Nvestigated synaptic plasticity in NAG neurons through the first 5 months of the animal’s life. Finally, we examined the effects of diet-induced obesity (DIO) in the organization of EPSCs and IPSCs onto NAG neurons. Our results support the idea that glutamatergic and GABAergic synaptic inputs onto NAG neurons are rapidly restructured to accommodate energy demands throughout the animals’ life.Baquero et al. ?Synaptic Distribution in Arcuate Nucleus NeuronsJ. Neurosci., June 3, 2015 ?35(22):8558 ?8569 ?Materials and MethodsAnimals. NPYhrGFP mice were purchased from The Jackson Laboratory for breeding. Litters of six to nine pups were born normally, and pups were genotyped to identify expression of green fluorescent protein (GFP) under the control of the mouse NPY promoter. NPYhrGFP mice were used in all Mikamycin IA site experiments and were weaned at P21. After weaning, only male mice were used in our studies. Mice were group-housed (4 per cage) and maintained under a 12 h light/dark cycle (lights on 7:00 A.M.). Standard chow diet (Purina lab chow no. 5001) and water were available ad libitum. To generate DIO mice, a separate cohort of NPYhrGFP mice were fed ad libitum with 60 High Fat Diet (HFD; Research Diets; catalog #D12492) after P30 for a minimum of 12 weeks (Lee et al., 2013). Animals for all studies were killed 1 h after lights on. Animal procedures were approved by the Oregon Health and Science University Institutional Animal Care and Use Committee. Drugs. Biocytin and GABA were purchased from Sigma-Aldrich. Bicuculline, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino5-phosphonovaleric acid (APV), baclofen were obtained from Tocris Bioscience, and tetrodotoxin (TTX) from Alomone Labs. Electrophysiology. All recordings were performed in NPY-GFP neurons in the ARH at the following ages: P13 15, P21 23, 9 ?0 weeks, and 17?8 weeks. Coronal slices containing ARH were prepared as previously described (Qiu et al., 2010). Briefly, brain slices (300 M) containing ARH were maintained with constant flow (1? ml/min) of artificial CSF (aCSF) containing the following (in mM): 124 NaCl, 5 KCl, 2.6 NaH2PO4, 1 MgSO4, 1 CaCl2, 26 NaHCO3, 10 HEPES, 10 glucose; oxygenated (95 O2, 5 CO2) osmolarity 305 at 32?3 . IPSCs were recorded at a holding potential of 60 mV in whole-cell patch-clamp mode. Pipettes have a resistance of 2?4 M when filled with a cesium chloride-based solution containing the following (in mM): 140 CsCl, 5 MgCl2, 1 BAPTA, 5 ATP, 0.3 GTP, biocytin 2 , pH 7.35 with CSOH, osmolarity 295. APV (50 M) and CNQX (10 M) were added into the bath to isolate IPSCs. TTX (1 M) was added into the bath to isolate miniature IPSCs (mIPSCs). EPSCs were recorded with a cesium-methanesulfonate-based solution containing the following (in mM): 125 CsMeSO3, 10 CsCl, 5 NaCl, 2 MgCl2, 10 HEPES, 1 EGTA, 5 ATP, 0.3 GTP, biocytin 2 , pH 7.35 with CSOH, osmolarity 295. Pipettes have a resistance of 2?4 M and neurons were held at 60 mV in whole-cell patch-clamp mode. Bicuculline (5 M) was added into the bath to block IPSCs. TTx (1 M) was added into the bath to isolate miniature EPSCs (mEPSCs). Series resistance values were generally 20 M during the experiments. Data analysis was performed using clampfit 10 and only synaptic events 5 pA were analyzed. Following IPSC or EPSC recordings, pipettes were purchase Cyclosporine slowly withdrawn from the neurons for postrecording immunohistochemistry studies. For current-clamp experiments, microelectrodes had resistances of 3? M and were filled with.Nvestigated synaptic plasticity in NAG neurons through the first 5 months of the animal’s life. Finally, we examined the effects of diet-induced obesity (DIO) in the organization of EPSCs and IPSCs onto NAG neurons. Our results support the idea that glutamatergic and GABAergic synaptic inputs onto NAG neurons are rapidly restructured to accommodate energy demands throughout the animals’ life.Baquero et al. ?Synaptic Distribution in Arcuate Nucleus NeuronsJ. Neurosci., June 3, 2015 ?35(22):8558 ?8569 ?Materials and MethodsAnimals. NPYhrGFP mice were purchased from The Jackson Laboratory for breeding. Litters of six to nine pups were born normally, and pups were genotyped to identify expression of green fluorescent protein (GFP) under the control of the mouse NPY promoter. NPYhrGFP mice were used in all experiments and were weaned at P21. After weaning, only male mice were used in our studies. Mice were group-housed (4 per cage) and maintained under a 12 h light/dark cycle (lights on 7:00 A.M.). Standard chow diet (Purina lab chow no. 5001) and water were available ad libitum. To generate DIO mice, a separate cohort of NPYhrGFP mice were fed ad libitum with 60 High Fat Diet (HFD; Research Diets; catalog #D12492) after P30 for a minimum of 12 weeks (Lee et al., 2013). Animals for all studies were killed 1 h after lights on. Animal procedures were approved by the Oregon Health and Science University Institutional Animal Care and Use Committee. Drugs. Biocytin and GABA were purchased from Sigma-Aldrich. Bicuculline, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino5-phosphonovaleric acid (APV), baclofen were obtained from Tocris Bioscience, and tetrodotoxin (TTX) from Alomone Labs. Electrophysiology. All recordings were performed in NPY-GFP neurons in the ARH at the following ages: P13 15, P21 23, 9 ?0 weeks, and 17?8 weeks. Coronal slices containing ARH were prepared as previously described (Qiu et al., 2010). Briefly, brain slices (300 M) containing ARH were maintained with constant flow (1? ml/min) of artificial CSF (aCSF) containing the following (in mM): 124 NaCl, 5 KCl, 2.6 NaH2PO4, 1 MgSO4, 1 CaCl2, 26 NaHCO3, 10 HEPES, 10 glucose; oxygenated (95 O2, 5 CO2) osmolarity 305 at 32?3 . IPSCs were recorded at a holding potential of 60 mV in whole-cell patch-clamp mode. Pipettes have a resistance of 2?4 M when filled with a cesium chloride-based solution containing the following (in mM): 140 CsCl, 5 MgCl2, 1 BAPTA, 5 ATP, 0.3 GTP, biocytin 2 , pH 7.35 with CSOH, osmolarity 295. APV (50 M) and CNQX (10 M) were added into the bath to isolate IPSCs. TTX (1 M) was added into the bath to isolate miniature IPSCs (mIPSCs). EPSCs were recorded with a cesium-methanesulfonate-based solution containing the following (in mM): 125 CsMeSO3, 10 CsCl, 5 NaCl, 2 MgCl2, 10 HEPES, 1 EGTA, 5 ATP, 0.3 GTP, biocytin 2 , pH 7.35 with CSOH, osmolarity 295. Pipettes have a resistance of 2?4 M and neurons were held at 60 mV in whole-cell patch-clamp mode. Bicuculline (5 M) was added into the bath to block IPSCs. TTx (1 M) was added into the bath to isolate miniature EPSCs (mEPSCs). Series resistance values were generally 20 M during the experiments. Data analysis was performed using clampfit 10 and only synaptic events 5 pA were analyzed. Following IPSC or EPSC recordings, pipettes were slowly withdrawn from the neurons for postrecording immunohistochemistry studies. For current-clamp experiments, microelectrodes had resistances of 3? M and were filled with.
