Complementary Modular Microcircuits of the Rat Medial Entorhinal Cortex

Abstract

The parahippocampal region is organized into different areas, with the medial entorhinal cortex (MEC), presubiculum and parasubiculum prominent in spatial memory. Here, we also describe a region at the extremity of the MEC and bordering the subicular complex, the medial-most part of the entorhinal cortex. While the subdivisions of hippocampus proper form more or less continuous cell sheets, the superficial layers of the parahippocampal region have a distinct modular architecture. We investigate the spatial distribution, laminar position, and putative connectivity of zinc-positive modules in layer 2 of the MEC of rats and relate them to the calbindin-positive patches previously described in the entorhinal cortex. We found that the zinc-positive modules are complementary to the previously described calbindin-positive patches. We also found that inputs from the presubiculum are directed toward the zinc-positive modules while the calbindin-positive patches received inputs from the parasubiculum. Notably, the dendrites of neurons from layers 3 and 5, positive for Purkinje Cell Protein 4 expression, overlap with the zinc modules. Our data thus indicate that these two complementary modular systems, the calbindin patches and zinc modules, are part of parallel information streams in the hippocampal formation.

Keywords: acetylcholinesterase; calbindin; mMEC; medial entorhinal cortex; modularity; parasubiculum; presubiculum; zinc.

FIGURE 1

Rat parahippocampal region. Overview of the rat parahippocampal region in sagittal, horizontal, and tangential sections. (A) Posterior view of the left cortical hemisphere of a rat brain indicating the locations of medial entorhinal cortex (MEC), lateral entorhinal cortex (LEC), parasubiculum (PaS), perirhinal cortex (Per), and post-rhinal cortex (Por). (B) Brain slice prepared by a tangential cut through entorhinal cortex and adjacent regions, unfolded and gently flattened showing the positions of the subiculum (Sub), retrosplenial granular cortex (Rsg), retrosplenial agranular cortex (Rsa), occipital cortex (Occ) and the triangular region (^∗) in addition to the MEC, LEC, PaS, Per, and Por. Sections stained for acetylcholinesterase activity (AChE) indicating the parahippocampal regions in a saggital (C), horitzontal (D), and tangential (E) section. Scale bars = 1 mm. D, dorsal; V, ventral; L, lateral; M, medial; R, rostral; C, caudal. Orientation in (A) also applicable to (B,E). © 2014 The American Association for the Advancement of Science, (A) is adapted from Ray et al. (2014). Reprinted with permission from AAAS.

FIGURE 2

Medial-most part of the medial entorhinal cortex (mMEC), a transitional region between medial entorhinal cortex and parasubiculum. The region between the parasubiculum and calbindin patch region in the MEC is markedly different from the rest of the MEC and is termed as the medial-most MEC. (A) The mMEC is sandwiched between the MEC and PaS (red, Wfs1) on the medial end, and lacks calbindin patches (green) as illustrated in the tangential section. (B) A horizontal section shows that the mMEC, like the PaS extends from layer 1 to layer 3 of the MEC. (C) Same section processed for calbindin (green) and zinc (black) showing the boundary between mMEC and PaS. (D) Same section processed for calbindin (green) and AChE (black) showing the boundaries of mMEC. Scale bars: 250 μm. D, dorsal; V, ventral; M, medial; L, lateral; R, rostral; C, caudal. Orientation in (A) also applies to (C,D).

FIGURE 3

Medial-most part of the medial entorhinal cortex layout. The mMEC shows an organization of parallel stripes in tangential sections near the surface (upper panels). This pattern comes about by sectioning, from medial to lateral, through by a patch of calretinin positive neuropil located above a group of calretinin positive neurons, then a region with predominantly parvalbumin positive neuropil and then a narrow stripe of calbindin patches at the border to the parasubiculum. The same tangential section processed for calbindin (green, A), calretinin (red, B) and parvalbumin (white, C) show that they form adjacent bands in the mMEC (D). (E–H) The same horizontal section processed for calbindin (green, E), calretinin (red, F) and parvalbumin (white, G) shows that the mMEC (H) contains a lower density of calbindin positive neurons, a relatively thick layer of parvalbumin positive neuropil and a patch of calretinin positive neurons extending their dendrites to the surface of layer 1. Scale bars: 250 μm (D also for A–C), (H also for E–G). D, dorsal; V, ventral; M, medial; L, lateral; R, rostral; C, caudal. Orientation in (A) applies to upper four panels (A–D) and orientation in (G) applies lower four panels (E–H).

FIGURE 4

Cellular structure of layer 2 of medial entorhinal cortex. Calbindin (CB) and reelin-positive cells differ in spatial distribution and innervation by basket cells. (A) Tangential section through upper layer 2 of MEC stained for calbindin immunoreactivity. CB-positive neurons cluster and form characteristic patches. (B) In contrast reelin positive cells have a more uniform distribution and avoid the center of calbindin patches. (C–E) Cholecystokinin (CCK) positive terminals preferentially target calbindin-positive cells. Tangential section stained for CB (C) and CCK (D) immunoreactivity; enlarged overlay view with CCK positive terminals indicated by arrows in (E). (F–H) Parvalbumin (PV) positive terminals target calbindin-positive and calbindin-negative cells. Tangential section stained for CB (F) and PV (G) immunoreactivity; enlarged overlay view (H) with PV-positive terminals indicated by arrows. Scale bars: B = 500 μm (also for A); G = 50 μm (also for C,D,F). H = 25 μm (also for E). D, dorsal; V, ventral; L, lateral; M, medial. Orientation in (A) also applicable to (B).

FIGURE 5

Complementary modular systems in MEC. Relation of acetylcholinesterase (AChE) and zinc-positive modules to calbindin patches. (A) Tangential section stained for AChE, showing discrete clusters of staining. (B) Same section as in (A) co-stained for calbindin, showing calbindin patches. (C) High magnification view of overlay from (A,B) showing that calbindin patches overlap with AChE innervation. (D) Tangential section stained for synaptic zinc ions, showing discrete clusters of staining. (E) Same section as in (D) co-stained for calbindin, showing calbindin patches. (F) High magnification view of overlay from (D,E) showing that zinc and calbindin form complementary modules. Scale bars: A,C,D,F = 500 μm (A also for B, D also for E). D, dorsal; V, ventral; L, lateral; M, medial. Orientation in (A) applicable to all. © 2014 The American Association for the Advancement of Science, (A–C) is adapted from Ray et al. (2014). Reprinted with permission from AAAS.

FIGURE 6

Purkinje cell protein 4 (PCP4) is complementary to calbindin patches and overlaps with zinc modules. Apical dendrites from layers 3 and 5 Purkinje cell protein 4-positive cells (PCP4) bundle together in layer 2. Same section of layer 2 of the entorhinal cortex processed for CB (A), PCP4 (B) immunoreactivity and synaptic zinc ions (C). (D,E) Inset from (A–C) showing that PCP4 is complementary to CB-patches (D) and overlaps with zinc-modules. Scale bars: C = 500 μm (also for A,B); E = 250 μm (also for D). D, dorsal; V, ventral; L, lateral; M, medial. Orientation in (A) applicable to all.

FIGURE 7

Parasubiculum provides inputs to calbindin patches while presubiculum provides inputs to complementary modules. Anterograde injections performed in the PaS and PrS target complementary modules in layer 2 of the MEC. (A) Tangential section of layer 2 of the MEC showing that axons of neurons from the presubiculum (red) largely avoid the CB patches (green). (B,C) Inset region from (A) showing calbindin patches (B) and complementary regions innervated by axons (C) from a BDA anterograde injection in the presubiculum. (D) Schematic illustrating that presubiculum neurons target modules complementary to the CB patches in layer 2 of MEC. (E–G) Tangential section illustrating an anterograde injection (BDA, red) performed in the parasubiculum showing selective targeting of CB patches (green) in layer 2 of MEC (F,G). (H) Schematic illustrating that the parasubiculum targets CB patches in layer 2 of the MEC. Scale bars: A,C,E,G = 250 μm (C also for B, G also for F). D, dorsal; V, ventral; L, lateral; M, medial. Orientation in (E) applicable to all. (E–G) Adapted from Tang et al. (2016).

FIGURE 8

Modularity of presubiculum and parasubiculum. The pre and parasubiculum provides inputs to the zinc modules and calbindin patches, respectively, and also have a modular layout themselves. (A–C) Tangential sections showing modules in the presubiculum, with the modules being devoid of CB (green, A) and synaptic zinc (C) but being enriched for cholinergic activity (B). (D–F) Tangential sections showing the presence of large modules in the parasubiculum, enriched for PV (green, D) and synaptic zinc (F) but lacking PCP4 (E). Scale bars: A,C,D,F = 1 mm (C also for B, D also for E). D, dorsal; V, ventral; L, lateral; M, medial. Orientation in (E) applicable to all. (D) Adapted from Tang et al. (2016).

FIGURE 9

Modular structures in the parahippocampal region. Overview of modular structures in the superficial layers of MEC and neighboring regions. (A) Overview of modular structures in the superficial layers of MEC and the neighboring, mMEC, PaS, and PrS. Sections stained for different markers were aligned at the entorhinal/parasubicular border and modular structures drawn. (B) Schematic illustration of modular structures in the dorsal MEC and parasubiculum. D, dorsal; V, ventral; L, lateral; M, medial. © 2006 Elsevier, (B) adapted from Witter and Moser (2006). Adapted with permission from Elsevier.