A Direct Comparison of Afferents to the Rat Anterior Thalamic Nuclei and Nucleus Reuniens: Overlapping But Different

Photomicrographs depicting the delineation of sublayers VIa and VIb in cresyl violet- and parvalbumin-stained sections. a–h, j–m, The images also show how retrogradely labeled cells from Fast Blue in nucleus reuniens are preferentially localized in layer VIb in cases #9 (a–h) and #3 (j–m). Notice that the parvalbumin stain is stained with a red fluorophore in case #9 and green in case #3. a, b, A parvalbumin-stained frontal section (red) with Fast Blue-labeled cells plotted (green/yellow color) in b. c, d, A 20× zoom scan of the frontal cortical portion indicated by the white stippled box in b. c shows the parvalbumin label, while d shows both parvalbumin and Fast Blue labels. e, f, Frontal brain sections as in a and b, but with the sublaminar differentiation of layer VI indicated in f. Inserted asterisks indicate the borders between layers VIb/VIa and layers VIa/V, as well as the cingulum bundle/layer VIb border. g, The laminar delineations are based on cytoarchitectonic criteria. Nissl-stained section from the same case (#9) is shown, with the sublaminar borders indicated by asterisks as in f. This brain section is adjacent to the section in e and f, and the indicated laminar borders in this case were made by overlaying the image with the cresyl violet-stained image. h, Enlargement of the portion indicated in g with a black stippled box. i, Center of the injection site in case #9. j–m, Images from case #3, showing parvalbumin (green) with Fast Blue (k, m) or without Fast Blue (j, l) in rostral anterior cingulate/prelimbic cortices (j, k) and a more caudal portion of the anterior cingulate cortex (l, m). Inserted asterisks indicate the border between layers VIa/VIb and VIb/cingulum bundle. Note that retrogradely labeled cells in b are manually plotted whereas in all other images the original scans are shown. Images are adjusted for contrast, brightness, and intensity. 3V, Third ventricle; mt, mammillothalamic tract; SMT, submedial thalamic nucleus; ACC, anterior cingulate cortex; RE, nucleus reuniens; CM, central medial thalamic nucleus; pRE, perireuniens. Scale bars, 200 μm.

Photomicrographs showing the distribution of labeled cells resulting from a CTB injection in the anteromedial thalamic nucleus (red cells) and a Fast Blue injection in the nucleus reuniens (blue cells; case #7). a–e, Scans (5×) illustrating cell labeling along the anteroposterior axis showing all portions of neocortex substantially labeled by both tracers. Stippled white lines indicate laminar borders, and arrows indicate regional borders. Cells labeled from the Fast Blue tracer in nucleus reuniens are densely concentrated in layer VIb in the ACC, PL, IL, and secondary motor cortices (M2), as well as the deep cell layer of the DP. At anterior portions of the anterior cingulate cortex only, appreciable Fast Blue labeling is also present in layer VIa. Cells labeled by CTB (anteromedial nucleus injection) were more concentrated in the anterior cingulate cortex and distributed in both layer VIa and layer VIb, but in some sections had a clear preference for layer VIa, avoiding the deepest portion of layer VIb. Some Fast Blue- and CTB-labeled cells are also present in layer V. In this case, cell labeling by both CTB and Fast Blue extends into the rostral retrosplenial cortex. c₁–c₃, Zoom images (10×) of the box in c. c₄–c₆, Zoom images (20×) of the box in c₃. d₁–d₃, Zoom images (10×) of the box in d. d₄–d₆, Zoom images (20×) of the box in d₃. In all zoom panels, the first image to the left shows Fast Blue-labeled cells, the second shows CTB-labeled cells, and the third image is an overlay of the two previous images. Double-labeled cells are present and can clearly be seen in the 20× zoom images. f, Line drawing of the injection site. For photomicrographs of injections, see Figure 1. Images are adjusted for contrast, brightness, and intensity. A13, A13 dopamine cells; ACC, anterior cingulate cortex; AD, anterodorsal thalamic nucleus; AM, anteromedial thalamic nucleus; AV, anteroventral thalamic nucleus; CM, central medial thalamic nucleus; DA, dorsal hypothalamic areas aRSC, anterior retrosplenial cortex; DP, dorsal peduncular cortex; IL, infralimbic cortex; IAM, interanteromedial thalamic nucleus; LO, lateral orbitofrontal cortex; M2, secondary motor cortex; PaXi, paraxiphoid thalamic nucleus; PL, prelimbic cortex; pRE, perireuniens; RE, nucleus reuniens; Rh, rhomboid nucleus; SMT, submedial thalamic nucleus; VM, ventromedial thalamic nucleus; ZI, zona incerta. Scale bars, 200 μm.

Density of cortical labeled neurons (per square millimeter): the plots show the mean and SEM from the seven highlighted cases. a, Density of retrogradely labeled cells. Densities are given for the IL/DP, PL, as well as the aACC and pACC. b, Cortical cell counts showing the number of cells in layer VIa as a proportion of the total cell counts in layer VIa and VIb combined. Cell counts are given for the same areas as in a, except for IL/DP. c, Density of retrogradely labeled cells in retrosplenial cortex (RSC). ATN, Anterior thalamic injections; RE, nucleus reuniens injections. ***p < 0.001; **p < 0.01.

Photomicrographs showing retrograde cell labeling in case #1 with a Fast Blue injection involving all anterior thalamic nuclei combined with a CTB injection in nucleus reuniens. a, Plentiful Fast Blue labeling in layer VI of retrosplenial cortex following the ATN injection, while no CTB-labeled cells are visible. The schematic drawing in the top right corner indicates a–p levels of the image. b, Retrograde cell labeling in the mammillary bodies where only Fast Blue (ATN injection) is visible, with no cells labeled following the nucleus reuniens injection. lMB, Lateral mammillary nucleus; mMB medial mammillary nuclei; dRSC, dysgranular retrosplenial cortex; gRSC, granular retrosplenial cortex; SUM, supramammillary nucleus. Scale bars, 200 μm.

Photomicrographs depicting the distribution of label resulting from a CTB injection in the AM (red cells) and a Fast Blue injection centered in RE (case #7). a–c, A 5× scan of cell labeling in subiculum. Labeled cells resulting from the Fast Blue tracer in nucleus reuniens are distributed across all septotemporal and proximodistal portions (with a slight preference for distal portions at septal levels), whereas the CTB label (from the anteromedial thalamic nucleus) shows a clear preference for the proximal subiculum and often avoids the most ventral subiculum, aside from the ventral portion of the intermediate subiculum (c). b₁–b₃, Zoom images (20×) of the dorsal white box in b. b₄–b₆, Zoom images (20×) of the black/white ventral box in b. c₁–c₃, Zoom images (20×) of the most dorsal white box in c. c₄–c₆, Zoom images (20×) of the slightly more ventral white box in c. Comparing c₁–c₃ and c₄–c₆, the proximodistal differences between the two cell populations are evident. c₇–c₉, Zoom images (20×) of the most ventral white box in c. Double-labeled cells are visible in the 20× zoom images, highlighted by arrows pointing to representative double-labeled cells. In all zoom panels, the first image shows Fast Blue-labeled cells, the second image shows CTB-labeled cells, and the third image is an overlay of the two. Images are adjusted for contrast, brightness, and intensity. d, Line drawings depicting the center of the two injections in case #7. A13, A13 dopamine cells; AD, anterodorsal thalamic nucleus; AM, anteromedial thalamic nucleus; AV, anteroventral thalamic nucleus; CM, central medial thalamic nucleus; dSUB, dorsal subiculum; IAM, interanteromedial thalamic nucleus; iSUB, intermediate subiculum; PaS, parasubiculum; PaXi, paraxiphoid thalamic nucleus; pRE, perireuniens; PrS, presubiculum; RE, nucleus reuniens; Rh, rhomboid nucleus; SMT, submedial thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; vSUB, ventral subiculum; ZI, zona incerta. Scale bars, 200 μm.

Photomicrographs of retrogradely labeled cells in dorsal and intermediate subiculum in five different cases. Case numbers are indicated in each frame. a, b, Injection sites are restricted to nucleus reuniens and the anterior thalamic nuclei. c–e, Other areas were involved in the injection sites (Table 1). In case #3, the Fast Blue injection (blue cells) is in nucleus reuniens and the CTB injection (red cells) is in the anterior thalamic nuclei. In all other cases, this is reversed. a, Labeled cells resulting from both tracer injections are intermixed in the deep cellular layers. Nevertheless, a tendency for laminar separation is visible with cells projecting to nucleus reuniens (blue) being positioned in the deepest portion. b, At very caudal levels of the intermediate subiculum (where in coronal sections, all cells belong to the deep cell layer), the cells targeting nucleus reuniens (blue) are located in the deepest portion, whereas cells targeting the anterior thalamic nuclei are more equally distributed, but avoid the deepest portion, creating a differentiation across these deepest layers. c–e, Independent of the density of the additional cell label in the superficial cell layer, a sublaminar differentiation is present in the deepest layer. c and e also show the more superficial subiculum label observed in a subset of reuniens injection cases. The insert in d is a 20× zoom image of the same section, showing the morphologic details. Images are adjusted for contrast, brightness, and intensity. Cx, Neocortex; POS, postsubiculum; dSUB, dorsal subiculum. Scale bars, 200 μm.

Density of labeled neurons in subiculum (per square millimeter). Plots showing mean density and SEM of retrograde labeled cells in five highlighted cases. Densities are given for the dorsal (dSUB), intermediate (iSUB), and ventral (vSUB) subiculum. ATN, anterior thalamic injections; RE, nucleus reuniens injections. ***p < 0.001.

Plots showing the density of double-labeled cells in cortex and subiculum (per square millimeter). a, Plots showing the mean density and SEM of double-labeled cells in infralimbic/dorsal peduncular cortices (IL/DP), prelimbic cortex (PL), rostral anterior cingulate cortex (aACC) and caudal anterior cingulate cortex (pAAC). b, Plots showing the mean density and SEM of double-labeled cells in the dSUB, iSUB, and vSUB. **p < 0.01; *p < 0.05.