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Neuropeptide Y-mediated sex- and afferent-specific neurotransmissions contribute to sexual dimorphism of baroreflex afferent function

Background Molecular and cellular mechanisms of neuropeptide-Y (NPY)-mediated gender-difference in blood pressure (BP) regulation are largely unknown. Methods Baroreceptor sensitivity (BRS) was evaluated by measuring the response of BP to phenylephrine/nitroprusside. Serum NPY concentration was determined using ELISA. The mRNA and protein expression of NPY receptors were assessed in tissue and single-cell by RT-PCR, immunoblot, and immunohistochemistry. NPY was injected into the nodose while arterial pressure was monitored. Electrophysiological recordings were performed on nodose neurons from rats by patch-clamp technique. Results The BRS was higher in female than male and ovariectomized rats, while serum NPY concentration was similar among groups. The sex-difference was detected in Y1R, not Y2R protein expression, however, both were upregulated upon ovariectomy and canceled by estrogen replacement. Immunostaining confirmed Y1R and Y2R expression in myelinated and unmyelinated afferents. Single-cell PCR demonstrated that Y1R expression/distribution was identical between A- and C-types, whereas, expressed level of Y2R was ∼15 and ∼7 folds higher in Ah- and C-types than A-types despite similar distribution. Activation of Y1R in nodose elevated BP, while activation of Y2R did the opposite. Activation of Y1R did not alter action potential duration (APD) of A-types, but activation of Y2R- and Y1R/Y2R in Ah- and C-types frequency-dependently prolonged APD. N-type ICa was reduced in A-, Ah- and C-types when either Y1R, Y2R, or both were activated. The sex-difference in Y1R expression was also observed in NTS. Conclusions Sex- and afferent-specific expression of Neuropeptide-Y receptors in baroreflex afferent pathway may contribute to sexual-dimorphic neurocontrol of BP regulation.


Surgical ovariectomy
The surgery was performed following protocols described in details previously [1]. Briefly, anesthetized animals (combination of xylazine 10 mg/kg and ketamine 75 mg/kg) were placed in a lateral position, and both flanks were shaved and cleaned using chlorhexidine scrub and disinfected with 70% ethanol and povidone-iodine (7.5%). A 2.0-cm incision was made on the left lateral side along a line spanning from the 2 nd to the 5 th lumbar vertebra, using a scalpel blade. The left ovary and associated fat were located and externalized by gentle retraction. After removal of the ovary, the peritoneal cavity, muscle layers, and skin were closed successively with 4-0 absorbable sutures and then penicillin (80000 Units) was given via intramuscular injection. The same procedure was repeated for removal of the right ovary. After recovering from anesthesia, the animals were monitored for at least 30 min to ensure that there was no bleeding from the surgery, and then were returned to the animal facility. Four weeks after ovariectomy, a part of them were administrated 17β-estradiol (17β-E2) via subcutaneous injection (10 μg/kg, once daily) delivered in 200 μl sesame oil [2] for another 3 weeks, which were sacrificed for experimental use.

Arterial baroreflex sensitivity
The arterial was cannulated to connect the physiological pressure transducer (AD Instruments MLT 844, Norway), which was used for measuring the change of mean arterial blood pressure (MAP) and heart rate (HR) automatically at the same time. Phenylephrine (PE; Sigma Chemical) or sodium nitroprusside (SNP; Sigma Chemical) was injected by indwelling venous catheter to induce transient increase or decrease in BP with three different doses (2, 5, and 10 μg/kg) respectively. After the HR and arterial blood pressure responses to the first drug reached plateau, the second drug would be administrated. The calculation method of baroreflex sensitivity was described as Min Lin [3]. The maximal change of HR (ΔHR) and the value between the maximal MABP and the baseline MABP (ΔMABP) were recorded for all the groups. The averaged ratio of HR and MABP (ΔHR/ΔMABP) was designed as the symbol of BRS, which was applied to each dose of PE and SNP. All the data processing were applied by using the software of Labchart 7.

Measurement of serum NPY concentration
After animals were anaesthetized, the blood of the three group rats were collected immediately and allowed samples to clot at 4°C overnight before centrifugation for 20 minutes at 1000×g. Then the supernatant were collected to measure the NPY concentration by using NPY enzymelinked immunosorbent Assay Kit (Cloud-clone Corp., Houston, USA) according to the manufacturer's instructions.

Aortic depressor nerve (ADN) labeling
The fluorescent labeling of ADN with dye Dil was performed and the procedures were described as previously reported [4][5][6]. Briefly, after the anesthesia with the cocktail mentioned above and proper treatments of the skin on the surgical area, a ~2 cm incision was made under aseptic conditions along the left ventral side of the neck. A blunt dissection of the underlying musculature exposed the left carotid artery and surrounding nerve fibers. Under higher magnification, the left ADN, which exclusively contains baroreceptor fibers arising from the aortic arch, was identified. The ADN was separated from the Vagus and sympathetic nerves and placed in a 5 mm long sterile silicon trough. A few crystals of the lipophilic fluorescent dye DiI was placed on the ADN. The nerve, dye crystals and trough were coated with ~0.3 ml of a peripheral nerve encapsulant (Kwik-Sil, WPI). The area was rinsed with sterile saline and the skin was closed using vicryl suture. The animal fully recovered before returning to the animal facility. At least 4 weeks passed before the animal was considered fully healed and available for experimentation.

Western blot analysis of Y1R and Y2R protein
Each group consisted of ten animals that were euthanized with an excessive dose of pentobarbital sodium. Both sides of the nodose ganglions were removed immediately and frozen on dry ice. Ten pairs of tissue were collected for Western blots. The tissues were lysed by using lysis buffer (Beyotime Biotechnology Institute, China) containing 1% protease inhibitor solution (Beyotime). The lysate was centrifuged for 15 min to collect supernatant. The protein concentration was determined using BCA Protein Assay Kit (Beyotime Biotechnology Institute). The samples were boiled for 5 min, followed by loading on 12% SDS-PAGE gel (100 μg of protein, 15 μl per well) for electrophoresis using 110 V for 90 min. The protein on the gel was transferred onto NC membranes at 300 mA for 75 min, which was blocked by 5% nonfat dry milk diluted by PBS at room temperature for 3 hours. The membrane was probed with primary antibody (Y1 receptor rabbit polyclonal antibody, Alomone, 1:200 or Y2 receptor rabbit polyclonal antibody, Alomone, 1:200) at 4°C overnight and secondary antibody (goat anti-rabbit, Abcam, 1:8000) for 60 min at room temperature. GAPDH was used as the internal control. Bound bands were visualized and analyzed using Odyssey Infrared Imaging System (LI-COR Biosciences) and Odyssey v1.2 software.

Real-time quantitative PCR in tissue level
For SYBR Green RT-PCR, 4 SD rats were used to harvest total nodose ganglion mRNAs in each group. Total RNAs were extracted using the TRIzol® Reagent (Invitrogen) according to the manufacturer's instructions. The cDNAs were synthesized using the Reverse Transcription Kit (Applied Biosystems). Quantitative PCR reactions were run on an ABI 7500 fast Real-Rime PCR System (Applied Biosystems). The primers (Invitrogen) were used as follows: 5'-GGAGACCATGTGCAAACTGA-3' (forward) and 5'-CACCCTCTTGGGTTGATGAT-3' (reverse) for Y 1 R; 5'-AGCCTTTCCACCCTGCTAAT -3' (forward) and 5'-CCTTCGCTGATGGTAATGGT-3' (reverse) for Y 2 R. GAPDH was used as an internal control. The primer sequences for GAPDH were: 5'-AA GAAGGTGGTGAAGCAGGC -3' (forward) and 5'-TCC ACCACCCAGTTGCTGTA-3' (reverse). The 2 −ΔΔCt method was applied for the data analysis and the data were normalized and converted into relative mRNA expression.
For single-cell RT-PCR, after recording the AP for identifying the subtypes of VGNs as mentioned above, individual identified neuron was aspirated into a largediameter pipette (3-7 μm) with ~2 μl of DEPC treated water in the tip and then immediately transferred into 5 μl of DEPC treated water at the bottom of the 0.2 ml EP tube, which was then stored at -80°C immediately for further analyze. SuperScript™ III CellsDirect cDNA Synthesis System (Invitrogen) was used to obtain cDNA. The other experimental conditions and analytic method are as the same with the tissue RT-PCR. The mRNA expression is based on the threshold cycles (Ct) and the appearance of GAPDH Ct proved the cells are successfully transferred into the EP tube.

Immunohistochemical analysis
Isolated one vagal ganglion of each group and section the embedded vagal ganglion into a thickness (8 μm) using the cryostat (LEICA cm 1850). Sections were placed onto glass slides and fixed with pre-cooled 4% paraformaldehyde for 10 min. Rinse the slides two times in phosphate buffered saline (PBS). Pour off the fixative and allow PBS to evaporate from the sections for 20 min at room temperature or stored at -80°C for later use. Sections were selected and rinsed in PBS for 3 min immediately, and incubated in PBS containing 1% BSA and 0.4% Triton X-100 (Sigma, St. Louis, MO, USA) for 1 h at 37°C. After blocking in 10% normal goat serum at 37°C for 2 h, sections were incubated with primary antibodies consisting Y1R (Alomone, 1:100) or Y2R antibody (Alomone, 1:100) in PBS for 2 h, Subsequently further incubated with HCN1 (Abcam, 1:100) diluted in PBS from 594 rabbit and 488 mouse (Life Molecular Probe, 1:100) for 1 h at 37°C. Sections were then washed three final times in PBS briefly and coverslipped using anti-quenching agent with DAPI diluted by PBS. Imaging was under confocal microscope (Olympus Fluo-view 300).

Direct injection of NPY and its agonists into nodose ganglia
Rat were anesthetized (initial injection xylazine: ketamine 10: 75 mg/kg) and the femoral arterial was cannulated to connect the physiological pressure transducer (AD Instruments MLT 844, Norway), which was used to record the baseline arterial blood pressure before neck surgery. Rats were placed in a supine position and the neck was shaved and disinfected with 70% ethanol and povidone-iodine (7.5%). A 4.0 cm midline longitudinal cut was made and isolated one side of nodose ganglion under stereomicroscopy (×40) to make sure separate the Vagus nerve from the carotid, which was cleaned to avoid any connective tissue to reducing resistance when injecting (Supplementary Figure S1 NG injection A). During recordings, surgical stage of anesthesia was monitored by tail pinch, and if necessary, rats were re-injected with anesthetic (xylazine: ketamine 5: 75 mg/kg). After the surgery baseline MABP was stable, placed a clamp to apply slight tension on the Vagus nerve and punctured the nodose ganglion using a precision glass syringe (HAMILTON) affixed with a 30G half -inch stainless steel syringe needle with a 35° beveled tip with 2 μl grugs in saline. Rapid drop and recovery of blood pressure was detected if we have punctured vehicle into the nodose ganglion (Supplementary Figure  6A-top) and nerves were functionally intact. To testify that nodose ganglion was not punctured through it, NG was embedded after injected Evans blue and placed in a -80°C freezer for 1 hour before being sectioned into a thickness (20 μm) using the cryostat (LEICA cm 1850). Images were captured by scanning electron microscopy (Nikon, ECLIPSE 80) directly to observe the pinhole size (Supplementary Figure S1 NG injection B-D). Nodose injection recording were repeated to analysis the value of ΔMABP by using the software of Labchart 7 to detect the drugs effect.

Isolated neurons for electrophysiology
Isolation procedure was described in details previously [7][8][9]. Briefly, after both sides of nodose ganglia of female rat were surgically removed, they were cut into six pieces of each ganglion and then placed in cold nodose complete medium (NCM) composed of Mito™ + serum extender (v/v, 1:20, Collaborative Biomedical Products, Bedford, USA), Dulbecco's modified Eagle's medium F-12 (Gibco, Grand Island, USA) supplemented with 5% fetal bovine serum (HyClone, Logan, USA) and 1% penicillin-streptomycin-neomycin antibiotic mixture (PSN; Gibco, Grand Island, USA). The ganglia were transferred to an the 1 st enzyme solution containing 20 units/ml of Papain (Worthington) in Earle's balance salt solution (Sigma, San Louis, USA) at 37°C for 20-22 min. The enzyme solution was replaced with the 2 nd enzyme solution containing 2 mg/ml of Dispase II (Roche) and 1 mg/ml of Collagenase type-II (Worthington) in Earle's balance salt solution (Sigma) at 37 °C for additional 30-35 min depending upon the animal age. The enzyme solution was replaced with NCM containing 1.5 mg/ml albumin (Bovine, Sigma) and the ganglia were dissociated by trituration with fire-polished pipettes. Cell suspension was placed on poly-D-lysine (Sigma, San Louis, USA)coated glass coverslips and cultured at 37°C moisture environment for at least 4 h before recording.
Whole-cell current-and voltage-clamp patch recordings [8,9] were conducted using an Amplifier 200B or 700B (Axon Instruments, Union City, USA). Borosilicate glass pipettes (BF150-86-10; Sutter, Novato, USA) were pulled (P-97, Sutter, USA) and polished (F-83, Narishige, Japan) down to a resistance of 1.2-1.8 MΩ. Following correction for all offsets, a giga-ohm seal was formed and the pipette capacitance was compensated. Total cell capacitance (30-50 pF) and electrode access resistance (3)(4)(5) were also compensated (60-80%). Recordings were low pass filtered to 10 KHz and digitized at 50 KHz. Experimental protocols, data collection, and preliminary analysis were performed using pCLAMP 10.3 and Digidata 1440A (Axon Instruments, Union City, USA). Corrections for liquid junction potentials were taken into consideration before final data analysis. The single and repetitive AP firings elicited by brief pulse and step depolarization current injects were collected, respectively, from each tested neurons for neuronal identification and discharge profile analysis before and after applications [9].

Vagus-nodose slice for electrophysiology
Slices of nodose ganglia with intact vagal axons were prepared in a manner previously described [8,9]. Briefly, adult SD rats (250-300 g) of either gender were used for the slice preparation. The unrestrained rats were placed in an airtight induction chamber for inhalation of the anesthetic Metofane (Methoxyflurane, Schering-Plough Animal Health Corp, Kenilworth, NJ, USA). Upon lack of reflex response to tail pinch the animals were immediately sectioned at the mid-auxiliary region, preserving at least 2 cm of the Vagus nerve. The nodose ganglia with attached Vagus were excised under stereomicroscopy (×40). The tissue was immediately placed in chilled (4°C) recording solution. Slicing exposed the interior of the ganglion capsule and the tissue was placed in a solution of Earle's balance salt solution (Sigma) containing type II Collagenase (1.0 mg/ml) at 37°C for 40 -45 min followed by the solution containing Trypsin-3X (5 mg/ml) for another 20 -22 min. The tissue was moved to the bath perfusion chamber and allowed to recover for 1 h prior to recording. The identical wholecell patch techniques were used as described above. After tightened seal, electric evoked currents by directly stimulation of Vagus [10] was collected under cell-attach configuration in AP bath solution with peptide solution for Ca 2+ current recording, and neuron type could be identified upon the afferent fiber CV. And then whole-cell Ca 2+ currents could be collected by completely bath perfusion with Ca 2+ current recording solution. Cell was held at −100 mV and voltage was stepped from−80 mV to +30 mV for 400 ms with 10 mV increments and 1 s interval between steps.

Action potential and Ca 2+ current recordings
Whole-cell patch [8,9] was conducted using an Amplifier 200B or 700B (Axon Instruments, Union City, USA). Borosilicate glass pipettes (BF150-86-10; Sutter, Novato, USA) were pulled (P-97, Sutter, USA) and polished (F-83, Narishige, Japan) down to a resistance of 1.2-1.8 MΩ. Following correction for all offsets, a giga-ohm seal was formed and the pipette capacitance was compensated. Total cell capacitance (30-50 pF) and electrode access resistance (3)(4)(5) were also compensated (60-80%). For isolated neuron identification and testing the effect of Y1R or Y2R activation on discharge profiles, a single AP and repetitive discharge were elicited under current-clamp mode by a brief current pulse and step depolarization. After control recordings, drug was applied through bath perfusion at flow rate no more than 1 ml/min and induced change in membrane potential was recorded. Recordings were low pass filtered to 10 KHz and digitized at 50 KHz. Experimental protocols, data collection, and preliminary analysis were performed using pCLAMP 10.3 and Digidata 1440A (Axon Instruments, Union City, USA). Corrections for liquid junction potentials were taken into consideration before final data analysis. For Ca 2+ current recording, slice preparation was employed and the afferent fiber types were firstly identified by conduction velocity (CV) in cell-attached mode with AP recording solution followed by completely perfusion with extracellular solution for Ca 2+ recording, and then ruptured the membrane and Ca 2+ current recording [8,10].