Journal: Scientific Reports

Article Title: Population diversity and function of hyperpolarization-activated current in olfactory bulb mitral cells

doi: 10.1038/srep00050

Figure Lengend Snippet: A In the presence of synaptic and ion channel blockers (see methods) the cell was voltage-clamped at −50 mV and held between −50 and −140 mV in steps 10 mV for 2 seconds. Subsequently ZD7288 was applied, thus the traces examples are the ZD7288 sensitive current exclusively. B Activation curve of the tail current (measured at arrow in A) obtained from recordings as in A. Sigmoid fitting indicates half maximal activation at V ½ = −88 ± 2, n = 7. The activation (C1) and deactivation (C2) times were determined by single exponential fitting (red). C3 Pooled data showing activation (grey) and deactivation (black) time constants (n = 7). D The reversal potential of I h was determined to −32 ± 2 mV (n = 7) by linear extrapolation to the peak of the tail current from clamping at potentials between −85 and −35 mV after full activation of the current for 2 seconds. E Plot showing the correlation between the voltage-sensitive I h and the instantaneous (I inst ) current (r > 0.9, n = 5).

Article Snippet: ZD7288 (4-( N -ethyl- N -phenylamino)-1,2-dimethyl-6-(methylamino)- pyridinum chloride (Tocris) was used at a concentration of 10–40 μM.

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: Population diversity and function of hyperpolarization-activated current in olfactory bulb mitral cells

doi: 10.1038/srep00050

Figure Lengend Snippet: A Example membrane voltage traces highlighting the effect of ZD7288 on the sag potential. The current injection steps are −356 pA and −712 pA (mean ± S.D., sag ctrl : 7.37 ± 3 mV vs. sag ZD : 0.03 ± 0.12 mV, n = 12, 10 – 40 μM ZD7288, p < 0.05). Dashed red box show the regions of the control and ZD7288 traces that have been enlarged to observe the bridge test pulse. B Plot of the peak vs. the steady state voltage under control and following application of ZD7288. Block of the sag was observed for all recorded voltages (n = 4 cells). C Left: Example of the sag potential recorded in current-clamp and the current recorded in I h isolation cocktail under voltage-clamp conditions in the same cell (at −100 mV) Right: Plot of the relation between sag amplitude and the voltage-clamp recorded I h current in the same cell (r = −0.78, p < 0.05). The data point corresponding to the example traces is represented in the graph by the open circle.

Article Snippet: ZD7288 (4-( N -ethyl- N -phenylamino)-1,2-dimethyl-6-(methylamino)- pyridinum chloride (Tocris) was used at a concentration of 10–40 μM.

Techniques: Membrane, Injection, Control, Blocking Assay, Isolation

Journal: Scientific Reports

Article Title: Population diversity and function of hyperpolarization-activated current in olfactory bulb mitral cells

doi: 10.1038/srep00050

Figure Lengend Snippet: A1 Example current-voltage relation of a sag (left) and a no sag expressing mitral cell (right). A2 Raster plots of spiking in cells shown in A1 during injection of depolarizing current up to 450 pA in steps of 50 pA. B Plot of the coefficient of variation of the mean inter-spike interval (CV ISI ) against current injection amplitudes ranging from 135 – 450 pA for the sag and no sag groups (mean ± S.E.M. of n = 15 sag cells; n = 17 no sag cells, p-values < 0.05 are marked with *). C Left: Example traces of spiking in a sag expressing mitral cell during a 1500 ms sustained current injection of 350 pA before (black) and after (red) ZD7288 application (left). Right: Summary of the CV ISI in control and in the presence of ZD7288 (n = 8 cells).

Article Snippet: ZD7288 (4-( N -ethyl- N -phenylamino)-1,2-dimethyl-6-(methylamino)- pyridinum chloride (Tocris) was used at a concentration of 10–40 μM.

Techniques: Expressing, Injection, Control

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 3 | The dose-response emetic effect of the HCN blockers in the lease shrew. Different groups of least shrews were given varying doses of ZD7288 (i.p., n 8 shrews per group or i. c.v., n 6) or ivabradine hydrochloride (i.p., n 6 shrews per group), and were observed for the next 30 min (A, C, E) The frequency of emesis was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (B, D, F) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. 0 mg/kg.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques:

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 4 | Immunohistochemical analysis of c-Fos following emesis induced by systemic administration of the HCN channel blocker ZD7288. Least shrews were sacrificed 90 min post vehicle treatment, or after the first vomiting occurred post systemic administration (1 mg/kg, i. p.) of ZD7288 (n 4 shrews per group). Shrew brainstem section (20 μm) and intestinal jejunum sections (25 μm) were stained with rabbit c-Fos antibody and Alexa Fluor 594 donkey anti-rabbit secondary antibody. Nuclei were stained with DAPI in blue. (A, C) Representative tile-scanned images show a robust c-Fos induction in the brainstem dorsal vagal complex (DVC) in response to ZD7288 (1 mg/kg, i. p.). Scale bar, 100 μm. (B, D) Representative single filed images (20x) show c-Fos expression evoked by ZD7288 observed in the DVC emetic nuclei, the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX), within the brainstem. Scale bar, 200 μm. (E–H) Representative images (20x) show low c-Fos expression induced by ZD7288 in the enteric nervous system (ENS) of the intestinal jejunum. Scale bar, 100 μm.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 5 | Quantified data for ZD7288-induced c-Fos expression in the least shrew brainstem dorsal vagal complex, containing the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX), as well as the enteric nervous system (ENS) embedded in the wall of jejunum. Values represent the mean number of c-Fos positive cells in each region of interest per section and are presented as mean ± SEM (n 4 shrews per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control (treated with vehicle of ZD7288), Unpaired t-test.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Control

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 6 | Immunohistochemical analysis of c-Fos expression in NeuN-positive neurons of ENS. Co-staining of jejunum sections from ZD7288 (1 mg/kg, i. p.)- treated shrews with rabbit c-Fos and mouse anti-NeuN antibodies followed by Alexa Fluor 594 donkey anti-rabbit and 488 donkey anti-mouse secondary antibodies. NeuN, a neuronal marker of intrinsic primary afferent neurons of the enteric nervous system. Nuclei were stained with DAPI in blue. (A–D) Representative images (60x) show c-Fos expression seen in neurons of the enteric nervous system (ENS), which is embedded in the lining of the intestine. Scale bar, 100 μm.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Immunohistochemical staining, Expressing, Staining, Marker

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 7 | ZD7288 increases c-Fos expression of serotonin neurons in the least shrew brainstem dorsal vagal complex. (A–F) Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in the synthesis of neuronal serotonin. Representative images (20x) showing anti- TPH2, anti-c-Fos and merged immunofluorescence staining in vehicle controls and shrews administered with ZD7288 (1 mg/kg, i. p.) (n 3 shrews per group). Scale bars 100 μm. (G–R) Representative higher magnification images (×60) showing c-Fos expressing evoked by ZD7288 (1 mg/kg, i. p.) localized in TPH2 positive serotonin neurons of the brainstem dorsal vagal complex, composed of the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX). Scale bars 10 μm.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Staining

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 8 | Immunohistochemical analysis of ERK1/2 phosphorylation following the HCN channel blocker ZD7288-induced emesis. Least shrews were sacrificed 15 min after vehicle or ZD7288 injection (1 mg/kg, i. p.) (n 3 shrews per group). Brainstem sections (20 μm) and intestinal jejunum sections (25 μm) were stained with rabbit anti-phospho-ERK1/2 antibody and Alexa Fluor 594 donkey anti-rabbit secondary antibody. Nuclei were stained with DAPI in blue. (A, C) Representative tile scan images show a strong upregulation of ERK1/2 phosphorylation (pERK) in the brainstem dorsal vagal complex (DVC) in response to ZD7288. Scale bar, 200 μm. (B, D) Representative images show ERK1/2 phosphorylation evoked by ZD7288 seen in the DVC throughout three emetic nuclei, the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX). Scale bar, 100 μm. (E) Statistical analysis of integrated density of ERK1/2 phosphorylation evoked by ZD7288 (1 mg/kg, i. p.) in the brainstem dorsal vagal complex. **p < 0.01 vs. Control, Unpaired t-test.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Immunohistochemical staining, Phospho-proteomics, Injection, Staining, Control

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 10 | Effects of modulators of cell membrane Ca2+ channels on the HCN channel blocker ZD7288-induced emesis. Different groups of least shrews were given an injection of either the corresponding vehicle, or varying doses of: 1) The L-type Ca2+ channel (LTCC) inhibitor nifedipine (s.c.) (n 6 shews per group); 2) the TRPV1R agonist resiniferatoxin (RTX) (s.c.) (n 8); 3) store-operated Ca2+ entry blocker MRS 1845 (i.p.) (n 8); 4) T-type Ca2+ channel inhibitor Z944 (i.p.) (n 9), 30 min prior to ZD7288 injection (1 mg/kg, i. p.). Emetic parameters were recorded for the next 30 min (A, C, E, G) The frequency of emesis was analyzed with Kruskal- Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (B, D, F, H) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. 0 mg/kg.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Membrane, Injection

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 9 | Effect of ERK1/2 inhibitor U0126 on the HCN channel blocker ZD7288-induced emesis. Different groups of shrews were given vehicle or varying doses of the ERK1/2 inhibitor U0126 (i.p.) (n 7 shrews per group), 30 min prior to ZD7288 (1 mg/kg, i. p.) administration. Shrews were observed the next 30 min. (A) The frequency of emesis was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (B) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. ***p < 0.001 vs. 0 mg/kg.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques:

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 11 | Effects of intracellular Ca2+ channel modulators on the HCN channel blocker ZD7288-induced emesis. Thirty minutes prior to an injection of ZD7288 (1 mg/kg, i. p.), different groups of least shrews were given an injection (i.p.) of either the corresponding vehicle, or varying doses of: 1) the ryanodine receptor (RyR) antagonist dantrolene (n 8 shrews per group) (A, B), and 2) the inositol-1, 4, 5-triphosphate receptor IP3R antagonist 2-APB (n 6) (C, D). Emetic parameters were recorded for the next 30 min post ZD8288 injection. (A, C) The frequency of emesis was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (B, D) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. *p < 0.05, **p < 0.01, ****p < 0.0001 vs. 0 mg/kg.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Injection

Journal: Frontiers in pharmacology

Article Title: The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew ( Cryptotis parva ).

doi: 10.3389/fphar.2021.647021

Figure Lengend Snippet: FIGURE 12 | Efficacy of receptor-selective antiemetics against the HCN channel blocker ZD7288-induced emesis. Different groups of least shrews were given an injection of either the corresponding vehicles (0 mg/kg), or varying doses of 5-HT3R antagonist palonosetron (s.c.) (n 6 shrews per group) (A and B), NK1R antagonist netupitant (i.p.) (n 6) (C and D), or the D2/3R antagonist sulpride (s.c.) (n 6) (E and F), 30 min prior to ZD7288 administration (1 mg/kg, i. p.). Emetic parameters were recorded for the next 30 min (A, C) The frequency of emesis was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (E) The frequency of emesis was analyzed with Unpaired t-test and presented as mean ± SEM. (B, D, F) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. *p < 0.05, **p < 0.01, ***p < 0.001 vs. 0 mg/kg.

Article Snippet: The following drugs were used in the present studies: ZD7288, U0126, MRS 1845, Z944 and RTX were purchased from Tocris (Ellisville, MO); ivabradine hydrochloride, nifedipine and sulpride from Sigma/RBI (St. Louis, MO); dantrolene and 2- APB from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Injection