Journal: Frontiers in Neuroscience

Article Title: GluN2A/ERK/CREB Signaling Pathway Involved in Electroacupuncture Regulating Hypothalamic-Pituitary-Adrenal Axis Hyperactivity

doi: 10.3389/fnins.2021.703044

Figure Lengend Snippet: EA reduces hypothalamic GluN2A expression after surgery. Quantification of hypothalamic (A) GluN1, (B) GluN2A, and (C) GluN2B mRNA levels in the NC, HT, and HT + EA groups ( n = 6 for each group). (D–F) Expression of phosphorylated GluN2A (pGluN2A) and total GluN2A protein in the hypothalamus was analyzed by western blot ( n = 6 for each group). (G,H) Representative images and quantification for Co-labeled CRH and GluN2A-positive cells in the PVN ( n = 4 for each group). Data are expressed as mean ± SEM. *vs. NC group (** p < 0.01, *** p < 0.001); #vs. HT group ( # p < 0.05, ## p < 0.01, ### p < 0.001). One-way analysis of variance (ANOVA).

Article Snippet: The separated groups of mice were administrated NMDA (M3262, Sigma, United States; an agonist of GluN2A-containing NMDA receptors, 0.4 nmol/μL, 0.5 μL/side) , PEAQX (HY-12294A, Med Chem Express, China; a preferential antagonist of GluN2A-containing NMDA receptors, 1 μg/μL, 0.25 μL/side) , MEK1/2 Inhibitor IV (444967, Sigma, United States; an inhibitor of ERK, 0.1 nmol/μL, 0.5 μL/side) , 666-15 (HY-101120, Med Chem Express, China; an antagonist of CREB, 0.1 nmol/μL, 0.5 μL/side) , or normal saline into the bilateral hypothalamus (4–6 animals in each group) for functional research.

Techniques: Expressing, Western Blot, Labeling

Journal: Frontiers in Neuroscience

Article Title: GluN2A/ERK/CREB Signaling Pathway Involved in Electroacupuncture Regulating Hypothalamic-Pituitary-Adrenal Axis Hyperactivity

doi: 10.3389/fnins.2021.703044

Figure Lengend Snippet: Hypothalamic CRH expression was downregulated after EA treatment in NMDA-administered mice. (A) Cannula implantation was performed and fluorescent dye (DiIC18) was injected into the hypothalamus through the cannula to determine the accuracy of drug injection (Administration location: AP 0.6 mm, ML ± 0.2 mm, DV 4.5 mm). For the GluN2A agonist, different doses of NMDA (0.08 nmol/μL, 0.4 nmol/μL, and 2 nmol/μL) were injected into the bilateral hypothalamus. (B) Quantification of hypothalamic CRH mRNA. (C,D) Representative WB images and quantification for CRH protein ( n = 6 for each group) 24 h after NMDA injection. Quantification of CRH (E) mRNA and (F,G) protein in the NS, NMDA, NMDA + EA, and EA groups (n = 5 for each group). Data are expressed as mean ± SEM. *vs. NS group (* p < 0.05, ** p < 0.01, *** p < 0.001); #vs. NMDA group ( # p < 0.05, ## p < 0.01).

Article Snippet: The separated groups of mice were administrated NMDA (M3262, Sigma, United States; an agonist of GluN2A-containing NMDA receptors, 0.4 nmol/μL, 0.5 μL/side) , PEAQX (HY-12294A, Med Chem Express, China; a preferential antagonist of GluN2A-containing NMDA receptors, 1 μg/μL, 0.25 μL/side) , MEK1/2 Inhibitor IV (444967, Sigma, United States; an inhibitor of ERK, 0.1 nmol/μL, 0.5 μL/side) , 666-15 (HY-101120, Med Chem Express, China; an antagonist of CREB, 0.1 nmol/μL, 0.5 μL/side) , or normal saline into the bilateral hypothalamus (4–6 animals in each group) for functional research.

Techniques: Expressing, Injection

Journal: Frontiers in Neuroscience

Article Title: GluN2A/ERK/CREB Signaling Pathway Involved in Electroacupuncture Regulating Hypothalamic-Pituitary-Adrenal Axis Hyperactivity

doi: 10.3389/fnins.2021.703044

Figure Lengend Snippet: The GluN2A, ERK, and CREB antagonist reverses HPA axis hyperactivity induced by surgery. The GluN2A antagonist PEAQX (1 μg/μL, 0.25 μL/side) was administered to both sides of the hypothalamus. Hypothalamus was dissected 24 h after the PEAQX administration. (A–D) Representative bands and quantification of hypothalamic pERK, pCREB, and CRH protein among the NS, HT + NS, HT + PEAQX, and HT + EA groups ( n = 4 for each group). The ERK inhibitor MEK1/2 Inhibitor IV (0.1 nmol/μL, 0.5 μL/side) was administered stereotaxically into the hypothalamus. (E–G) Representative bands and quantification of pCREB and CRH protein in the NS, HT + NS, HT + MEK, and HT + EA groups ( n = 5 for each group). 666-15 (an antagonist of CREB, 0.1 nmol/μL, 0.5 μL/side) was administrated to investigate the relationship between CREB and HPA axis. (H,I) Western Blot analysis of hypothalamic CRH protein in the NS, HT + NS, HT + 666-15, and HT + EA groups ( n = 5 for each group). (J) Relative hypothalamic CRH mRNA expression after surgical trauma and drugs administration ( n = 5 for each group). (K,L) ACTH and CORT levels in the peripheral serum of mice ( n = 5 for each group). Data are expressed as mean ± SEM. *vs. NS group (* p < 0.05, ** p < 0.01, *** p < 0.001); #vs. HT group ( # p < 0.05, ## p < 0.01, ### p < 0.001).

Article Snippet: The separated groups of mice were administrated NMDA (M3262, Sigma, United States; an agonist of GluN2A-containing NMDA receptors, 0.4 nmol/μL, 0.5 μL/side) , PEAQX (HY-12294A, Med Chem Express, China; a preferential antagonist of GluN2A-containing NMDA receptors, 1 μg/μL, 0.25 μL/side) , MEK1/2 Inhibitor IV (444967, Sigma, United States; an inhibitor of ERK, 0.1 nmol/μL, 0.5 μL/side) , 666-15 (HY-101120, Med Chem Express, China; an antagonist of CREB, 0.1 nmol/μL, 0.5 μL/side) , or normal saline into the bilateral hypothalamus (4–6 animals in each group) for functional research.

Techniques: Western Blot, Expressing

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 , b ) A CSF-c neuron retrogradely labelled following BDA injection in the lateral margin. The cilium (arrowheads) is α-tubulin immunoreactive. ( c ) In vitro preparation with the central canal lumen exposed. A CSF-c neuron was patched and a Ringer-filled pressure pipette was placed close to its bulb-like ending. ( d ) A short (80 ms) fluid-pulse elicited receptor- or action potential responses. ( e ) The neuron responded with action- or receptor potentials at short and constant latency to all stimuli (20 p.s.i., 80 ms). ( f ) The response latency was around 20 ms irrespective of the duration of the fluid pulse (10–80 ms; means±s.e.m.; n =10). ( g ) Fluid-pulses of 5–15 p.s.i. elicited subthreshold receptor potentials of increasing amplitude, while a 20 p.s.i. pulse triggered an action potential response. ( h ) The mean receptor potential amplitude increased for all cells tested ( n =6) in response to increasing fluid pulse magnitude. Means±s.e.m.; Student's t-test: ** P <0.01 and *** P <0.001, significant difference compared with value at 5 p.s.i. ( i ) The response to fluid movement remains after application of GABA and glutamate receptor antagonists, gabazine (20 μM) and NBQX (40 μM)/AP5 (100 μM), respectively. ( j ) A CSF-c neuron showing spontaneous sodium-mediated action potentials (blocked by TTX, 1.5 μM), as well as spontaneous GABA- and glutamate-mediated postsynaptic potentials (blocked by gabazine (20 μM) and NBQX (40 μM)/AP5 (100 μM), respectively. ( k ) By changing the holding potential in the presence of gabazine (20 μM), AP5 (100 μM), NBQX (40 μM) and TTX (1.5 μM), the receptor potential decreased in amplitude to finally become reversed at +30 mV. ( l ) Motoneurons (upper trace) or glia-like CSF-c cells (lower trace) did not show any response to pressure pulse stimulation. BDA, biotinylated dextran amine; cc, central canal. Scale bars, 10 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: Injection, In Vitro, Transferring

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 ) Whole-cell patch recording of a CSF-c neuron, showing mechanosensitivity to a fluid-pulse (20 p.s.i., 80 ms). ( b ) The same neuron firing spontaneous action potentials in control conditions (pH 7.4) in the presence of gabazine (20 μM), AP5 (100 μM) and NBQX (40 μM). ( c – e ) Lowering pH to 6.9 increased action potential frequency ( c ; red), and more so at pH 6.5 ( e ; orange) Also, the membrane potential was depolarized by ∼5 mV at lowered pH. ( f ) Action potential frequency during 1 min in CSF-c neurons at pH 7.4, 6.9 and 6.5, respectively. The values are means±s.e.m., normalized to basal activity at pH 7.4 ( n =15). Student's t -test: *** P <0.001, significant difference compared with control, pH 7.4. ( g ) Cell-attached patch recording of a CSF-c neuron showing the response to fluid-pulse stimulation. ( h – j ) This mechanosensitive CSF-c neuron in addition responded by increased action potential frequency to lowered pH (6.9), also evident in the cell-attached recording configuration. ( k – m ) In addition, subthreshold depolarizing receptor potentials appeared with lowered pH (pH 6.9).

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, Membrane, 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 ) 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

Journal: Frontiers in Pharmacology

Article Title: Therapeutic effects of traditional Chinese medicine Hua-Feng-Dan in a rat model of ischemic stroke involve renormalization of gut microbiota

doi: 10.3389/fphar.2025.1485340

Figure Lengend Snippet: Hua-Feng-Dan ameliorates the size of the brain infarct and the neurological impairments. Animals were subjected to middle cerebral artery occlusion or sham surgery, then left untreated (MCAO, Sham) or treated with nimodipine (NMDP) or Hua-Feng-Dan at a low dose (HFD-L, 0.162 g/kg), intermediate dose (HFD-M, 0.324 g/kg) or high dose (HFD-H, 0.648 g/kg). (A) Representative photographs of ischemic halves of brains after TTC staining. (B) Extent of cerebral infarction (n = 3). (C) Brain water content (n = 4). (D) Neurological score (n = 20). Quantitative data are mean ± SD. *** P < 0.001 vs. Sham group; ## P < 0.01, ### P < 0.001 vs. MCAO group; based on one-way ANOVA and Tukey’s multiple-comparisons post hoc test.

Article Snippet: Rats were randomized to receive, by oral gavage, saline instead of drug treatment or one of the following drugs dissolved in saline: the calcium channel blocker nimodipine (NMDP, batch 211274, Yabao Pharmaceutical, Shanghai, China), which is widely used to ischemic cerebrovascular disease; and Hua-Feng-Dan.

Techniques: Staining

Journal: Frontiers in Pharmacology

Article Title: Therapeutic effects of traditional Chinese medicine Hua-Feng-Dan in a rat model of ischemic stroke involve renormalization of gut microbiota

doi: 10.3389/fphar.2025.1485340

Figure Lengend Snippet: Hua-Feng-Dan alleviates histopathology injury and apoptosis of nerve cells after ischemic stroke in rats. Animals were subjected to middle cerebral artery occlusion or sham surgery, then left untreated (MCAO, Sham) or treated with nimodipine (NMDP) or Hua-Feng-Dan at a low dose (HFD-L, 0.162 g/kg), intermediate dose (HFD-M, 0.324 g/kg) or high dose (HFD-H, 0.648 g/kg). (A) Thin sections of brain tissue after hematoxylin-eosin staining (n = 3). Blue arrows represent macrophage infiltration or vascular stasis; red arrows, necrotic neurons or pyramidal cells; green arrows, glial cell proliferation; yellow arrows, vacuoles or septa; and black arrows, crumpled neurons or pyramidal cells. Scale bar, 100 μm. (B) Thin sections of brain tissue after terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining (n = 3). Scale bar, 100 μm. (C) TUNEL positive cells density (n = 3).

Article Snippet: Rats were randomized to receive, by oral gavage, saline instead of drug treatment or one of the following drugs dissolved in saline: the calcium channel blocker nimodipine (NMDP, batch 211274, Yabao Pharmaceutical, Shanghai, China), which is widely used to ischemic cerebrovascular disease; and Hua-Feng-Dan.

Techniques: Histopathology, Staining, End Labeling, TUNEL Assay

Journal: Frontiers in Pharmacology

Article Title: Therapeutic effects of traditional Chinese medicine Hua-Feng-Dan in a rat model of ischemic stroke involve renormalization of gut microbiota

doi: 10.3389/fphar.2025.1485340

Figure Lengend Snippet: Hua-Feng-Dan restores the integrity of the intestinal barrier after ischemic stroke in rats. Animals were subjected to middle cerebral artery occlusion or sham surgery, then left untreated (MCAO, Sham) or treated with a high dose of Hua-Feng-Dan (HFD, 0.648 g/kg) and nimodipine (NMDP). (A) Representative photographs of colon H&E staining (n = 3). Red arrows indicate the presence of lymphocytic infiltration; blue arrows, edema or edematous mucosal epithelial cells; black arrows, necrosis of mucosal epithelial cells; yellow arrows, dilated intestinal glands; and purple arrows, vasodilation. Scale bar = 200 and 100 μm. (B–D) Comparison of levels of three indices of intestinal barrier permeability: lipopolysaccharide (LPS), diamine oxidase (DAO) and d -lactate ( d -LA). (E–G) Comparison of levels of three pro-inflammatory factors: tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. (H) Comparison of levels of total cholesterol (T-CHO) as an index of dyslipidemia. (I, J) Comparison of levels of oxidative stress factors total superoxide dismutase (T-SOD) and malondialdehyde (MDA). Data are mean ± SD, n = 6 (excluding H&E staining). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. MCAO group (based on one-way ANOVA and Tukey’s multiple-comparisons post hoc test).

Article Snippet: Rats were randomized to receive, by oral gavage, saline instead of drug treatment or one of the following drugs dissolved in saline: the calcium channel blocker nimodipine (NMDP, batch 211274, Yabao Pharmaceutical, Shanghai, China), which is widely used to ischemic cerebrovascular disease; and Hua-Feng-Dan.

Techniques: Staining, Comparison, Permeability