A Novel Excitatory Paraventricular Nucleus to AgRP Neuron Circuit that Drives Hunger methods
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Characterization of mice
Pdyn-ires Cre, Avp-ires-Cre, Crh-ires-Cre, and Pacap-ires-Cre mice were crossed to R26-loxSTOPlox-L10-GFP mice, sacrificed and sectioned at 30 µm. Brain sections were washed in 0.1 M phosphate-buffered saline with Tween 20, pH 7.4 (PBST, 2 changes) and then incubated in the primary antiserum (anti-GFP, Abcam (1:1000), rabbit anti-hrGFP. After several washes in PBS, sections were incubated in Alexa fluorophore secondary antibody (Molecular Probes, 1:200) for 2 hours at room temperature. After several washes in PBS, sections were mounted onto gelatin-coated slides and fluorescent images were captured with Olympus VS120 Slide Scanner microscope.
Characterization of mice
In situ mRNA hybridization experiments were performed for Pdyn-ires-Cre, Trh-ires-Cre and Pacap-ires-Cre mice (details below). Antibody staining experiments were performed for Avp-ires-Cre and Crh-ires-Cre mice. Briefly, Avp-ires-Cre; R26-loxSTOPlox-L10-GFP mice were co-localized with anti-AVP, Sigma (1:500), rabbit anti-AVP and anti-GFP, Abcam (1:1000), chicken anti-GFP. 48 hrs ICV colchicine treatment (Sigma, 10 ug), Crh-ires-Cre; R26-loxSTOPlox-L10-GFP mice were co-localized with anti-CRF, Peninsula Laboratories (1:2500), rabbit anti-CRF and anti-GFP, Abcam (1:1000), chicken anti-GFP.
Viral injections
Stereotaxic injections were performed as previously described. Mice were anesthetized with xylazine (5 mg/kg) and ketamine (75 mg/kg) diluted in saline (350 mg/kg) and placed into a stereotaxic apparatus (KOPF Model 963). After exposing the skull via small incision, a small hole was drilled for injection. A pulled-glass pipette with 20-40 mm tip diameter was inserted into the brain and virus was injected by an air pressure system. A micromanipulator (Grass Technologies, Model S48 Stimulator) was used to control injection speed at 25 nl/min and the pipette was withdrawn 5 min after injection. For retrograde rabies tracing, AAV-FLEX-TVA-mCherry, serotype 8 (titer 1.1 × 10 12 genomes copies per ml) and AAV-FLEX-RG, serotype 8 (titer 1.4 × 10 12 genomes copies per ml) were injected unilaterally into the ARC (200 nl, coordinates, bregma: AP:-1.50 mm, DV:-5.80 mm, L: -0.20 m...
Electrophysiology and circuit mapping
To assess the effect of CNO on to TRH and PACAP neurons, 5- to 7-week-old Pacap-ires-Cre and Trh-ires-Cre mice were injected with AAV8-DIO-hM3Dq-mCherry into the PVH 2-3 weeks prior to recording. CNO was applied to bath solution through perfusion as previously reported. After acquisition of stable whole-cell recordings for 2-5 min, aCSF solution containing 5 µM CNO was perfused into the brain slice preparation.
Electrophysiology and circuit mapping
For brain slice preparation, 6-10 weeks old mice were anesthetized with isoflurane before decapitation and removal of the entire brain. Brains were immediately submerged in ice-cold, carbogen-saturated (95% O2, 5% CO2) high sucrose solution (238 mM sucrose, 26 mM NaHCO3, 2.5 mM KCl, 1.0 mM NaH2PO4, 5.0 mM MgCl2, 10.0 mM CaCl2, 11 mM glucose). Then, 300 µM thick coronal sections were cut with a Leica VT1000S Vibratome and incubated in oxygenated aCSF (126 mM NaCl, 21.4 mM NaHCO3, 2.5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2, 10 mM glucose) at 34°C for 30 minutes. Then, slices were maintained and recorded at room temperature (20-24°C). The intracellular solution for voltage clamp recording contained the following (in mM): 140 CsCl, 1 BAPTA, 10 HEPES, 5 MgCl2, 5 Mg-ATP, 0.3 Na2GTP, and 10 lidocaine N-ethyl bromide (QX-314), pH 7.35 and 290 mOsm. The intrace...
Electrophysiology and circuit mapping
To assess the effect of PACAP 1-38 (100 nM) and PACAP 6-38 (200 nM) onto AgRP neurons, we performed whole cell current clamp recordings on to 5-8 weeks old Npy-hrGFP mice. Synaptic blockers (1 mM kynurenate and 100 µM picrotoxin) were added in aCSF to synaptically isolate AgRP neurons.
Fos analysis
Animals ( Trh-ires-Cre; Npy-hrGFP; n=3 and Pacap-ires-Cre; Npy-hrGFP; n=3 mice), were handled for 10 consecutive days before the assay to reduce stress response, and then injected with CNO (0.3 mg kg -1; i.p.) at 9am. 150 min later, the animals were sacrificed with 7% chloral hydrate diluted in saline (350 mg/kg) for histological assay. The mice were perfused and brains were sectioned as described above. Assessment of Fos induction was performed using a previously developed method modified for fluorescent colocalization with hrGFP in AgRP neurons. Brain sections were processed for immunohistochemical detection of Fos and hrGFP and counting.
Fos analysis
Brain sections were washed in 0.1 M phosphate-buffered saline with Tween 20, pH 7.4 (PBST, 2 changes) and then incubated in the primary antiserum (anti-GFP, Abcam (1:1000), rabbit anti-hrGFP and anti-Fos, Calbiochem (1:10000). After several washes in PBS, sections were incubated Alexa fluorophore secondary antibodies (Molecular Probes, 1:200) for 2 hours at room temperature. After several washes in PBS, sections were mounted onto gelatin-coated slides and fluorescent 10X images were taken initially using a Zeiss AxioImager Z.1 microscope. Colocalization and quantification was further determined using fluorescent 20X images taken using a Zeiss AxioImager Z.1 microscope. Data are expressed as the percentage of all AgRP neurons (i.e., all hrGFP-positive neurons) that were double-positive for Fos and hrGFP.
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Pdyn-ires Cre, Avp-ires-Cre, Crh-ires-Cre, and Pacap-ires-Cre mice were crossed to R26-loxSTOPlox-L10-GFP mice, sacrificed and sectioned at 30 µm. Brain sections were washed in 0.1 M phosphate-buffered saline with Tween 20, pH 7.4 (PBST, 2 changes) and then incubated in the primary antiserum (anti-GFP, Abcam (1:1000), rabbit anti-hrGFP. After several washes in PBS, sections were incubated in Alexa fluorophore secondary antibody (Molecular Probes, 1:200) for 2 hours at room temperature. After several washes in PBS, sections were mounted onto gelatin-coated slides and fluorescent images were captured with Olympus VS120 Slide Scanner microscope.
In situ mRNA hybridization experiments were performed for Pdyn-ires-Cre, Trh-ires-Cre and Pacap-ires-Cre mice (details below). Antibody staining experiments were performed for Avp-ires-Cre and Crh-ires-Cre mice. Briefly, Avp-ires-Cre; R26-loxSTOPlox-L10-GFP mice were co-localized with anti-AVP, Sigma (1:500), rabbit anti-AVP and anti-GFP, Abcam (1:1000), chicken anti-GFP. 48 hrs ICV colchicine treatment (Sigma, 10 ug), Crh-ires-Cre; R26-loxSTOPlox-L10-GFP mice were co-localized with anti-CRF, Peninsula Laboratories (1:2500), rabbit anti-CRF and anti-GFP, Abcam (1:1000), chicken anti-GFP.
Stereotaxic injections were performed as previously described. Mice were anesthetized with xylazine (5 mg/kg) and ketamine (75 mg/kg) diluted in saline (350 mg/kg) and placed into a stereotaxic apparatus (KOPF Model 963). After exposing the skull via small incision, a small hole was drilled for injection. A pulled-glass pipette with 20-40 mm tip diameter was inserted into the brain and virus was injected by an air pressure system. A micromanipulator (Grass Technologies, Model S48 Stimulator) was used to control injection speed at 25 nl/min and the pipette was withdrawn 5 min after injection. For retrograde rabies tracing, AAV-FLEX-TVA-mCherry, serotype 8 (titer 1.1 × 10 12 genomes copies per ml) and AAV-FLEX-RG, serotype 8 (titer 1.4 × 10 12 genomes copies per ml) were injected unilaterally into the ARC (200 nl, coordinates, bregma: AP:-1.50 mm, DV:-5.80 mm, L: -0.20 mm) of 5-6 weeks old mice. 21 days later SADΔG-EGFP (EnvA) rabies (titer 10 7 ) was unilaterally injected into the ARC (400 nl, coordinates, bregma: AP:-1.50 mm, DV:-5.80 mm, L: -0.20 mm). For electrophysiology experiments, AAV-DIO-ChR2(H134R)-mCherry, serotype 8 (titer 1.3 × 10 12 genomes...
To assess the effect of CNO on to TRH and PACAP neurons, 5- to 7-week-old Pacap-ires-Cre and Trh-ires-Cre mice were injected with AAV8-DIO-hM3Dq-mCherry into the PVH 2-3 weeks prior to recording. CNO was applied to bath solution through perfusion as previously reported. After acquisition of stable whole-cell recordings for 2-5 min, aCSF solution containing 5 µM CNO was perfused into the brain slice preparation.
For brain slice preparation, 6-10 weeks old mice were anesthetized with isoflurane before decapitation and removal of the entire brain. Brains were immediately submerged in ice-cold, carbogen-saturated (95% O2, 5% CO2) high sucrose solution (238 mM sucrose, 26 mM NaHCO3, 2.5 mM KCl, 1.0 mM NaH2PO4, 5.0 mM MgCl2, 10.0 mM CaCl2, 11 mM glucose). Then, 300 µM thick coronal sections were cut with a Leica VT1000S Vibratome and incubated in oxygenated aCSF (126 mM NaCl, 21.4 mM NaHCO3, 2.5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2, 10 mM glucose) at 34°C for 30 minutes. Then, slices were maintained and recorded at room temperature (20-24°C). The intracellular solution for voltage clamp recording contained the following (in mM): 140 CsCl, 1 BAPTA, 10 HEPES, 5 MgCl2, 5 Mg-ATP, 0.3 Na2GTP, and 10 lidocaine N-ethyl bromide (QX-314), pH 7.35 and 290 mOsm. The intracellular solution for current clamp recordings contained the following (in mM): 128 K gluconate, 10 KCl, 10 HEPES, 1 EGTA, 1 MgCl2, 0.3 CaCl2, 5 Na2ATP, 0.3 NaGTP, adjusted to pH 7.3 with KOH.
To assess the effect of PACAP 1-38 (100 nM) and PACAP 6-38 (200 nM) onto AgRP neurons, we performed whole cell current clamp recordings on to 5-8 weeks old Npy-hrGFP mice. Synaptic blockers (1 mM kynurenate and 100 µM picrotoxin) were added in aCSF to synaptically isolate AgRP neurons.
Animals ( Trh-ires-Cre; Npy-hrGFP; n=3 and Pacap-ires-Cre; Npy-hrGFP; n=3 mice), were handled for 10 consecutive days before the assay to reduce stress response, and then injected with CNO (0.3 mg kg -1; i.p.) at 9am. 150 min later, the animals were sacrificed with 7% chloral hydrate diluted in saline (350 mg/kg) for histological assay. The mice were perfused and brains were sectioned as described above. Assessment of Fos induction was performed using a previously developed method modified for fluorescent colocalization with hrGFP in AgRP neurons. Brain sections were processed for immunohistochemical detection of Fos and hrGFP and counting.
Brain sections were washed in 0.1 M phosphate-buffered saline with Tween 20, pH 7.4 (PBST, 2 changes) and then incubated in the primary antiserum (anti-GFP, Abcam (1:1000), rabbit anti-hrGFP and anti-Fos, Calbiochem (1:10000). After several washes in PBS, sections were incubated Alexa fluorophore secondary antibodies (Molecular Probes, 1:200) for 2 hours at room temperature. After several washes in PBS, sections were mounted onto gelatin-coated slides and fluorescent 10X images were taken initially using a Zeiss AxioImager Z.1 microscope. Colocalization and quantification was further determined using fluorescent 20X images taken using a Zeiss AxioImager Z.1 microscope. Data are expressed as the percentage of all AgRP neurons (i.e., all hrGFP-positive neurons) that were double-positive for Fos and hrGFP.
Machine-readable layer
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"headline": "A Novel Excitatory Paraventricular Nucleus to AgRP Neuron Circuit that Drives Hunger",
"datePublished": "2014",
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