Nine wild type P14 male Wistar rats were used for this study. This work was performed following the experimental procedures approved by the Home Office (UK) in the guidance on the operation of the Animals (Scientific Procedures) Act 1986 and European guidelines on animal experimentation. The animals were sacrificed respecting Schedule 1 methods (humane killing methods) listed in the Act, which covers all elements of animal research in the UK.

These reagents are used to obtain the stock solutions 5× Krebs, 5× Hepes and Sodium bicarbonate in order to prepare the “slicing” aCSF and the “recording” aCSF.

The final concentrations, in mmol, of the two aCSFs (previously described from Badin et al., 2013) are for the “slicing” aCSF: 120 NaCl, 5 KCl, 20 NaHCO₃, 2.4 CaCl₂, 2 MgSO₄, 1.2 KH₂PO₄ and 10 glucose; 6.7 HEPES salt and 3.3 HEPES acid; pH: 7.1. “Recording” aCSF: 124 NaCl, 3.7 KCl, 26 NaHCO₃, 2 CaCl₂, 1.3 MgSO₄, 1.3 KH₂PO₄ and 10 glucose; pH: 7.1.

Fix the (2) to the (1) using (3) (Figure 1K). Connect to one end of the (2) the 5 mm bore tubing (4) which is linked to the oxygen source through the straight barbed connector (5) (Figure 1L). Attach to the lower outlet of the valves the 3 mm bore tubing (Figure 1K). After, insert the three barbed Y connectors (5) in the other side of the 3 mm bore tubing. Attach the small tubes (1.5 mm bore) to each Y shaped connector, in order to have two tubes per each valve (Figure 1K). The free edges of these tubes are glued (6) to the 21G needles (7), which are individually inserted in (8) (Figure 1J), where the 25G needles (7) are also placed. The 25G needles allow the outflow of oxygen excess from (9). Once all the vials (9) (Figure 1I) are closed (Figures 1J,K) position them in the support (10), connect the apparatus to the oxygen source and start the experiment (Figure 1L).

Rats were sacrificed as previously described (Badin et al., 2013). Briefly, they were anesthetized with isoflurane, 1.5 ml 100% w/w, poured on the cotton layer inside the induction chamber. After the proper level of anesthesia was confirmed by the absence of the pedal reflex, the animals were decapitated and the brains were quickly and carefully extracted (Figures 1A–C). Shortly, after decapitation the skin covering the skull was cut with a surgical blade and the two sides held under the skull with the index and thumb finger (Figures 1A,B). The skull was subsequently cut along the midline with dissection scissors towards the olfactory bulbs and removed with the forceps retracting the two halves laterally (Figures 1B,C). The brain, once extracted, was cooled and kept hydrated with ice-cold “slicing” aCSF for 1–2 minute (min) and placed on filter paper where the cerebellum was removed with a surgical blade. Finally the brain was vertically glued in the center of the vibratome disc (Figure 1D), placed into the vibratome chamber which was filled with oxygenated ice cold “slicing” aCSF and surrounded by ice (Figure 1E). After regulating the cutting distance (Figure 1F), the brain was sectioned (frequency at 6–7 and speed at 6) in three consecutive coronal sections (300 μm thick) containing BF structures, (approximately Bregma 1.20–0.20 mm; Paxinos, 1998; Figures 1G, 2A). Following the rostro-caudal axis, the sections include the medial septum (MS), diagonal band of Broca (DBB) and, to a lesser extent, the substantia innominata (SI), containing the nucleus basalis of Meynert (NBM). Each slice was subsequently cut along the midline (Figure 1G), in order to obtain two hemisections, used respectively as control and treated preparations at the same anatomical level (Figure 2A). All hemisections were then individually placed in 5 ml glass vials (Figure 1H), where a disc of filter paper was previously inserted to prevent direct contact between the brain tissue and the glass, containing either 3 ml of “recording” aCSF alone for the control group (Figure 1I, green frame) or enriched with 2 μM of T30 for the treated group (Figure 1I, red frame). From top to bottom, the vials in both conditions contained the rostral, the intermediate and the caudal region of the BF (Figure 1I). The hemisections were positioned into their corresponding vials using two different brushes, one for the control group and the other for the treated counterpart, in order to avoid any contamination. Afterwards, the vials were sealed (Figures 1J–L) and carbogen (95% O₂–5% CO₂) was continuously provided during the experiment (Figure 1L). The procedure described in Figure 1 was performed within a time window of 10–15 min to minimize any degradation process of the tissue. The oxygen flow was frequently checked, to prevent hypoxia, and kept at the minimum to avoid fluctuation of the hemisections, which could be damaged by the oxygenating needle. After 5 h of incubation, the hemislices were separately transferred, with their respective brushes, into 1.5 ml tubes containing lysis buffer. The brain tissue was then homogenized with pestles, keeping the tubes on ice. To prevent any contamination between samples, each hemisection was processed with a different pestle. The brain lysate was then centrifuged at 1000 g for 5 min at 4°C. The supernatant was transferred to a new tube and stored at −80°C until use.

Protein concentration was determined with Pierce Assay as previously described (Garcia-Ratés et al., 2016). Aliquots containing 10 μg/μl of proteins were prepared mixing the brain lysate with loading buffer and the denaturing agent beta-mercaptoethanol. Samples were heated at 95°C for 5 min and stored at −80°C until use. Western blot procedure was performed as previously described (Garcia-Ratés et al., 2016). Briefly, proteins were separated through 4%–20% precast gels and blotted on PVDF membranes. After staining the membranes with Blot-FastStain to determine the transfer quality and the total protein content used for the statistical analysis, the blots were destained with warm distilled water until all the bands were removed. Then the blots were blocked in 5% blotting grade blocker in TBS for 1 h at room temperature (RT), with gentle agitation. After the blocking step, the membranes were rinsed 3 × 5 min with TBST and incubated overnight at 4°C with primary antibodies diluted with 1% blocking solution prepared with TBST. The next day, the membranes were thoroughly washed in TBST 5 × 5 min, then incubated for 1 h at RT with the secondary antibodies, diluted in TBST. After the incubation, the blots were rinsed 6 × 5 min with TBST and 10 min with TBS and probed for protein revelation using chemiluminescent substrates. The primary antibodies, all diluted at 1:1000, were: mouse anti-phosphorylated Tau, mouse anti-Amyloid beta and rabbit anti-α7-nAchR. The secondary antibodies, all HRP conjugated, were goat anti mouse (1:2000) and goat anti rabbit (1:5000).

Protein values were normalized to total protein expression (Supplementary Figure S1; Aldridge et al., 2008; Zeng et al., 2013) and analyzed with ImageJ software (NIH, Bethesda, MD, USA). Differences in protein levels between control and treated group were determined using paired Student’s t-test and subsequently plotted with Graphpad software (Graphpad Prism 6, San Diego, CA, USA). All data were considered significant with a p-value < 0.05.

Table 1 indicates possible issues which can occur during the procedure, their related cause and suggestions to solve and avoid them.

This protocol provides several advantages: (1) it maintains the anatomical site-selectivity and the physiological milieu of the living brain; (2) slices can be easily obtained for studying a specific area; (3) depending on the characteristics of the investigated region, such as size and discernible boundaries, a microdissection could be performed after the treatment, providing a more precise read out of the investigated area; (4) direct intervention, precision dosing and an untreated contralateral side from the same slice offers an accurate control; and (5) the potential reduction in the number of living animals ultimately used in behavioral studies, based on the ability of multiplexing the desired experimental conditions. The limitations are: (1) the thickness and integrity of the tissue; (2) the incubation time; and (3) severing of connections in the rostro-caudal plane. All these aspects could affect biochemical, molecular or immunohistochemical analysis. However, the integrity of the tissue can be determined through immunohistochemical detection of several markers specific for neuronal viability and electrophysiology techniques as previously described (Cho et al., 2007; Badin et al., 2013).

After 5 h, T30 exposure (Figure 2A) induced a heterogeneous expression of α7-nAChR (Figure 2B), p-Tau (Figure 2C) and Aβ (Figure 2D) along the BF rostro-caudal axis. The α7 receptor, showed a 38% increase in the rostral portion (slice 1, p = 0.0310) in the treated group (Figure 2B), whereas in the intermediate slice there was no difference in the two groups (slice 2, p = 0.1195; Figure 2B). In the caudal section its levels were reduced by 36% compared to the control group (slice 3, p = 0.0476; Figure 2B). Phosphorylated Tau displayed higher levels, 63%, in the rostral hemisection upon T30 application, (slice 1, p = 0.0158; Figure 2C), while the other slices did not show any change between treatments (slice 2, p = 0.1014; slice 3, p = 0.6405; Figure 2C). Aβ levels were significantly increased by 30% in the anterior and intermediate portion in the treated hemisections (slice 1, p = 0.0136; slice 2, p = 0.0109), whereas in the posterior slice no difference was observed in the treated side above the control (slice 3, p = 0.1231; Figure 2D). Figure 2E recapitulates the expression patterns of the three markers upon T30 administration compared to the control value (black dotted line). Furthermore, along the rostro-caudal plane, the peptide induced two different trends in the protein levels, one promoting a continuous reduction of α7-nAChR and p-Tau and the second displaying a similar Aβ overexpression over the control (Figure 2E).