Digital Atlas of Anatomical Subdivisions and Boundaries of the Rat Hippocampal Region

The rat hippocampal region is frequently studied in relation to learning and memory processes and brain diseases. The region is complex, consisting of multiple subdivisions that are challenging to delineate anatomically. Published atlases of the rat brain typically lack the underlying histological criteria necessary to identify boundaries, and textbooks descriptions of the region are often inadequately illustrated and thus difficult to relate to experimental data. An overview of both anatomical features and criteria used to delineate boundaries is required to assign location to experimental material from the hippocampal region. To address this issue, we have developed a web-based atlas application in which images of histological sections are integrated with new and up-to-date criteria for subdividing the rat hippocampus formation, fasciola, and associated parahippocampal regions. The atlas application consists of an interactive image viewer with high-resolution images of an extensive series of sections stained for NeuN, calbindin, and parvalbumin, and an index of structures with detailed descriptions of the criteria used to define the boundaries. Images can be inspected with a graphical overlay of selected subregions. Bi-directional links between images and the index of structures are provided. In summary, we provide a novel content-rich digital atlas resource facilitating identification of morphological features relevant for delineating the anatomical subdivisions of the rat hippocampal region. The atlas application is available at http://www.rbwb.org

Dual Transneuronal Tracing in the Rat Entorhinal-Hippocampal Circuit by Intracerebral Injection of Recombinant Rabies Virus Vectors

Dual transneuronal tracing is a novel viral tracing methodology which employs two recombinant viruses, each expressing a different reporter protein. Peripheral injection of recombinant pseudorabies viruses has been used as a powerful method to define neurons that coordinate outputs to various peripheral targets of motor and autonomic systems. Here, we assessed the feasibility of recombinants of rabies virus (RV) vector for dual transneuronal tracing in the central nervous system. First, we examined whether two different RV-vectors can double label cells in vitro, and showed that efficient double labeling can be realized by infecting targeted cells with the two RV-vectors within a short time interval. The potential of dual transneuronal tracing was then examined in vivo in the entorhinal-hippocampal circuit, using the chain of projections from CA3 pyramidal cells to CA1 pyramidal cells and subsequently to entorhinal cortex. Six days after the injection of two RV-vectors into the left and right entorhinal cortex respectively, double-labeled neurons were observed in CA3 bilaterally. Some double-labeled neurons showed a Golgi-like labeling. Dual transneuronal tracing potentially provides a powerful and sensitive method to study issues such as the amount of convergence and divergence within and between circuits in the central nervous system. Using this sensitive technique, we established that single neurons in CA3 are connected to the entorhinal cortex bilaterally with only one synaptic relay

What Does the Anatomical Organization of the Entorhinal Cortex Tell Us?

The entorhinal cortex is commonly perceived as a major input and output structure of the hippocampal formation, entertaining the role of the nodal point of cortico-hippocampal circuits. Superficial layers receive convergent cortical information, which is relayed to structures in the hippocampus, and hippocampal output reaches deep layers of entorhinal cortex, that project back to the cortex. The finding of the grid cells in all layers and reports on interactions between deep and superficial layers indicate that this rather simplistic perception may be at fault. Therefore, an integrative approach on the entorhinal cortex, that takes into account recent additions to our knowledge database on entorhinal connectivity, is timely. We argue that layers in entorhinal cortex show different functional characteristics most likely not on the basis of strikingly different inputs or outputs, but much more likely on the basis of differences in intrinsic organization, combined with very specific sets of inputs. Here, we aim to summarize recent anatomical data supporting the notion that the traditional description of the entorhinal cortex as a layered input-output structure for the hippocampal formation does not give the deserved credit to what this structure might be contributing to the overall functions of cortico-hippocampal networks

Projections of the insular cortex to orbitofrontal and medial prefrontal cortex. A tracing study in the rat

The dense fiber pathways that connect the insular cortex with frontal cortices are thought to provide these frontal areas with interoceptive information, crucial for their involvement in executive functions. Using anterograde neuroanatomical tracing, we mapped the detailed organization of the projections from the rat insular cortex to its targets in orbitofrontal (OFC) and medial prefrontal (mPFC) cortex. In OFC, main insular projections distribute to lateral and medial parts, avoiding ventral parts. Whereas projections from the primary gustatory cortex densely innervate dorsolateral OFC, likely corresponding to what in primates is known as the secondary gustatory cortex, these projections avoid mPFC. Instead, mPFC is targeted almost exclusively by projections from agranular fields of the insular cortex. Finally, “parietal” domains of the insular cortex project specifically to the dorsolateral OFC, and strongly innervate ventral portions of mPFC, i.e., the dorsal peduncular cortex

The Retrosplenial Cortex: Intrinsic Connectivity and Connections with the (Para)Hippocampal Region in the Rat. An Interactive Connectome

A connectome is an indispensable tool for brain researchers, since it quickly provides comprehensive knowledge of the brain's anatomical connections. Such knowledge lies at the basis of understanding network functions. Our first comprehensive and interactive account of brain connections comprised the rat hippocampal–parahippocampal network. We have now added all anatomical connections with the retrosplenial cortex (RSC) as well as the intrinsic connections of this region, because of the interesting functional overlap between these brain regions. The RSC is involved in a variety of cognitive tasks including memory, navigation, and prospective thinking, yet the exact role of the RSC and the functional differences between its subdivisions remain elusive. The connectome presented here may help to define this role by providing an unprecedented interactive and searchable overview of all connections within and between the rat RSC, parahippocampal region and hippocampal formation

Use of peroxidase substrate Vector VIP for multiple staining in light microscop

The study of the distribution of a fiber input to a particular brain area and the visualization of the anatomical relationships of that input with both projection- and interneurons, requires a triple-staining that allows the unequivocal distinction of each of the three components in one and the same histological section. In this regard, we investigated the properties of a recently introduced peroxidase chromogen, VIP (V-VIP; Vector Labs) in combination with two traditional substrates, standard diaminobenzidine (DAB, brown precipitate) and nickel-enhanced DAB (DAB-Ni, black). In rats, the anterograde tracer biotinylated dextran amine (BDA) and the retrograde tracer fluorogold (FG) were injected in the perirhinal cortex and hippocampus, respectively. Transported BDA was detected with an avidin-biotin-peroxidase complex, whereas the transported FG was detected via a PAP method. Tracing with BDA and FG was combined with parvalbumin- or calbindin-immunocytochemistry. We compared various combinations and staining sequences. The best results were obtained with a staining sequence comprising first the BDA stain with DAB-Ni as chromogen, second the FG protocol with the chromogen DAB and finally, parvalbumin- or calbinding-immunocytochemistry using the chromogen V-VIP. The order with which the chromogens were applied appeared to be critical. Partial or even total loss of V-VIP reaction product has been observed after standard dehydration in ethanol. As an alternative, a quick dehydration procedure in toluene yields much better staining. Colour separation is excellent and the sensitivity is high. This procedure may also be used for detection of any other combination of three different labels, taking the usual care to avoid cross-reactivity between antibodies

GABAA Receptor Subunit α3 in Network Dynamics in the Medial Entorhinal Cortex

Layer II of the medial entorhinal cortex (MEC LII) contains the largest number of spatially modulated grid cells and is one of the first regions in the brain to express Alzheimer’s disease (AD)-related pathology. The most common principal cell type in MEC LII, reelin-expressing stellate cells, are grid cell candidates. Recently we found evidence that γ-aminobutyric acid (GABA)A receptor subunits show a specific distribution in MEC LII, in which GABAA α3 is selectively associated with reelin-positive neurons, with limited association with the other principal cell type, calbindin (CB)-positive pyramidal neurons. Furthermore, the expression of α3 subunit decreases in mice between P15 and P25, which coincides with the emergence of stable grid cell activity. It has been shown that the α3 subunit undergoes specific developmental changes and that it may exert pro-inflammatory actions if improperly regulated. In this review article, we evaluate the changing kinetics of α3-GABAA receptors (GABAARs). during development in relation to α3-subunit expression pattern in MEC LII and conclude that α3 could be closely related to the stabilization of grid cell activity and theta oscillations. We further conclude that dysregulated α3 may be a driving factor in early AD pathology

Coexpression of vesicular glutamate transporters 1 and 2, glutamic acid decarboxylase and calretinin in rat entorhinal cortex

We studied the distribution and coexpression of vesicular glutamate transporters (VGluT1, VGluT2), glutamic acid decarboxylase (GAD) and calretinin (CR, calcium-binding protein) in rat entorhinal cortex, using immunofluorescence staining and multichannel confocal laser scanning microscopy. Images were computer processed and subjected to automated 3D object recognition, colocalization analysis and 3D reconstruction. Since the VGluTs (in contrast to CR and GAD) occurred in fibers and axon terminals only, we focused our attention on these neuronal processes. An intense, punctate VGluT1-staining occurred everywhere in the entorhinal cortex. Our computer program resolved these punctae as small 3D objects. Also VGluT2 showed a punctate immunostaining pattern, yet with half the number of 3D objects per tissue volume compared with VGluT1, and with statistically significantly larger 3D objects. Both VGluTs were distributed homogeneously across cortical layers, with in MEA VGluT1 slightly more densely distributed than in LEA. The distribution pattern and the size distribution of GAD 3D objects resembled that of VGluT2. CR-immunopositive fibers were abundant in all cortical layers. In double-stained sections we noted ample colocalization of CR and VGluT2, whereas coexpression of CR and VGluT1 was nearly absent. Also in triple-staining experiments (VGluT2, GAD and CR combined) we noted coexpression of VGluT2 and CR and, in addition, frequent coexpression of GAD and CR. Modest colocalization occurred of VGluT2 and GAD, and incidental colocalization of all three markers. We conclude that the CR-containing axon terminals in the entorhinal cortex belong to at least two subpopulations of CR-neurons: a glutamatergic excitatory and a GABAergic inhibitory

From Rapid Place Learning to Behavioral Performance: A Key Role for the Intermediate Hippocampus

Rapid place encoding by hippocampal neurons, as reflected by place-related firing, has been intensely studied, whereas the substrates that translate hippocampal place codes into behavior have received little attention. A key point relevant to this translation is that hippocampal organization is characterized by functional-anatomical gradients along the septotemporal axis: Whereas the ability of hippocampal neurons to encode accurate place information declines from the septal to temporal end, hippocampal connectivity to prefrontal and subcortical sites that might relate such place information to behavioral-control processes shows an opposite gradient. We examined in rats the impact of selective lesions to relevant parts of the hippocampus on behavioral tests requiring place learning (watermaze procedures) and on in vivo electrophysiological models of hippocampal encoding (long-term potentiation [LTP], place cells). We found that the intermediate hippocampus is necessary and largely sufficient for behavioral performance based on rapid place learning. In contrast, a residual septal pole of the hippocampus, although displaying intact electrophysiological indices of rapid information encoding (LTP, precise place-related firing, and rapid remapping), failed to sustain watermaze performance based on rapid place learning. These data highlight the important distinction between hippocampal encoding and the behavioral performance based on such encoding, and suggest that the intermediate hippocampus, where substrates of rapid accurate place encoding converge with links to behavioral control, is critical to translate rapid (one-trial) place learning into navigational performance