Decapitation

All experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986) and the Hungarian Act of Animal Care and Experimentation (1998, XXVIII, section 243/1998), and with the guidelines of the institutional ethical code. Male Wistar rats (postnatal day 14–20; Harlan UK, Bicester, UK, or Charles River Hungary, Budapest) or CD1 mice (postnatal day 16–18; Charles River, Hungary, Budapest) were deeply anaesthetized with isoflurane and decapitated. Following decapitation, the brain was quickly removed into ice-cold cutting solution. Transverse hippocampal slices 400–450 μm in thickness were prepared using a Leica VT1000S microtome (Leica, Nussloch, Germany), and kept in an interface-type holding chamber at room temperature for at least 60 min before recording in standard or modified ACSF. The standard ACSF was composed of 126 mm NaCl, 2.5 mm KCl, 1.25 mm NaH₂PO₄, 2 mm MgCl₂, 2 mm CaCl₂, 26 mm NaHCO₃, and 10 mm glucose, prepared with ultrapure water and bubbled with 95% O₂/5% CO₂ (carbogen gas), pH 7.2–7.4. All experiments were performed using rat hippocampal slices, except the investigation of the propagation of network activities from CA3 to CA1, which was performed in slices prepared from mice. Recordings were made in either an ‘Oslo’-style interface chamber or in commercially available submerged-type slice chambers (Luigs & Neumann, Ratingen, Germany, and MED64 probes, Alpha MED Sciences, Osaka, Japan). In preliminary experiments, we found that persistent oscillations in these conventional submerged-type slice chambers were only achieved with a flow rate exceeding 10 mL/min, similar to previous observations of hippocampal network activity (Wu et al., 2005 (link)). Adding a dye to the superfusion fluid to visualize the flow, we noticed that the solution tended to flow along the edges of these chambers. The chamber design was therefore modified in either of two ways. First, in order to reduce the volume of the chamber and direct the superfusion fluid over the slice, an inert plastic insert was used (Fig. 1A and B). These plastic inserts were used in all experiments in which the effect of flow rate on generation of network oscillations was investigated. The second modification allowed a double superfusion system to be used (Supertech Ltd, Pecs, Hungary; http://www.super-tech.eu). In this design, the slices were placed on a mesh glued between two plastic rings with a thickness of 2 mm. Two separate fluid inlets allowed ACSF to flow separately above and below the slice (Fig. 1C–F). This second design was only used to study the propagation of network activity from CA3 to CA1.

All procedures were approved by the local Animal Ethics Committee of Iwate University. The African lungfish P. aethiopicus and South American lungfish, L. paradoxa, were purchased from commercial suppliers. The fishes were anesthetized with tricaine methanesulfonate and euthanized by decapitation. Information pertaining to the animals is shown in Table 1. Juvenile and adult individuals of each lungfish were used. According to Mlewa and Green (2004) [29 (link)] and Jorgensen and Joss (2010) [30 ], P. aethiopicus individuals over 43 cm in body length (BL) reach sexual maturity. Thus, P. aethiopicus #1 (BL 50 cm) and L. paradoxa #1 (BL 65 cm) were regarded as adults, whereas P. aethiopicus #2–4 and L. paradoxa #3 (BL 35 cm or less) were regarded as juveniles [29 (link), 30 ]. Also, we confirmed during dissection whether they had functional genital organs or not.Table 1

Animals

	Animal No	Total body length (cm)	Body weight (g)	Sex	Application
P. aethiopicus	1	50.0	349.0	F	ISH (left)/RNA extraction (right)
	2	35.0	150.6	M	Dice CT
	3	31.5	100.0	unknown	ISH
	4	34.0	118.3	F	SEM
L. paradoxa	1	65.0	994.5	F	RNA extraction (left)/ISH (right)
	3	18.5	18.6	M	ISH

ISH in situ hybridization; Dice CT Diffusible iodine-based contrast-enhanced computed tomography; SEM Scanning Electron Microscopy

For histological examination, olfactory organs were dissected from the heads and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The specimens were cryoprotected in a sucrose gradient (10%, 20%, and 30% in 0.1 M PB), embedded in O.C.T. compound (Sakura Finetek, Tokyo, Japan), and sectioned sagittally using a cryostat. Sections (20 µm in thickness) were thaw mounted on MAS-coated slides (Matsunami, Osaka, Japan), air-dried, and processed for hematoxylin–eosin staining, immunohistochemistry, and in situ hybridization.

Animals were anesthetized with isoflurane. After decapitation, the brain was quickly transferred to the frozen cutting solution (containing, in mM, 15 KCl, 3.3 MgCl₂, 110 K-gluconate, 0.05 EGTA, 5 HEPES, 25 glucose, 26.2 NaHCO₃ and 0.0015 (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) with carbogen gas (95% O₂ and 5% CO₂). The brain was sliced into 250-µm thick sections using a vibratome (VT1200S, Leica), and transferred into artificial cerebrospinal fluid (aCSF, containing, in mM, 124 NaCl, 3 KCl, 2 MgCl₂, 2 CaCl₂, 1.23 NaH₂PO₄, 26 NaHCO₃, 25 glucose) with carbogen gas (95% O₂ and 5% CO₂) at 35 °C for at least 1 h, then at room temperature covered with aluminum foil to avoid light exposure. An amplifier (Multiclamp 700B, Molecular Devices) and a digitizer (Axon Digidata 1550B, Molecular Devices) were used for patch clamp recording. The recording chamber was perfused with aCSF saturated with carbogen gas (95% O₂ and 5% CO₂) at room temperature. A glass pipette (GC150-10; Harvard Apparatus) was made with a puller (P-1000, Sutter Instrument) and its resistance was between 2.8 and 7 MΩ. The pipette was loaded with K-gluconate-based pipette solution (in mM, 138 K-gluconate, 8 NaCl, 10 HEPES, 0.2 EGTA-Na₃, 2 Mg-ATP, and 0.5 Na₂-GTP, pH 7.3 with KOH) for whole-cell recording, or aCSF for loose cell recording. Under an epifluorescent microscope (BX51WI, Olympus), 505 nm LED illumination (74 µW/mm², 100 ms, Niji, Blue Box Optics) was used to visualize native EYFP fluorescence from ACR2-EYFP. We identified LC-NA neurons by a combination of anatomical location, triangular cellular shape, and fluorescence observed in the recording area. In Fig. 2, the membrane potential was held at − 60 mV for measuring current deflection. In Fig. 3, the membrane potential was held from − 120 to − 40 mV in 20 mV steps with a duration of 700 ms. The voltage deflection was evaluated at a current holding of 0 pA. Clampex 11.0.3 (Molecular Devices) was used to record the data.