Retrograde neuronal labeling

Retrograde tracer solutions containing Cholera Toxin Subunit B- Alexa Fluor 488 or 546 conjugates (Life Technologies) (CTB; 0.8% w/vol in PB 0.1 M) were injected in 200-–250 g rats under ketamine/xylazine anesthesia. Briefly, animals were placed in a stereotaxic apparatus, and a small craniotomy (<1 mm²) was performed at the coordinates for targeting the MEC (see Burgalossi et al., 2011; Ray et al., 2014), dorsal PreS (lambda coordinates: 0.0 mm AP, 3.3 mm ML, −3.0 mm DV) or RS29 (lambda coordinates: −1.0 mm AP, 3.3 mm ML, −3.0 mm DV). Injections in RS29 (n = 4) were centered on, but not restricted to, the caudal portion of the RS29, bordering rostrally with the PreS. The border between PreS and RS29 were confirmed by calbindin staining (see Figure 1—figure supplement 2). We note that this region has been previously referred to as ‘area retrosplenialis 29e’ (Blackstad, 1956; Haug, 1976; Slomianka and Geneser, 1991) or ‘triangular region’ (Ding, 2013). PreS injections (n = 3) were centered on L2; to this end, prior to injection, PreS layer 2 was localized by electrophysiological mapping with low-resistance electrodes (1–3 MΩ), based on characteristic signatures of the multiunit spiking activity (see Results). Glass electrodes with a tip diameter of ~20–40 µm filled with CTB solution were then lowered into the target region. To avoid diffusion of the tracer during electrode penetration, the tip of the pipette was front-filled with a small amount of Ringer solution. Typically, small amounts of tracer solutions (~0.3–0.8 µl) were then slowly injected using positive pressure. After the injections, the pipettes were left in place for several minutes and slowly retracted. The craniotomy was closed by application of silicone (Kwik-cast, World Precision Instruments) and animals survived for 3–5 days before being euthanized and transcardially perfused. Both hemispheres were cut into 60–70 µm thick parasagittal slices and analyzed with epifluorescence and/or confocal microscopy. When necessary, immunochemical staining for calbindin was performed to outline the cytoarchitecture of the superficial PreS layers.