Recording miniature and spontaneous inhibitory postsynaptic currents

Voltage‐clamp recordings from lateral LHb neurons (from the posterior and middle segment, rather than from the rostral pole) were made at −50 mV with an internal medium consisting of (in mm): KCl 100, K‐gluconate 30, MgCl₂ 4, EGTA 0.2, HEPES 5, Na₂ATP 3.4, Na₃GTP 0.1, Na⁺ creatine phosphate 10; pH 7.3, 300 mOsm. As a result of the high chloride concentration in this medium, GABA_AR responses are inward at −50 mV and easier to detect. After patching, LHb neurons were allowed to recover for at least 10 min before another epoch of 10 min of sIPSCs was recorded. Recordings were done in the presence of the AMPA/kainate receptor antagonist NBQX (20 μm; Tocris) and the NMDA receptor antagonist D‐APV (100 μm; Tocris) to pharmacologically isolate GABA_AR responses. These responses were mediated by GABA_ARs as they were blocked by the GABA_AR antagonist (−)‐Bicuculline‐methiodide (20 μm; Tocris), as assessed in a subset of recordings.

mIPSCs were recorded in the presence of tetrodotoxin (TTX; 1 μm; Tocris) to abolish action potential‐dependent transmission. Furthermore, to allow for better detection of mIPSCs, we voltage clamped cells at −50 mV with a cesium‐based internal solution, consisting of (in mm): CsCl 130, NaCl 4, MgCl2 2, EGTA 1.1, HEPES 5, Na2ATP 2, sodium creatinephosphate 5, Na3GTP 0.6, spermine 0.1; pH 7.3, 300 mOsm.

To record GABA_AR transmission at EPN‐to‐LHb synapses, voltage‐clamp recordings were made at –50 mV using an internal medium consisting of (in mm): K‐Gluconate 140; KCl 5; HEPES 10; EGTA 0.2; MgCl2 2; Na2ATP 4; Na3GTP 0.3; creatine phosphate 10. Light pulses (470 nm, 1–10 ms) for opto‐stimulation of EPN nerve terminals were delivered with a LED (CoolLed, UK) illumination system. To isolate GABA_AR responses we added the AMPA/kainate receptor antagonist NBQX (20 μm; Tocris) and the NMDA receptor antagonist D‐APV (100 μm; Tocris) during the recording. To evoke GABA_BR responses we delivered a train of 10 pulses at 5, 10, or 20 Hz each sweep (0.1 Hz inter‐sweep interval). We utilized the same stimulation frequency for at least 10 consecutive sweeps and then averaged these sweeps. In this average trace, slow outward IPSCs were quantified as the delta current between the 50 ms baseline prior to opto‐stimulation and the current remaining between 40 and 50 ms after the tenth optogenetic pulse was delivered (when fast ionotropic GABA_AR‐dependent responses had ended). A cell was classified as having a GABA_BR outward IPSC in the event that there was at least a 5 pA delta current quantified in this way that could be blocked by the GABA_BR antagonist {"type":"entrez-protein","attrs":{"text":"CGP54626","term_id":"875260408"}}CGP54626 (10 μm; Tocris UK). To detect the presence or absence of GABA_BR currents specifically in LHb neurons innervated by EPN (i.e. EPN‐responsive LHb neurons), we only used cells in which single pulse optogenetic stimulation of EPN nerve terminals gave rise to at least 50 pA of fast ionotropic synaptic current at −50 mV (AMPAR and/or GABA_AR‐mediated). In the case of glutamine incubation experiments assessing effects on GABA_BR transmission, we preincubated the slice in ACSF with l‐Glutamine (2 mm; Sigma‐Aldrich/Merck) for at least 20 min prior to the recording and then maintained this presence of glutamine throughout the recording.

For experiments in which GABA_BR‐responses were evoked pharmacologically we first ensured, by means of optogenetic stimulation of EPN terminals, that the recorded lateral LHb neuron was innervated by the EPN. Subsequently, in the absence of opto‐stimulation, we recorded the holding current required to maintain the cell at −50 mV during a baseline period (5 min). Then we applied the GABA_BR agonist (+)‐Baclofen (100 μm; Sigma‐Aldrich/Merck) for 5 min, and subsequently the GABA_BR antagonist {"type":"entrez-protein","attrs":{"text":"CGP54626","term_id":"875260408"}}CGP54626 (10 μm). The pharmacological GABA_BR response was quantified as the delta holding current between the baseline period and the peak current upon baclofen application.