Animal procedures were carried out in accordance with the Animals (Scientific Procedures) Act, 1986 (UK). Coronal slices (250 μm thick) were obtained from 13- to 22-day-old transgenic mice selectively expressing enhanced green fluorescent protein (eGFP) in orexin cells, as previously described (González et al. 2008). Briefly, orexin-eGFP cells were identified in brain slices by epifluorescence, whole-cell recordings were made at 36°C using an EPC-10 amplifier (Heka, Lambrecht, Germany), and data were sampled using Patchmaster software (Heka, Lambrecht, Germany). Most recordings consisted of alternating 2 min-long current-clamp traces and ∼30 s-long voltage-clamp protocols (see below). Because of the latter, breaks can be seen in current-clamp traces in Figs 1 and 2, but the shown duration of these ∼30 s-long breaks is compressed for presentation clarity.
Figure 1. Effects of thyrotropin-releasing hormone (TRH) on the membrane potential of orexin neurones A, an eGFP-expressing orexin neurone during a whole-cell recording (left; scale bar, 30 μm). The cell was identified in a brain slice by epifluorescence (right). B, effect of 250 nm TRH on an orexin cell recorded using a KCl pipette solution. Breaks in this and subsequent current-clamp traces correspond to intervals (< 30 s) where the recording was paused to perform voltage-clamp analysis. C, effect of 250 nm TRH on an orexin cell recorded using a potassium gluconate pipette solution. D, effect of 250 nm TRH in the presence of 1 μm bath tetrodotoxin. E, lack of effect of 250 nm TRH free acid, a biologically inactive TRH analogue. F, mean firing rate of orexin neurones (n= 11) in the absence and presence of 250 nm TRH, **P < 0.001. G, TRH dose–response curve (EC50= 6.2 nm, see Methods). Each point corresponds to ≥ 3 cells.
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Figure 2. Effects of ion substitution and drugs on membrane potential responses to TRH A, effect of 250 nm TRH on an orexin cell in ‘low Ca2+’ extracellular solution (see Methods). B, effect of 250 nm TRH on an orexin cell in ‘low Na+’ extracellular solution (see Methods). C, lack of effect of the Na+/Ca2+ exchange blocker KB−R7943 (70 μm) on depolarization induced by 250 nm TRH. D, summary of mean depolarization elicited by 250 nm TRH in the presence of different solutions and drugs. **P < 0.005, each bar corresponds to at least 4 cells. n.s. = no significant difference (P > 0.2).
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Pipettes were pulled from borosilicate glass and had tip resistances of 3–5 MΩ when filled with intracellular solution containing (in mm): KCl 130, Hepes 10, EGTA 0.1, MgCl2 2, K2ATP 5, and NaCl 2 (pH 7.25 with KOH). This solution was used in most recordings, but in certain experiments (when stated in Results), we instead used ‘low-Cl−’ (potassium gluconate) or ‘low-K+’ pipette solutions. The potassium gluconate solution contained (in mm): potassium gluconate 120, KCl 10, EGTA 0.1, Hepes 10, K2ATP 4, Na2ATP 1, and MgCl2 2 (pH 7.3 with KOH). The ‘low K+’ solution contained (in mm): CsCl 112, TEA-Cl 20, MgCl2 2, Hepes 10, Na2ATP 5, and Cs-EGTA 0.2 (pH 7.25 with CsOH).
‘Control’ extracellular solution contained (in mm): NaCl 125, KCl 2.5, MgCl2 2, NaH2PO4 1.2, NaHCO3 21, CaCl2 2, and glucose 1. ‘Low Ca2+’ extracellular solution instead contained 9 mm MgCl2 and 0.3 mm CaCl2. ‘Low Na+’ extracellular solution contained (in mm): NMDG (N-methyl-d-glucamine)-Cl 125, KCl 2.5, MgCl2 2, NaH2PO4 1.2, NaHCO3 21, CaCl2 2, and glucose 1. Extracellular solutions were bubbled with 95% O2–5% CO2 during the experiments. To calculate membrane conductance, whole-cell current was recorded during voltage steps (Fig. 3A), and conductance was determined as the slope of the line of best fit to the current–voltage relationship between −120 and −70 mV (i.e. where the relationship was the most linear). The dose–response curve in Fig. 1G was obtained by fitting a modified Hill equation to the data:
where Vmax is the maximal change in membrane potential, EC50 is the concentration that gives half-maximal response, and h is the Hill coefficient. The fit shown in Fig. 1G was obtained using EC50= 6.2 nm, Vmax= 10.9 mV, and h= 1. Data were analysed using SciPy (http://www.scipy.org/) and plotted with Matplotlib (http://matplotlib.sourceforge.net/). Student's t test was used to determine statistical significance. Values are shown as mean ±s.e.m. Tetrodotoxin, KB-R7943 and ZD7288 were from Tocris, TRH and TRH free acid were from Phoenix Pharmaceuticals, and all other chemicals were from Sigma.
Figure 3. Effects of TRH on membrane current–voltage relationship of orexin neurones A, the voltage-clamp protocol used to obtain data in B. B, currents obtained in response to voltage steps using a ‘low Ca2+’ extracellular solution (see Methods). Grey bars (a and b) show where the steady-state values were measured to produce the plot in C. C, net current activated by TRH (b minus a), n= 4 cells.
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Mice were anaesthetized with sodium pentobarbital and perfused via the ascending aorta with 10 ml of Ca2+-free Tyrode solution (37°C) followed by 10 ml of fixative containing 4% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid in 0.16 m phosphate buffer, pH 6.9, 37°C, followed by 50 ml of the same, but ice-cold, fixative. Brains were dissected, immersed in fixative for 90 min, and rinsed for 24 h in 0.1 m phosphate buffer (pH 7.4) containing 10% sucrose. Brains were then cut in 1 mm slabs and rinsed in 0.1% sodium borohydride for 30 min prior to freezing. Coronal sections were cut on a cryostat (Microm, Heidelberg, Germany) at 14 μm thickness and thaw-mounted onto gelatine-coated glass slides. Conventional immunofluorescence was employed for orexin using monoclonal anti-orexin antibodies (1 : 400) raised in mouse; these antibodies were a gift from Drs K. Eriksson and E. Mignot and their specificity was confirmed by cell body staining restricted to the LHA, and by parallel immunofluorescence performed with three other different monocolonal antibodies raised against orexin, which produced identical staining patterns of LHA cell bodies and terminals throughout the brain. The Tyramide Signal Amplification (TSA) protocol (Perkin Elmer, Waltham, MA, USA) was used to visualize TRH by polyclonal anti-TRH antiserum (1 : 2000; raised in rabbit; gift of Dr T. Visser, see Klootwijk et al. 1995), as previously described (Broberger et al. 1999). For quantification, five sections at regularly spaced intervals were sampled from the LHA of four brains, and the total number of orexin-immunopositive cell bodies on each side were counted, as well as those in close apposition with TRH-immunopositive terminals. By ‘close apposition’ we mean that (a) there is no observable gap between terminal and cell body/dendrite, and (b) the density of terminals on cell body/dendrite is not lower than that in the surrounding neuropil. Images were captured by a Zeiss LSM 510 META confocal microscope.