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. 2012 Aug 15;590(16):3719-41.
doi: 10.1113/jphysiol.2012.227959. Epub 2012 May 8.

Properties of Subependymal Cerebrospinal Fluid Contacting Neurones in the Dorsal Vagal Complex of the Mouse Brainstem

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Properties of Subependymal Cerebrospinal Fluid Contacting Neurones in the Dorsal Vagal Complex of the Mouse Brainstem

Adeline Orts-Del'immagine et al. J Physiol. .
Free PMC article

Abstract

Cerebrospinal fluid (CSF) contacting neurones have been observed in various brain regions such as the hypothalamus, the dorsal nucleus of the raphe and around the central canal (cc) of the spinal cord but their functional role remains unclear. At the level of the spinal cord, subependymal cerebrospinal fluid contacting neurones (S-CSF-cNs) present a peculiar morphology with a soma close to the ependymal layer, a process projecting towards the cc and ending with a bud and a cilium. These neurones were recently shown to express polycystin kidney disease 2-like 1 (PKD2L1 or TRPP3) channels that are members of the polycystin subtype of the transient receptor potential (TRP) channel superfamily and that have been proposed as either chemo- or mechanoreceptors in several tissues. Using immunohistological techniques and whole-cell electrophysiological recordings in brain slices obtained from PKD2L1:EGFP transgenic adult mice, we looked for and determined the functional properties of S-CSF-cNs in the dorsal vagal complex (DVC), a hindbrain structure controlling autonomic functions such as blood pressure, energy balance and food intake. Here, we demonstrate that S-CSF-cNs received GABAergic and/or glycinergic synaptic entries and were also characterised by the presence of non-selective cationic channels of large conductance that could be detected even under whole-cell configuration. The channel activity was not affected by Psalmopoeus cambridgei toxin 1, a blocker of acid sensing ion channels (ASICs), but was blocked by amiloride and by a strong extracellular acidification. In contrast, extracellular alkalinisation and hypo-osmotic shocks increased channel activity. Based on these properties, we suggest that the single-channel activity recorded in medullar S-CSF-cNs is carried by PKD2L1 channels. Our study therefore reinforces the idea that PKD2L1 is a marker of S-CSF-cNs and points toward a role for S-CSF-cNs in the detection of circulating signals and of modifications in the extracellular environment.

Figures

Figure 1
Figure 1. In the dorsal vagal complex S-CSF-cNs are present and express PKD2L1 channels
A, acute coronal brainstem slice observed under infrared differential interference contrast (IR-DIC) illumination showing the characteristic structures of the dorsal vagal complex (DVC). AP, area postrema; NTS, nucleus tractus solitarii; ST, solitary tract; nX, dorsal motor nucleus of the vagus nerve; cc, central canal. B, in the same acute brainstem slice as in A, under 490 nm illumination, EGFP+ cells were observed close to the cc and in the parenchyma. Inset shows at higher magnification that EGFP+ cells close to the cc project a single process ending with a protrusion in the cavity (arrow) while cells in the parenchyma do not. C, selected representative micrographs illustrating 4 different neurones recorded in the whole-cell configuration with an intracellular solution containing 10 μm AlexaFluor 594 to reveal the cellular morphology. Star: round protrusion or bud in the CSF cavity; thin arrow: thin neurite resembling the axon; arrow head: shadow of the patch pipette. Note that images are presented in greyscale for a better visualisation. D, sagittal brainstem section showing that EGFP+ cells (left) in contact with the cc or in the parenchyma exhibit PKD2L1 positive immunoreactivity (middle). Right, merged image showing that all EGFP+ cells were also PKD2L1+. E, sagittal section showing the high density of PKD2L1+ cells along the cc of the brainstem. F, brainstem coronal section showing PKD2L1+ cells (green) inserted between vimentin positive ependymal cells (VIM, red). Nuclei were visualised using Hoechst coloration (blue). In AE, thin dashed line delineates cc. Open triangles: cells in contact with cc; filled triangles: cells in the parenchyma. Scale bars: A and B, 100 μm; inset in B and DF, 20 μm and C, 10 μm. Objective: 5× in A and B; 63×, B inset, CE; 20x in F. AC: epifluorescence imaging; DF: confocal Z projections.
Figure 2
Figure 2. PKD2L1:EGFP+ cells are neurones and exhibit both synaptic and unitary single-channel activities
A, one PKD2L1:EGFP+ subependymal CSF-cN (Left) was patched under IR-DIC illumination (middle) with a pipette solution containing 10 μm AlexaFluor 594. After cell dialysis, AlexaFluor 594 fluorescence confirmed that the identified PKD2L1:EGFP+ neurone was recorded (right). Arrow points to the neuronal somata. B, two representative S-CSF-cNs recorded in current-clamp mode at −60 mV (current injected to maintain the membrane potential at −60 mV: −12 pA left panel; −18 pA right panel) exhibiting either tonic (left panel) or phasic (right panel) action potential (AP) discharge in response to depolarising current injection (+20 pA, +40 pA). Note that while the tonic neurone responded with a higher AP discharge frequency when the injected current increased from +20 to +40 pA, the phasic neurone still generated a single AP. C, top, current-clamp trace from a S-CSF-cN recorded at resting potential (Vr = −53 mV) showing spontaneous depolarising events (arrows) as well as spontaneous AP discharge (star). Bottom, at −80 mV (Vh) in voltage-clamp mode, the same neurone exhibited spontaneous inward currents. D, expanded portions, selected from the recording in C, showing that S-CSF-cNs exhibited both fast inward currents resembling synaptic events (arrows) and currents with a square time course (filled circle) characteristic of single-channel activity. Dashed line indicates channel closed state.
Figure 6
Figure 6. In CSF-CNs extracellular acidification elicits ASICs activation and subsequent inhibition of PKD2L1
A, left, typical current response recorded at −80 mV (Vh) in one S-CSF-cN following 1 s pressure application of a citric acid solution at pH 2.8 (1 s, black bar). Black and grey traces represent respectively recordings in control and in the presence of 40 nm PcTx1, a blocker of homomeric ASIC1a and heteromeric ASIC1a/2b. Bottom, in the continuous presence of 2 mm amiloride, the pH 2.8 citric acid-mediated current was blocked as well as single channel openings. Right, summary bar graph for the average acid-mediated current in control (black bar, n = 14), in the presence of 40 nm PcTx1 (grey bar, n = 6) and in the presence of 2 mm amiloride (open bar, n = 4). ***P < 0.001; **P < 0.01. B, top, representative spontaneous current trace recorded in a S-CSF-cN at −80 mV (Vh) in the presence of 40 nm PcTx1 before, during and after pressure application of a citric acid, pH 2.8 solution (10 s, black bar). Note that although the acid-mediated fast inward current is strongly reduced in the presence of PcTx1, an acid-sensitive channel activity can still be recorded. Middle, expanded current traces selected from the recording at the top before (Control), during (Citric acid, pH 2.8) and after (Wash) extracellular acidification. Dashed line indicates channel closed state. Bottom, summary bar graphs of the average NPO (left) and unitary current amplitude (right) determined from 3 different cells before (Control, filled bars; n = 3) and in the presence of 40 nm PcTx1 (open bars, n = 3). C, representative current trace recorded at a holding potential of −80 mV (Vh) in one S-CSF-cN showing the reversible block of spontaneous channel activity following pressure application of 2 mm amiloride (20 s, black bar). Bottom, expanded current traces selected before (Control), during (Amiloride) and after (Wash) amiloride application from the recording at the top. Dashed line indicates channel closed state.
Figure 3
Figure 3. S-CSF-cNs possess functional synaptic GABAA and glycine receptors
A, left, representative current traces recorded at −80 mV in two S-CSF-cNs in response to pressure application of GABA (top; 1 mm, 50 ms, arrow) and glycine (bottom; 1 mm, 100 ms, arrow). In the presence of 10 μm gabazine (top) or 10 μm strychnine (bottom), pressure application of GABA or glycine, respectively, markedly reduced the elicited currents (grey traces). Right, spontaneous inward current activity recorded in voltage-clamp mode at −80 mV (Vh), in the presence of 1 mm kynurenic acid, a broad spectrum ionotropic glutamate receptors antagonist, to block fast glutamatergic transmission. Bottom, expanded portions selected from the current trace at the top showing that inward currents were of synaptic nature with a fast onset and an exponential decay. B, top, in the presence of 1 mm kynurenic acid and 10 μm gabazine, to block both glutamatergic and GABAergic transmission, pressure application of perfusion medium supplemented with 500 mm sucrose (10 s, black bar) evoked at −80 mV (Vh) a burst of synaptic events. Middle, expanded portions selected from the current trace at the top showing characteristic inward synaptic currents. Bottom, this sucrose-induced synaptic activity was completely blocked in the presence of 10 μm strychnine added to 1 mm kynurenic acid and 10 μm gabazine. C, top, in the presence of 1 mm kynurenic acid and 1 μm strychnine, to block both glutamatergic and glycinergic transmission, a similar protocol to that in B (30 s sucrose application, black bar) evoked a burst of synaptic events as can be better visualised on the selected expanded trace (middle). The sucrose-induced synaptic activity was completely blocked in the presence of 10 μm gabazine added to 1 mm kynurenic acid and 1 μm strychnine (bottom). D, summary bar graphs of average GABA (filled bar; n = 4) and glycine (open bar; n = 6) current amplitude (left) and decay time constant (right). In each cell, the decay time constant was obtained from the fit of the average current trace using a monoexponential function. **P < 0.01. E, representative average GABAergic (left) and glycinergic (right) synaptic currents with the superimposed fit of the current decay (grey line, with the decay time constant of the illustrated cell above the traces). Bottom, for a better comparison of the current decays, GABAergic (black line) and glycinergic (grey line) currents shown at the top were normalised to the current peak and superimposed.
Figure 4
Figure 4. S-CSF-cNs present a spontaneous channel activity with properties similar to PKD2L1
A, current traces recorded at a holding potential of −80 mV (Vh) in one S-CSF-cN showing spontaneous single-channel activity. The dashed lines represent channel closed state (C, closed), first opening level (O1) and second opening level (O2), from top to bottom, respectively. B, left, summary bar graphs of the average current amplitude for the opening of one channel (1st level, filled bar; n = 16 cells) or two channels (2nd level, open bar; n = 16 cells). Middle, average ratio of the current amplitude measured from the second opening level (A2) divided by the one measured at the first level (A1). This ratio is close to 2 suggesting the simultaneous opening of two channels. Right, summary bar graph for the average channel open probability (NPO) for the opening of one channel (1st level, filled bar; n = 16 cells) or two channels (2nd level, open bar; n = 16 cells). Note the very low occurrence of multiple opening events. ***P < 0.001. C, left, representative spontaneous single-channel current traces recorded at holding potentials between −80 mV and +40 mV (in 20 mV increments) in one S-CSF-cN. Dashed line indicates channel closed state. Middle, representative all-points amplitude distribution histograms obtained from the recordings at holding potential of +40, −40 and −80 mV (from top to bottom). Arrow indicates for each holding potential the unitary current amplitude. Right, plot of the single-channel current amplitude against the holding potential obtained from the current traces illustrated on the left and calculated from the amplitude distribution histogram as illustrated in the middle. The unitary current reversal potential (Erev,Channel) and the channel conductance (γ) were obtained from the linear fit of the experimental data points (black line). D, left, representative current traces recorded between −80 mV and −20 mV (20 mV increments) showing in the same S-CSF-cN the current response to GABA pressure application (1 mm, 50 ms, arrow) and the unitary current. Right, plot against the holding potential of the mean peak amplitude of the GABAA current (open circles, n = 6) and of the mean single-channel current amplitude (grey circles, n = 6) obtained from the current traces illustrated on the left. For a better comparison of the two current–potential relationships, in each cell the experimental data points were normalised against the current amplitude recorded at −80 mV (filled square). The reversal potential for GABAA current (Erev,GABA) and for unitary current (Erev,Channel) were obtained from the linear fit of the experimental data points. The values indicated on the graph correspond to the mean values ± SEM for Erev,GABA, Erev,Channel and single-channel conductance (γ) obtained from the fit of the raw data recorded in 6 different S-CSF-cNs. Note that on average Erev,GABA tended towards ECl whereas Erev,Channel was more positive.
Figure 5
Figure 5. Acidification blocks unitary currents in a graded manner and modulates S-CSF-cNs excitability
A, representative spontaneous current trace recorded in voltage-clamp mode at −80 mV (Vh) in a S-CSF-cN before, during and after pressure application of a 30 mm citric acid solution at pH 2.8 (10 s; black bar). Bottom, expanded current traces selected from the recording at the top before (Control), during (Citric acid, pH 2.8) and after (Wash) extracellular acidification. Note the complete block of channel activity during extracellular acidification. Dashed line indicates channel closed state. B, representative voltage trace recorded in current-clamp mode at resting membrane potential (Vr = −50 mV) in the same S-CSF-cN as in A before, during and after extracellular acidification (Citric acid, pH 2.8, 10 s; black bar). Bottom, expanded voltage traces selected from the regions labelled with numbers on the voltage trace at the top before (Control; 1), during (Citric acid, pH 2.8; 2) and after (Wash; 3 to 5) extracellular acidification. Note that extracellular acidification evoked AP discharge at the start of citric acid application (2), presumably due to the inward current observed in voltage-clamp mode (see A top trace) and an increased depolarising activity at the end of exposure to acid (3). Interestingly on traces 4 and 5, depolarising events with kinetics similar to postsynaptic potentials (arrows) can be observed along with ‘square-shaped’ events (filled circles). C, summary bar graph for the variation in open probability (NPO as a percentage) before (CTR, black bars), during (pH 2.8, open bars) and after (Wash, grey bars) pressure application of a pH 2.8 acid solution. Note the increased channel activity at the end of acidic exposure. NPO values were obtained from recordings in 9 cells and represent mean values calculated over 10 s recording periods at the times indicated under the bars. D, summary histograms for the percentage of inhibition in NPO following pressure application of citric acid solution with increased acidity: pH 6.3 (n = 4), pH 6.0 (n = 5), pH 5.5 (n = 4), pH 5.0 (n = 5) and pH 2.8 (n = 6). See also Table 2. For each pH condition, the NPO during application was compared against the corresponding control NPO and the percentage of inhibition calculated. ***P < 0.001; **P < 0.01; *P < 0.05, #full inhibition was observed for pH 2.8 and no statistical test could be performed.
Figure 7
Figure 7. Alkalinisation increases both single-channel activity and excitability in S-CSF-cNs
A, representative spontaneous current activity recorded in voltage-clamp mode at −80 mV (Vh) in a S-CSF-cN before, during and after pressure application of a pH 8.8 TAPS-based alkaline solution (30 s, black bar). Bottom, expanded current traces selected from the recording at the top before (Control), during (TAPS, pH 8.8) and after (Wash) extracellular alkalinisation. Dashed line indicates channel closed state. Note the increased channel activity during extracellular alkalinisation (Middle). B, representative voltage trace recorded in current-clamp mode at resting membrane potential (Vr = −45 mV) in the same S-CSF-cN as in A before, during and after extracellular alkalinisation (TAPS, pH 8.8; 30 s, black bar). Bottom, expanded voltage traces selected from the trace shown at the top before (Control), during (TAPS, pH 8.8) and after (Wash) extracellular alkalinisation. Note that extracellular alkalinisation induces an increase in APs discharge (middle).
Figure 8
Figure 8. Extracellular hypo-osmotic shock strongly increases single-channel activity
A, top, representative spontaneous current trace recorded in a S-CSF-cN at −80 mV (Vh) before, during and after pressure application of a hyperosmotic solution (3 min; black bar). Bottom left, expanded current traces selected from the recording at the top before (Control), during (Hyperosmotic shock) and after (Wash) exposure to the hyperosmotic solution. Dashed line indicates channel closed state. Bottom right, summary bar graphs of the average NPO determined before (CTR, black bar), during (Shock, open bar) and after (Wash, grey bars) pressure application of the hyperosmotic solution (n = 5 cells). B, top, representative spontaneous current traces recorded in a S-CSF-cN at −80 mV (Vh) before, during and after pressure application of a hypo-osmotic solution (3 min; black bar). Bottom left, expanded current traces selected from the recording at the top before (Control), during (Hypo-osmotic shock) and after (Wash) exposure to the hypo-osmotic solution. Dashed line indicates channel closed state. Bottom right, summary bar graphs of the average NPO determined before (CTR, black bar), during (Shock, open bar) and after (Wash, grey bars) pressure application of the hypo-osmotic solution (n = 3 cells). In A and B, only the first 9 min of recordings are illustrated and in the histograms, NPO represent mean values calculated over 3 min periods at the time indicated under the bars. **P < 0.01.

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