Article Figures & Data
Figures
- Figure 1.
Subicular neurons collateralize to innervate the RSP and MBs. A, Coronal photomicrographs of dorsal subiculum in a rat following FB injections in RSP and CTB in the MBs with pink double-labeled cells in the overlay panel indicating neurons that collateralize to both regions. Proximal-distal regions (R1–R4) were divisions used for subsequent quantification. B, Coronal section showing FB injection into RSP. C, Coronal section showing CTB injection into MBs. D, Coronal dorsal subiculum section after injections of FB into the RSP and FluoroGold into the MBs. The open arrowhead points to a single-labeled neuron projecting to MB, the closed arrowhead to single-labeled neuron projecting to RSP, the open diamonds indicate double-labeled neurons. CA1, hippocampal field CA1; LMB, lateral mammillary nucleus; MMB, medial mammillary nucleus; Rga and Rgb, granular RSP, subdivisions a and b, respectively (collectively, area 29); Rdg, dysgranular RSP (area 30). Scale bars: 500 µm.
- Figure 2.
Quantification of extent and location of collateralizing neurons in dorsal subiculum. Histogram illustrates the percentage of subiculum neurons projecting to RSP that colabel with MB tracer. For this analysis, dorsal subiculum was divided by proximal-distal (R1–R4) and AP locations (cell counts are presented in Extended Data Fig. 2-1). Photomicrographs depict dorsal subiculum (right hemisphere) at five AP levels (numbers indicate distance from bregma in millimeters), the borders are color coded to match the corresponding bars in the histogram. The photomicrographs show pink double-labeled cells that innervate both sites, red neurons projecting to MB, and blue neurons projecting to RSP. Additional, higher magnification panels show labeling in more detail; FB (blue) fills the cytoplasm while retrogradely transported CTB (red) remains in vesicles and so appears granular. The open arrowhead marks a single-labeled neuron projecting to MB, the closed arrowhead marks a single-labeled neuron projecting to RSP, the open diamonds indicate double-labeled neurons. Scale bar: 500 µm unless otherwise specified.
- Figure 3.
Characterization of collateral-collateral transport. A1, Photomicrograph of collateral-collateral transport following a CTB injection into the MBs. The section shows CTB terminal label in Layers II and III of granular RSP (area 29). The Nissl-stained overlay (A2) confirms the abrupt border with dysgranular cortex (area 30). B, Coronal section showing terminal label in dorsal pars lateralis (MMBl) and pars medianus (MMBmed) of the medial mammillary nucleus following a retrosplenial CTB injection. Note, pia artifact has been removed. C, Coronal section showing dense terminal label in the anterior thalamic nuclei. D, Pattern of both retrograde and light terminal label in the entorhinal cortex after a CTB injection into the MBs. Boxes, D2, and D3 correspond to higher magnification images of medial and lateral entorhinal cortex, respectively. E, Photomicrograph of dorsal subiculum following injection of an anterograde tracer (BDA). F, Coronal section of RSP showing pattern of BDA anterograde transport from dorsal subiculum. G, Coronal section from same level of RSP as depicted in F, illustrating pattern of CTB terminal label following CTB injection in MBs. AD, anterodorsal thalamic nucleus; AM, anteromedial thalamic nucleus; AV, anteroventral thalamic nucleus; LMB, lateral mammillary nucleus; MMBl, medial MB, pars lateralis; MMBm, medial MB, pars medialis. Scale bars: 500 µm unless otherwise specified.
- Figure 4.
Absence of collateral-collateral transport to RSP following a CTB injection into the MBs combined with lesion involving the postcommissural descending fornix. A, B, Nissl-stained sections, 1.56 mm behind bregma (according to Paxinos and Watson, 2005), showing postcommissural fornix lesion (A) and intact case (B), respectively. C, Coronal photomicrograph showing the very limited retrograde label in proximal dorsal subiculum after a postcommissural fornix lesion. D, Typical appearance of retrograde label in the dorsal subiculum in an intact case (CTB in MBs). E, Lack of terminal label in the RSP after postcommissural fornix lesion. The inset provides a comparison with an intact case. F, G, Retrogradely labeled neurons in Gudden’s ventral tegmental nucleus when the postcommissural descending fornix is lesioned (F) or intact (G) Note, while the label in F appears more restricted, it is denser. 3V, 3rd ventricle; opt, optic nerve. Scale bars: 500 µm.
- Figure 5.
Cross-species comparisons. A, Coronal section showing CTB injection into mouse MBs. B, Coronal section showing FB injection into mouse RSP. C, Coronal photomicrograph of dorsal subiculum. The numerous double-labeled (pink) cells innervate both sites. Inset depicts higher magnification of indicated region. The open arrowhead points to a single-labeled neuron projecting to MB, the closed arrowhead to a single-labeled neuron projecting to RSP, the open diamonds indicate double-labeled neurons. Associated cell counts are presented in Extended Data Figure 5-1. D1, Red terminal label in the granular RSP (area 29) from collateral-collateral transport, alongside scattered retrogradely labeled cells in RSP and the indusium griseum (IG). D2, A Nissl-stained overlay of section D1 shows the border between area 29 and area 30. The label is concentrated in deep Layer II and Layer III of area 29. IG, indusium griseum; LMB, lateral MBs; MMB, medial MBs; PM, premammillary nucleus. Scale bar: 500 µm unless otherwise specified.
- Figure 6.
Neurochemical characterization of collateral-collateral terminals. A1, Combined immunohistochemical signal for VGluT2 matching the distribution of CTB terminal label localized in superficial area 29. A2 shows at greater magnification the separate CTB and VGluT2 label, with the overlay showing colocalization within Layers II and III of area 29. B1, Combined immunohistochemical signal for neurotensin (NT) matching the distribution of CTB terminal label localized in superficial area 29. B2 shows at greater magnification the separate CTB and NT label, with the overlay showing colocalization within Layers II and III of area 29. Scale bar: 500 µm unless otherwise specified. Note, pia artifact has been removed. Neurochemicals that did not colocalize with the CTB-positive terminals are shown in Extended Data Figure 6-1.
- Figure 7.
Schematic depictions of described hippocampal network connectivity. A, Ipsilateral and crossed collaterals from the subiculum reach the MBs and RSP (area 29). Note, the subiculum projections to area 29 remain ipsilateral while collaterals to MB can remain ipsilateral or cross hemispheres. B, Updated hippocampal-limbic network (Papez circuit) showing the ventral (subcortical), dorsal (cingulate), and new “collateral” routes. ATN, anterior thalamic nuclei; MTT, mammillothalamic tract.
Extended Data
Extended Data Figure 2-1
Numbers of CTB- and FB-positive cells within different proximal-distal positions (R1–R4) of the dorsal and intermediate subiculum of the rat, including the number of double-labelled cells. The case numbers and hemisphere of cell counts (R or L) are shown, along with the percentage of subicular cells projecting to the RSP that are double labelled. Download Figure 2-1, DOCX file.
Extended Data Figure 5-1
Numbers of CTB- and FB-positive cells within of the dorsal and intermediate subiculum of the mouse, including the number of double-labelled cells. The case numbers and hemisphere of cell counts (R or L) are shown, along with the percentage of subicular cells projecting to the RSP that are double labelled. Download Figure 5-1, DOCX file.
Extended Data Figure 6-1
Series of coronal immunofluorescence images at the level of the RSP in an animal with a CTB injection in the MBs. Left column, Green immunofluorescent label associated with antibodies for VGluT1, GAD67, PV, calretinin (CR), calbindin (CB), and cholecystokinin (CCK). Middle column, CTB terminal label in the RSP (area 29, Layers II and III) highlighting the collateralizing subiculum projections that were present in the same section as depicted in the left column. Right column, The section overlay shows how the distribution of these neurochemicals do not match the termination sites of the collateral projections from the subiculum to area 29. Scale bar: 500 µm. Download Figure 6-1, TIF file.
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