frequency increase of events (5–15 pA) in response to acidic and alkaline pH [mean ± SEM; n = 7; Student's paired t test: ***p < 0.001 significant difference vs pH 7.4 at pH 6.5 (p = 2.34 × 10−7; t6 = 25.59) and at 8 (p = 4.48 × 10−8; t6 = 33.79)]. G, Unitary current deflections recorded at acidic and alkaline pH. TTX (1.5 μm) was present in B, E–G.
- Figure 5.
Response to acidic and alkaline pH is mediated by different channels. A, Whole-cell current clamp in the presence of gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm). Application of APETx2 (1 μm) abolished the response to acidic pH but not to alkaline pH in the same cell. B, Recordings from a hypothalamic CSF-c neuron in voltage-clamp mode. No current events were seen at pH 7.4 (black traces) in the presence of gabazine (20 μm), AP5 (50 μm), CNQX (40 μm), and TTX (1.5 μm). Inward current deflections appeared after a decrease or increase in the extracellular pH. The current events recorded in acidic pH (6.5) were completely blocked in the presence of APETx2 (1 μm; red trace), whereas those recorded in alkaline pH (8.0; blue trace) remained. C, Mean frequency increase of events (5–15 pA) in response to acidic and alkaline pH in the presence of APETx2 [mean ± SEM; n = 3; Student's paired t test: no significant difference vs pH 7.4 at 6.5 (p = 1, t2 = 0), but a significant difference vs pH 7.4 at 8 (**p < 0.01, p = 0.007, t2 = 11.55)]. D, Hypothalamic somatostatin-immunopositive CSF-c neurons (green) do not express the PKD2L1 channel (magenta). Scale bar, 20 μm. E, Spinal somatostatin-CSF-c neurons (green) coexpress PKD2L1 (arrows; magenta). Scale bar, 20 μm. cc, Central canal. F, Whole-cell current clamp in the presence of gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm) showing the response to both acidic (pH 6.5; red trace) and alkaline (pH 8.0; blue trace) pH. The connexin hemichannel blocker lanthanum (100 μm) abolished the alkaline response but not the acidic response. G, Mean frequency increase of action potential firing in response to acidic and alkaline pH before and after application of lanthanum chloride (100 and 70 μm) [before application (control): means ± SEM; n = 5; Student's paired t test: ***p < 0.001 significant difference vs pH 7.4 at 6.5 (p = 1.31 × 10−4, t4 = 14.51) and at 8 (p = 9.43 × 10−5, t4 = 15.77)]. In the presence of lanthanum, a complete (100 μm) or partial (70 μm) blockade of the spiking response to alkaline pH was seen [means ± SEM; n = 5; Student's paired t test: ***p < 0.001 significant difference at pH 8 vs control in the presence of lanthanum at 100 μm (p = 9.4 × 10−5, t4 = 15.77; as well as at 70 μm (p = 9.3 × 10−4, t4 = 8.76)]. A tendency for a small, nonsignificant (n.s.) frequency reduction was also observed at acidic pH (6.5) versus control in the presence of lanthanum at 100 μm (p = 0.06, t4 = 2.58). H, The recorded CSF-c neuron in F intracellularly labeled with Neurobiotin expressed somatostatin. Scale bars, 10 μm. I, Whole-cell current clamp of a hypothalamic CSF-c neuron in control condition and in the presence of gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm) showing that this cell did not respond to either acidic (pH 6.5; red trace) or alkaline (pH 8.0; blue trace) pH. J, The recorded CSF-c neuron in I intracellularly labeled with Neurobiotin did not express somatostatin. Scale bars, 10 μm.
- Figure 6.
The same hypothalamic CSF-c neuron is sensitive to both fluid movement and pH changes. A, In vitro preparation of the hypothalamus with CSF-c neurons protruding into the third ventricle. An aCSF-filled pressure pipette was placed close to a bulb-like ending of a recorded hypothalamic CSF-c neuron. Scale bar, 20 μm. B, A short fluid-pulse (80 ms) elicited receptor potential responses and action potentials while holding the membrane potential at −65 mV and −55 mV, respectively, in the presence of gabazine (20 μm), CNQX (40 μm), and AP5 (50 μm). C, Receptor potential elicited by fluid pulse stimulation (20 psi, 80 ms) was blocked by application of the ASIC3 blocker APETx2 (1 μm). D, Complete blockade of responses after application of APETx2 (n = 4). E, In the same hypothalamic CSF-c neuron as in C, exposure to acidic (pH 6.5) as well as alkaline (pH 8.0) pH depolarized the membrane potential (10–12 mV) and triggered action potentials (with GABA and glutamate receptor antagonists present).
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