Skip to main content
. 2016 Jun 10;5:e14592. doi: 10.7554/eLife.14592

Figure 1. Anatomical organization and projection targets of superficial PreS neurons.

(A) Top, parasagittal section through the dorsal PreS stained for calbindin (Cb, green) and NeuN (red). Scale bar = 500 μm. Bottom, outline of the PreS (grey) from the section shown above. RS29 indicates the subfield of RS cortex which was targeted for retrograde tracing experiments. See Figure 1—figure supplement 1 for more details. (B) Superimposed staining for calbindin (green) and NeuN (red) showing the clustering of neuronal somata in L2 of PreS and the more homogeneous distribution of cells in L3. Right, close-up magnification of the single channels for panel 1 (red, NeuN; green, calbindin). Scale bars: 100 μm (left) and 50 μm (right). (C) Parasagittal section through PreS stained for calbindin (green) showing retrogradely-labeled neuronal somata following injection of CTB-Alexa 555 (red) in ipsilateral MEC (‘ipsi-MEC’). Left panels, single channels; right panel, overlay. Scale bars: 200 μm. (D) Bar-graph showing the % of retrogradely-labelled (CTB-positive) neurons in L2 and L3 of PreS, following tracer injection in ipsi-MEC (as shown in C; 4497 total counted neurons, n = 4 brains). Error bars indicate SEM. (E) Parasagittal section through PreS stained for calbindin (green) showing retrogradely-labeled neuronal somata following injection of CTB-Alexa 555 (red) in contralateral PreS (‘contra-PreS’). Scale bar: 50 μm. Right panel, close-up magnification of the inset shown on the left, showing three retrogradelly-labelled neurons (red) positive for the marker calbindin (green). Scale bar: 10 μm. (F) Bar-graph showing the % of calbindin-positive (Cb+) and calbindin-negative (Cb-) L2 neurons, which were retrogradely-labelled following tracer injection in contra-PreS (as shown in E; 159 total counted neurons, n = 3 brains). Error bars indicate SEM. (G) Left panels, close-up magnification PreS L2 neurons following injection of CTB-Alexa 555 (red) in contralateral RS29 and stained for calbindin (green). One calbindin-positive (asterisk) and two calbindin-negative neurons (arrowheads) are indicated. Scale bar: 10 μm. Right, bar-graph showing the % of calbindin-positive (Cb+) and calbindin-negative (Cb-) L2 neurons, which were retrogradely-labelled following tracer injection in contra-RS29 (896 total counted neurons, n = 4 brains). Error bars indicate SEM.

DOI: http://dx.doi.org/10.7554/eLife.14592.002

Figure 1.

Figure 1—figure supplement 1. Immunohistochemical analysis and outline of the PreS.

Figure 1—figure supplement 1.

(A )Parasagittal section through the dorsal PreS (~3.0 mm lateral from the midline) stained for calbindin (Cb, green) and NeuN (red). Top panels show single channels, bottom panel shows the overlay. Scale bar = 500 μm. (B) Outline of the PreS (grey) from the section shown in (A). RS29 indicates the subfield of RS cortex which was targeted for retrograde tracing experiments (see Materials and methods, Figure 1G and Figure 1—figure supplement 2). (B–D) and (E–F) same as in (A) but for more lateral sections (~3.3 and 3.7 lateral from the midline, respectively; Paxinos and Watson, 2007). Scale bars = 500 μm. PreS, presubiculum; RS29, retrosplenial cortex area 29; WM, white matter; Sub, subiculum; PaS, parasubiculum; MEC, medial entorhinal cortex.
DOI: http://dx.doi.org/10.7554/eLife.14592.003
Figure 1—figure supplement 2. Neuroanatomical markers outlining the rostral and caudal PreS borders.

Figure 1—figure supplement 2.

(A) Parasagittal section through the dorsal PreS stained for calbindin (Cb, green) and paravalbumin (PV, red). Top panels show single channels, bottom panel shows the overlay. Scale bar = 500 μm. (B) Parasagittal section through the dorsal PreS stained for calbindin (Cb, green) and Wolframin (Wfs-1, red). Top panels show single channels, bottom panel shown the overlay. Scale bar = 500 μm. (C) Parasagittal section through the dorsal PreS processed for zinc histochemistry. Scale bar = 500 μm. (D) Outline and location of the PreS (indicated in grey) from the section shown in (A). The arrowheads point to the rostral and caudal PreS borders, which can be assessed based on differential expression of the neuroanatomical markers shown in (A–C). PreS, presubiculum; RS29, retrosplenial cortex area 29; WM, white matter; Sub, subiculum; PaS, parasubiculum.
DOI: http://dx.doi.org/10.7554/eLife.14592.004
Figure 1—figure supplement 3. Layer distribution of retrogradely-labeled neurons in the contralateral PreS.

Figure 1—figure supplement 3.

(A) Left panel, parasagittal section through the dorsal PreS showing retrogradely-labelled neurons following injection of CTB in the contralateral PreS. Note the presence of retrogradely-labelled neurons within PreS L2 and the sparser labeling within RS29. Right panel, overlay with the calbindin-staining (Cb, green). Scale bar = 200 μm. (B) High-magnification picture from the section shown in (A) (left panel) showing the presence of retrogradely-labeled neurons (red) within the contralateral PreS L2. Scale bar = 50 μm. (C) Distribution of retrogradely-labeled neurons across the PreS layers following injection of CTB in the contralateral PreS (n = 3 experiments). Error bars indicate SEM. (D) Representative injection site for the experiment shown in (A–B). Red, CTB; green, calbindin (Cb). Scale bar = 200 μm. (E) same as in (A) but for CTB injection in the contralateral RS29. Note the presence of retrogradely-labelled neurons within PreS L2 (see also panel F below) and the denser labeling within RS29. Scale bar = 200 μm. (F) High-magnification picture from the section shown in (E). Scale bar = 50 μm. (G) same as in (C) but for CTB injection in the contralateral RS29 (n = 4 experiments). Error bars indicate SEM. (H) Representative injection site for the experiment shown in (E–F). Red, CTB; green, calbindin (Cb). Scale bar = 200 μm. PreS, presubiculum; RS29, retrosplenial cortex area 29; WM, white matter; Sub, subiculum; PaS, parasubiculum.
DOI: http://dx.doi.org/10.7554/eLife.14592.005