doi:10.1146/annurev.physiol.68.040504.094635. effect was dependent on glutamate release from afferent terminals and required presynaptic 5-HT3Rs. Decreasing the glucose concentration also decreased both basal and 5-HT3R agonist-induced increase in the frequency of spontaneous glutamate inputs onto NTS-CA neurons. Low glucose also blunted 5-HT-induced inward currents in nodose ganglia neurons, which are the cell body of vagal afferents. The effect of low glucose in both nodose ganglia cells and in NTS slices was mimicked Rabbit Polyclonal to GPR152 by the glucokinase inhibitor glucosamine. This study suggests that NTS-CA neurons are glucosensing through a presynaptic mechanism that is dependent on vagal glutamate release, 5-HT3R activity, and glucokinase. (NIH). Eight to sixteen-week-old mice (both males and females) were utilized for these studies. No difference was noted between male and female mice for the parameters analyzed and so the data were pooled. The hindbrain was removed and placed for 1 min in chilly (0C4C) artificial cerebral spinal fluid (aCSF). The trimming plane was aligned vertically, sectioning caudal to rostral RGFP966 when mounted in a vibrating microtome (Lieva VT-1000S). Coronal slices (250 m solid) from your TH-EGFP mice (Matsushita et al. 2002) were cut with RGFP966 a sapphire knife (Delaware Diamond Knives) and preserved the raphe nucleus and caudal NTS. This allowed us to conserve serotonergic inputs into the NTS. Slices were submerged in RGFP966 a perfusion chamber and all recording were performed at 31C35C. Whole cell recordings were made using an external aCSF bath answer made up of (in mM): 125 NaCl, 3 KCl, 1.2 KH2PO4, 1.2 MgSO4, NaHCO3, 10, 5, RGFP966 2, or 1 dextrose, and 2 CaCl2, bubbled with 95% O2-5% CO2; 30C34C; pH?=?7.3, adjusted to 300C310 mosmol using sucrose. Internal recording solution contained (in mM) 10 NaCl, 125 KCl, 11 EGTA, 1 CaCl2, 2 MgCl2, and 10 HEPES, pH?=?7.3, 295C300 mosmol. External bath answer was used in patch electrodes for cell-attached recordings. Neurons were recorded from NTS within 200 m rostral or caudal from obex and medial to the ST. Patch electrodes, 3C5 M, were guided to neurons using both fluorescence (FITC) and differential interference contrast (DIC) optics (Olympus BX51). Voltage clamp and current clamp recordings were made with an Axopatch 700B and pClamp10 software (Axon Devices). Only neurons not exceeding holding currents of 100 pA at holding potential (VH)?=??60 mV for the 10 min control period (input resistance 120 M) were studied further. Current clamp recordings were made at resting membrane potentials, and current injections were not used to hold the membrane at set potentials. Only neurons with an access resistance less than 20 M were used for analysis. All RGFP966 membrane potentials reported were corrected for junction potential (~14 mV). All drugs were obtained from Tocris Cookson or Sigma Aldrich. Cell cultures. Nodose ganglion neurons were dissected from male and female mice (6C16 wk) and cultured much like previously reported protocols (24). Cells were managed in Neurobasal A media (Invitrogen). Nodose cells were recorded on at room temperature. Locally administered drugs were applied to nodose cells for 2 s with a pressure of 2C3 psi using a picospritzer III (Parker). Input resistance ranged from 0.9 to 18.5 M. The response rate of neurons to 5-HT was highly variable, ranging from 10 to 70% of the neurons tested, depending on the culture. Statistics. All data are offered as means with errors bars as SE. Differences in drug effects were tested by repeated measured ANOVA, using Tukeys post hoc analysis unless normally noted. Differences were considered statistically significant for values 0.05 unless otherwise stated (Sigmaplot 11.2). Mice. TH-EGFP mice were on a C57Bl/6J background. Transgenic mice were housed on a 12-h light/12-h dark cycle at room heat in the Department of Integrative Physiology and Neuroscience animal vivarium. Mouse chow and water were provided ad libitum. Genotyping and breeding of mice were as explained previously (1). All animal procedures were conducted with the approval of the Institutional Animal Care and Use Co mMittee at WSU in accordance with the US General public Health Service Policy on Humane Care and Use of Laboratory Animals (PHS Policy) and the National Institute of Health (NIH). RESULTS Low glucose decreases action potential firing of TH-EGFP.