Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) Fluid-pulse stimulation (20 p.s.i., 80 ms) elicited action potential responses in control conditions (black) in the presence of gabazine (20 μM), AP5 (100 μM) and NBQX (40 μM). Application of the ASIC3 blocker APETx2 (2 μM) abolished responses (blue), which partially recovered upon washout (green). ( b ) Raster plot showing reliable responses to pressure stimulations (20 p.s.i., 80 ms) in control, and which were completely abolished by application of APETx2. Responses reappeared upon washout. ( c ) The receptor potential elicited by fluid pulses (20 p.s.i., 60 ms) was also blocked by APETx2 in the presence of TTX (1.5 μM). ( d ) Complete blockade of responses after application of APETx2 ( n =6). Means±s.e.m.

Article Snippet: Corresponding experiments were also performed in the presence of the ASIC3 antagonist APETx2 (1 μM) or the somatostatin receptor sst 2 antagonist CYN-154806 (2 μM; Tocris).

Techniques: Control

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) CSF-c neuron showing mechanosensitivity to a fluid pressure pulse (80 ms, 20 p.s.i.). ( b , c ) In the same neuron, the action potential frequency increased at pH 6.9 in the presence of gabazine (20 μM), AP5 (100 μM) and NBQX (40 μM). ( d ) Application of APETx2 (2 μM) abolished the response to fluid-pulse stimulation. ( e , f ) APETx2 also abolished the response to pH lowering. ( g ) Firing of CSF-c neurons before and after application of APETx2. The values are means±s.e.m. during 1 min of recording, normalized to basal activity at pH 7.4 ( n =5). Student's t -test: *** P <0.001, significant difference at pH 6.9 compared with 7.4 only in control conditions in the absence of APETx2. ( h ) Decreases in pH resulted in depolarizing potentials also in the presence of TTX (1.5 μM), that reversed at positive holding potentials. ( i ) Addition of APETx2 (1 μM) completely blocked the response to lowered pH in the presence of TTX (1.5 μM).

Article Snippet: Corresponding experiments were also performed in the presence of the ASIC3 antagonist APETx2 (1 μM) or the somatostatin receptor sst 2 antagonist CYN-154806 (2 μM; Tocris).

Techniques: Activity Assay, Control

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) The amplitude of the receptor current increases at hyperpolarised holding potentials and decreases upon depolarization in the presence of gabazine (20 μM), AP5 (100 μM), NBQX (40 μM) and TTX (1.5 μM). ( b ) The receptor current reversed at ∼+25 mV ( n =5). ( c ) The receptor current evoked by fluid pulse was eliminated in the presence of the ASIC3 blocker APETx2 (1 μM) and returned after washout. The receptor currents in a – c were elicited by a fluid pulse of 60 ms, 20 p.s.i. ( n =6). ( d ) No current events were seen at pH 7.4 in the presence of gabazine (20 μM), AP5 (100 μM), NBQX (40 μM) and TTX (1.5 μM). After a decrease in extracellular pH to 6.5, inward current deflections appeared that decreased in amplitude and frequency at more depolarized holding potentials and were reversed in sign at +35 mV ( n =5). ( e ) At pH 6.5 discrete current deflections were recorded, which may correspond to single-channel openings. ( f ) The inward currents recorded at pH 6.5 were completely blocked in the presence of APETx2 (1 μM; n =3). The data in b are represented as means±s.e.m.

Article Snippet: Corresponding experiments were also performed in the presence of the ASIC3 antagonist APETx2 (1 μM) or the somatostatin receptor sst 2 antagonist CYN-154806 (2 μM; Tocris).

Techniques:

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) Illustration of arrangement for ventral root recordings with suction electrodes in the intact, isolated spinal cord preparation (VR-L, VR-R, left and right side ventral root, respectively). ( b ) Bilateral ventral root recording during NMDA (100 μM)-induced fictive locomotion in the isolated lamprey spinal cord, during control conditions (pH 7.4; black traces) and during lowered pH of 6.9 (red traces) and 6.5 (orange traces). ( c ) Decreases in extracellular pH prolonged the cycle period. The mean period was determined during 20 cycles for each of the conditions and normalized to the value during control conditions (% of control; n =7). ( d ) Bilateral ventral root recording during control conditions (pH 7.4; black traces), during pH 6.9 (red traces) and in the presence of the ASIC3 blocker APETx2 (1 μM; blue traces). ( e ) Application of APETx2 blocked the effect of lowered extracellular pH (6.9) on the cycle period, which recovered upon washout (mean values calculated for 20 cycles during each condition; n =2 preparations). ( f ) Effect of somatostatin (10 nM, 100 nM and 1μΜ) on the cycle period of the locomotor activity. Somatostatin significantly increased the period at all tested concentrations ( n =19). ( g ) Bilateral ventral root recording in the isolated spinal cord, during control conditions (pH 7.4; black traces), during pH 6.5 (orange traces) and following application of the somatostatin receptor sst 2 antagonist CYN-154806 (2 μM; green traces). ( h ) Application of CYN-154806 lead to a shortening of the period length at control pH 7.4 (dark green; n =4). In the presence of the antagonist, a decrease of pH (here to 6.5; light green) had no effect on the cycle period. The data are represented as means±s.e.m. ( c , f,h ) and ±s.d. ( e ); Student's t -test: *** P <0.001; ** P <0.01, significant difference compared with control; NS: non-significant, NS P >0.5.

Article Snippet: Corresponding experiments were also performed in the presence of the ASIC3 antagonist APETx2 (1 μM) or the somatostatin receptor sst 2 antagonist CYN-154806 (2 μM; Tocris).

Techniques: Isolation, Control, Activity Assay

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) Ciliated CSF-c neurons respond to fluid movements and to lowered pH through activation of ASIC3. ( b ) Schematic illustration of a cross-section of the lamprey spinal cord, with a CSF-c neuron by the central canal (cc) and lateral axonal projections in the grey matter providing input to the locomotor network and projections in close apposition to a stretch receptor neuron at the spinal cord margin.

Article Snippet: Corresponding experiments were also performed in the presence of the ASIC3 antagonist APETx2 (1 μM) or the somatostatin receptor sst 2 antagonist CYN-154806 (2 μM; Tocris).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: In silico screening of GMQ-like compounds reveals guanabenz and sephin1 as new allosteric modulators of acid-sensing ion channel 3

doi: 10.1101/704445

Figure Lengend Snippet: (A) Example traces of the effect for the 5 different selected drugs (500 µM) on pH 7-induced rASIC3 activation together with example trace showing the effect of the prototypic rASIC3 modulator GMQ (500 µM). In all cases drugs were applied at pH 7. (B-C) Bar plots showing the effect of the selected drugs and GMQ on transient (peak) (B) and the sustained current (expressed as the ratio I 5s /I peak ) (C). (D) Example traces of the effect for the 5 different selected drugs (500 µM) on pH 6-induced rASIC3 activation together with example traces showing the effect of known rASIC3 modulators, APETx2 (1 µM) and GMQ (500 µM). In all cases drugs were applied for 30 s prior to pH 6-induced rASIC3 activation. (B-D) Bar plots showing the effect of the selected drugs, APETx2 and GMQ on transient (peak) (B) and sustained current (C), and inactivation time constant (D) of rASIC3 activation. All values were normalised to baseline pH 7 or pH 6 rASIC3 activation and expressed as means ± SEM (n = 6-11, paired t-test, *p <0.05 and ***p ≤ 0.005 vs baseline pH 7 or pH 6 activation).

Article Snippet: All the small molecules used in this study were purchased from Sigma (Gilliangham, UK) except for tizanidine (Tocris, Bristol, UK) and APETx2 (Smartox, Saint egrève, France).

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) Fluid-pulse stimulation (20 p.s.i., 80 ms) elicited action potential responses in control conditions (black) in the presence of gabazine (20 μM), AP5 (100 μM) and NBQX (40 μM). Application of the ASIC3 blocker APETx2 (2 μM) abolished responses (blue), which partially recovered upon washout (green). ( b ) Raster plot showing reliable responses to pressure stimulations (20 p.s.i., 80 ms) in control, and which were completely abolished by application of APETx2. Responses reappeared upon washout. ( c ) The receptor potential elicited by fluid pulses (20 p.s.i., 60 ms) was also blocked by APETx2 in the presence of TTX (1.5 μM). ( d ) Complete blockade of responses after application of APETx2 ( n =6). Means±s.e.m.

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2 (1–2 μM, Merck Chemicals Ltd., Nottingham, UK), the GABA A receptor antagonist gabazine (20 μM, Tocris, Ellisville, MO, USA), the NMDA receptor antagonist AP5 (100 μM, Tocris), the AMPA receptor antagonist NBQX (40 μM, Tocris) and TTX (1.5 μM; Sigma).

Techniques: Control

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) CSF-c neuron showing mechanosensitivity to a fluid pressure pulse (80 ms, 20 p.s.i.). ( b , c ) In the same neuron, the action potential frequency increased at pH 6.9 in the presence of gabazine (20 μM), AP5 (100 μM) and NBQX (40 μM). ( d ) Application of APETx2 (2 μM) abolished the response to fluid-pulse stimulation. ( e , f ) APETx2 also abolished the response to pH lowering. ( g ) Firing of CSF-c neurons before and after application of APETx2. The values are means±s.e.m. during 1 min of recording, normalized to basal activity at pH 7.4 ( n =5). Student's t -test: *** P <0.001, significant difference at pH 6.9 compared with 7.4 only in control conditions in the absence of APETx2. ( h ) Decreases in pH resulted in depolarizing potentials also in the presence of TTX (1.5 μM), that reversed at positive holding potentials. ( i ) Addition of APETx2 (1 μM) completely blocked the response to lowered pH in the presence of TTX (1.5 μM).

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2 (1–2 μM, Merck Chemicals Ltd., Nottingham, UK), the GABA A receptor antagonist gabazine (20 μM, Tocris, Ellisville, MO, USA), the NMDA receptor antagonist AP5 (100 μM, Tocris), the AMPA receptor antagonist NBQX (40 μM, Tocris) and TTX (1.5 μM; Sigma).

Techniques: Activity Assay, Control

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) The amplitude of the receptor current increases at hyperpolarised holding potentials and decreases upon depolarization in the presence of gabazine (20 μM), AP5 (100 μM), NBQX (40 μM) and TTX (1.5 μM). ( b ) The receptor current reversed at ∼+25 mV ( n =5). ( c ) The receptor current evoked by fluid pulse was eliminated in the presence of the ASIC3 blocker APETx2 (1 μM) and returned after washout. The receptor currents in a – c were elicited by a fluid pulse of 60 ms, 20 p.s.i. ( n =6). ( d ) No current events were seen at pH 7.4 in the presence of gabazine (20 μM), AP5 (100 μM), NBQX (40 μM) and TTX (1.5 μM). After a decrease in extracellular pH to 6.5, inward current deflections appeared that decreased in amplitude and frequency at more depolarized holding potentials and were reversed in sign at +35 mV ( n =5). ( e ) At pH 6.5 discrete current deflections were recorded, which may correspond to single-channel openings. ( f ) The inward currents recorded at pH 6.5 were completely blocked in the presence of APETx2 (1 μM; n =3). The data in b are represented as means±s.e.m.

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2 (1–2 μM, Merck Chemicals Ltd., Nottingham, UK), the GABA A receptor antagonist gabazine (20 μM, Tocris, Ellisville, MO, USA), the NMDA receptor antagonist AP5 (100 μM, Tocris), the AMPA receptor antagonist NBQX (40 μM, Tocris) and TTX (1.5 μM; Sigma).

Techniques:

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) Illustration of arrangement for ventral root recordings with suction electrodes in the intact, isolated spinal cord preparation (VR-L, VR-R, left and right side ventral root, respectively). ( b ) Bilateral ventral root recording during NMDA (100 μM)-induced fictive locomotion in the isolated lamprey spinal cord, during control conditions (pH 7.4; black traces) and during lowered pH of 6.9 (red traces) and 6.5 (orange traces). ( c ) Decreases in extracellular pH prolonged the cycle period. The mean period was determined during 20 cycles for each of the conditions and normalized to the value during control conditions (% of control; n =7). ( d ) Bilateral ventral root recording during control conditions (pH 7.4; black traces), during pH 6.9 (red traces) and in the presence of the ASIC3 blocker APETx2 (1 μM; blue traces). ( e ) Application of APETx2 blocked the effect of lowered extracellular pH (6.9) on the cycle period, which recovered upon washout (mean values calculated for 20 cycles during each condition; n =2 preparations). ( f ) Effect of somatostatin (10 nM, 100 nM and 1μΜ) on the cycle period of the locomotor activity. Somatostatin significantly increased the period at all tested concentrations ( n =19). ( g ) Bilateral ventral root recording in the isolated spinal cord, during control conditions (pH 7.4; black traces), during pH 6.5 (orange traces) and following application of the somatostatin receptor sst 2 antagonist CYN-154806 (2 μM; green traces). ( h ) Application of CYN-154806 lead to a shortening of the period length at control pH 7.4 (dark green; n =4). In the presence of the antagonist, a decrease of pH (here to 6.5; light green) had no effect on the cycle period. The data are represented as means±s.e.m. ( c , f,h ) and ±s.d. ( e ); Student's t -test: *** P <0.001; ** P <0.01, significant difference compared with control; NS: non-significant, NS P >0.5.

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2 (1–2 μM, Merck Chemicals Ltd., Nottingham, UK), the GABA A receptor antagonist gabazine (20 μM, Tocris, Ellisville, MO, USA), the NMDA receptor antagonist AP5 (100 μM, Tocris), the AMPA receptor antagonist NBQX (40 μM, Tocris) and TTX (1.5 μM; Sigma).

Techniques: Isolation, Control, Activity Assay