2.1.2. Whole-cell patch clamp recordings

One week after stereotaxic Retrobead tracer injection into the PVN, rats were deeply anesthetized with isoflurane (5% in oxygen via inhalation) and then decapitated. The brain was quickly removed and immersed in ice-cold pre-oxygenated artificial cerebrospinal fluid (ACSF). A tissue block containing the anterior BST was excised, and coronal slices (350–400 μm thick) were cut with a vibratome (Leica VT1000S, Leica, Germany). Visual landmarks including the anterior commissure, lateral ventricles, and optic chiasm were used to select slices through the appropriate rostrocaudal level of the anterior BST [i.e., approximately 0.2–0.5 mm caudal to bregma, based on the most recent version of Swanson's rat brain atlas (Swanson, 2018); see Fig. 1]. Slices were incubated at 37°C for 0.5–1 h, then kept at room temperature before being transferred to a recording chamber perfused with oxygenated ACSF (95% O₂/5% CO₂; pH 7.25–7.3) at 31–32°C. ACSF solution composition (in mM) was 126 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 1 MgSO₄, 2 CaCl₂, 24 NaHCO₃, and 10–20 glucose.

Subnuclear organization of the rat alBST.

Klüver-Barrera-stained coronal tissue sections through two representative rostrocaudal levels of the alBST. Myelinated fibers are blue, neuron cell bodies are pink/purple. Images are adapted from our previous publication (Bienkowski et al., 2013), with BST subnuclei outlined in yellow and labeled according to the most recent version of Swanson's rat brain atlas (Swanson, 2018). 3V, third ventricle; aco, anterior commissure; al, anterolateral subnucleus; am, anteromedial subnucleus; BAC, bed nucleus anterior commissure; dm, dorsomedial subnucleus, fu, fusiform subnucleus; fx, fornix; ju, juxtacapsular subnucleus; LV, lateral ventricle; mg, magnocellular subnucleus; ov, oval subnucleus; pr, principal subnucleus; ps, parastrial subnucleus of the hypothalamus; st, stria terminalis; v, ventral subnucleus. The approximate (~) rostro-caudal level of each section (relevant to bregma) is indicated; the more caudal section is at the top, and the more rostral section at the bottom. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

A Zeiss Axioskop microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a 40× water immersion objective and a digital video camera (CoolSnap, Photometrics, Tucson, Az) was used to visualize retrogradely-labeled neurons for whole-cell recordings. Within each slice, the ventral region of the alBST was identified by its medial-lateral position (i.e., vertically aligned with the lateral ventricle, medial to the internal capsule), by its close ventral proximity to the anterior commissure, and by the presence of red retrobead-labeled neurons. Patch electrodes were filled with an internal solution containing (in mM): 105 Cs-gluconate, 2 MgCl₂, 10 NaCl, 10 HEPES, 10 phosphocreatine, 4 ATP-Mg, 0.3 GTP, and 10 BAPTA; pH 7.25. In addition, Alexa 568 (0.075%; Molecular Probes, Eugene, OR) was added to the intracellular solution to fill each recorded neuron for later morphological identification, as previously described (Povysheva et al., 2006). Electrodes had 5–10 MΩ open-tip resistance. Voltage and current recordings were performed with a Multi-Clamp 700A amplifier (Axon Instruments, Union City, CA). Voltage recordings were performed in bridge-balance mode. Signals were filtered at 2 KHz, and acquired at a sampling rate of 10 kHz using a Digidata 1440 digitizer and Clampex 10.2 software (Molecular Devices Corporation, Sunnyvale, CA). Access resistance and capacitance were compensated on-line. Access resistance typically was 10–20 MΩ and remained relatively stable during experiments (≤30% increase) for cells included in data analysis. Membrane potential was corrected for the liquid junction potential of −13 mV.

The synthetic GLP1 analogue Exendin-4 (Ex-4; 200–600 nM; Bachem, Torrance, CA) was bath-applied to activate GLP1Rs, while the specific antagonist Exendin-9 (Ex-9; 900 nM; Bachem) was used to block GLP1Rs (Goke et al., 1993) (Thorens et al., 1993). Additional pharmacological agents were bath applied at the following concentrations: tetrodotoxin (TTX; 0.5 μM; Sigma) to block voltage-gated Na⁺ channels; AP-5 (D-2-amino-5-phospho-pentanoic acid; 50 μM; Ascent Scientific LTD, Bristol, UK) to block NMDA (N-methyl-D-aspartate) receptors; NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline; 20 μM; Ascent Scientific) to block AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and kainate receptors; gabazine (10 μM; Ascent Scientific LTD, Bristol, UK) to block GABA_A receptors.

Patch-clamp recordings in identified ventral alBST neurons were made in the presence or absence of Ex-4. Spontaneous firing frequency was estimated during 2 min intervals before and after bath application of Ex-4, with the latter interval beginning 1 min after application. Spontaneous inhibitory post-synaptic currents (sIPSCs) were recorded at a holding potential of +12 mV in the presence of NBQX and AP-5. Miniature inhibitory post-synaptic currents (mIPSCs) were recorded at a holding potential of +12 mV in the presence of TTX to inhibit action potential-mediated inhibitory postsynaptic currents. Spontaneous excitatory post-synaptic currents (sEPSCs) were recorded at a holding potential of −70 mV. Miniature excitatory post-synaptic currents (mEPSCs) were recorded at a holding potential of −70 mV in the presence of TTX to inhibit action potential-mediated excitatory postsynaptic currents. Spontaneous and miniature events were analyzed using the MiniAnalysis Program (Synaptosoft, Decatur, GA). Peak events were first detected automatically using an amplitude threshold of 1.5 times the average RMS noise, which approximated 3 pA for recordings at a holding potential of −70mV. More than 500 events per cell were included in each analysis.

To characterize neuronal membrane properties, hyper- and depolarizing current steps were applied for 500 ms in increments of 5–10 pA at 0.5 Hz. Input resistance was measured from the slope of a linear regression fit to the voltage-current relation in a voltage range hyperpolarized from resting potential. The membrane time constant was determined by single-exponential fitting to the average voltage responses activated by hyperpolarizing current steps of 5–15 pA. Action potential (AP) properties were quantified using the first action potential evoked through application of depolarizing current steps. AP threshold was measured at the level of voltage deflection exceeding 10 mV/1 ms. Peak AP amplitudes of the AP and afterhyperpolarization were measured relative to AP threshold. AP duration was measured at the base (AP threshold level). Action potential frequency was calculated in Hz as a ratio between number of action potentials and current step duration at 60 pA above the rheobase.

Neurons were filled with Alexa 568 during whole-cell recordings. A subset of these were maintained for at least 30 min to ensure extensive dendritic labeling. After recording, slices were fixed in ice-cold 4% paraformaldehyde for at least 72 h, then transferred into an anti-freeze solution (ethylene glycol and glycerol in 0.1 M phosphate buffer) and stored at −20°C. A few representative labeled neurons were reconstructed three-dimensionally using an Olympus Fluoview BX61 confocal microscope and Fluoview software (Olympus America Inc, Melville, NY).