Hippocampal anatomy, Altered

The hippocampus is a brain region located in the medial temporal lobe in humans and other mammals (West, 1990). Developmentally it is derived from neuronal and glial cells in the neural tube and differentiates in the proencephalon and telencephalon.  The hippocampus is a cortical structure, but only contains 3-layers, distinct from the 6-layered neocortical structures. For this reason, it is known as archicortex or paleocortex meaning old cortex. In humans, at the macro level, the structure is identified as early as fetal week 13 and continues to mature until 2 to 3 years of age (Kier et al., 1997), with continuing slow growth thereafter until adult ages (Utsunomiya et al., 1999).  In rodents, the hippocampus begins to form in mid-gestation, with the CA fields forming in advance of the dentate gyrus (Altman and Bayer, 1990a; 1990b). The primary structural and functional development of the hippocampus occurs in the third trimester of pregnancy in humans, whereas in rodents, much of the maturation of the CA fields and almost all dentate gyrus occurs in the first 2-3 postnatal weeks.

The structure of the hippocampus has been divided into regions that include CA1 through CA4 and the dentate gyrus. The principal cell bodies of the CA field are pyramidal neurons, those of the dentate gyrus are granule cells.

The major input pathway to the hippocampus is from the layer 2 neurons of the entorhinal cortex to the dentate gyrus via the perforant path forming the first connection of the trisynaptic loop of the hippocampal circuit (Figure 2). Direct afferents from the dentate gyrus (mossy fibers) then synapse on CA3 pyramidal cells which in turn send their axons (Schaeffer Collaterals) to CA1 neurons to complete the trisynaptic circuit. Information from the CA fields then passes through the subiculum entering the fiber pathways of the alveus, fimbria, and fornix and is routed to other areas of the brain (Amaral and Lavenex, 2006). Through the interconnectivity within the hippocampus and its connections to amygdala, septum and cortex, the hippocampus plays a pivotal role in several learning and memory processes, including spatial behaviors. The primary input pathway to the CA regions of the hippocampus is from the septum by way of the fornix and direct input from the amygdala. Reciprocal outputs from the hippocampus back to these regions and beyond also exist. 

Figure 2. Trisynaptic circuit of hippocampal formation. For further details see Amaral and Lavenex, 2006.

At the cellular level, the components of the mammalian hippocampus undergo typical stages of neurodevelopment. With each developmental time window, distinct patterns of gene transcription and protein expression appear, corresponding to cell proliferation, differentiation, migration, synapse formation, and terminal neuronal/glial maturation, culminating in the structural formation of a neuronal network (Mody et al., 2001; Laeremans et al., 2013).  The principal neurons of the CA fields develop in advance of the principal cells of the dentate gyrus and the genes and proteins controlling the distinct phases are expressed at different stages in these two sub-regions (Altman and Bayer, 1990 a, b; Laeremans et al., 2013). In the rodent brain, almost all neurons show extensive growth and differentiation on axons and dendrites during the first postnatal week. These cellular changes are marked by rapid protein expression specific for different neuronal and glial subtypes including cytoskeletal proteins (e.g. cofilin, actins, tubulins etc..), production of cell adhesion molecules, and extracellular matrix formation which are critical structural elements of a neuronal network.

As neurons mature, they extend dendritic processes that lengthen and branch, the ends of which broaden to form a spine head. Dendritic spines form the postsynaptic structural component of most excitatory synapses in the mammalian brain, including hippocampus. The spine head has a greater potential for connectivity and synapse formation (Dailey and Smith, 1996; Fiala et al., 1998; Hardy, LR and Redmond, 2008, Pfeiffer et al., 2018).   The postsynaptic density-95 (PSD-95) is one of the key proteins involved in dendritic spine maturation, clustering of synaptic signalling proteins, and ultimately mediating synaptic transmission. It also plays a critical role in regulating dendrite outgrowth and branching and formation dendritic spines.

As the hippocampus matures during the postnatal period hippocampal circuits become more active and exhibit increased activity-dependent plasticity. Many genes and proteins are upregulated during this phase of development, especially molecules involved in the axon guidance (e.g. BDNF/CREB) (Hinkemeyer et al., 2003; Shen and Cowan., 2010), dendritic spine formation (e.g. Neuroligin, Ephrins) and synaptogenesis. Increased expression of vesicle associated proteins (e.g., SNAP-25), synaptic vesicle proteins (e.g., synaptophysin, synapsin I) and proteins involved in sodium and calcium-mediated transmitter release occurs during this period. These changes are accompanied by a parallel increase in neurotrophins and neurotransmitters, receptors and ion channels (Sudhof, 2018; Zhong et al., 2020; Rizo and Rosenmund, 2008).  Therefore, any alterations in the expression of these proteins (Figure 3) may result in changes of synapse formation, followed by alteration of neuronal networks within the hippocampus. 

Figure 3. The main structural components of the synapse: (A) a presynaptic and a postsynaptic neuron, separated by a synaptic cleft; (B) the dendritic spines and (C) the proteins involved in synaptic formation and transmission, including synaptic vesicle (SV), presynaptic and postsynaptic proteins present in excitatory and inhibitory synapse. Adapted from Serrano et al., 2022.

The dendritic spine represents the primary site of synaptic activity at the postsynaptic site. A variety of proteins present in the presynaptic terminal and the postsynaptic dendritic spine are expressed at different times during synaptogenesis, perturbation of which can negatively impact synaptic formation and structure at the macro- and ultra- structural level.

The use of genetically modified mouse models has been widely applied to delineate a host of different proteins involved in the structural development of the hippocampus (Joo et al., 2020). With this approach, changes in neuronal morphology, synapse and network formation in the hippocampus are contrasted in animals lacking this protein vs ‘wild type’ mice where the protein has been maintained. These comparisons have adopted a variety of techniques, several described below. In KE4, differential expression of proteins identified in these model systems is taken as evidence of altered structure.

However, it is essential to consider the timing of events during development, when their detection is optimal (Hevner, 2007; Garman et al., 2001; Zgraggen et al., 2012). Some macrolevel structural changes may be transient yet still significantly impact downstream events. In the case of knockout models, it is also important to recognize that in most cases, the protein has been removed for the entire lifespan of the animal, in the brain and elsewhere in the body, a scenario distinct from a chemical perturbation.

Data in support of this key event have been collected using a wide variety of standard biochemical, molecular, cellular, histological and anatomical methods (e.g., morphometrics, protein quantification using different types of cellular staining, immunohistochemistry, and imaging procedures) at different stages of hippocampus development. Many of methods applied are routine neurohistopathology procedures like those recommended in EPA and OECD developmental neurotoxicity guidelines (US EPA, 1998; OCED, 2007). The quantification of cell body and neurite proteins can be carried out by performing immunocytochemistry and automated high content imaging analyses in in vitro and in vivo preparations (Harrill and Mundy, 2011, Meng et al., 2002), Pistollato et al., 2020). Subtle cytoarchitectural features depend on more specialized birth dating procedures and staining techniques. At the micro- and ultra- structural levels, changes in neuronal and glial morphology, alterations in synapse structure, dendritic spine formation (size, shape, number,  distribution of head/neck ratios  in hippocampal cultures or in vivo studies) and dendritic morphology (branching points, length etc..) can be assessed in Golgi-Cox impregnated neurons (Bongmbaa et al., 2011), two-photon microscopy (Ehrlich et al., 2007), transmission electron (TEM) and fluorescent microscopy  ( Runge et al., 2020; Pchitskaya et al., 2020)

Two-photon time-lapse images can be used to visualise dendrites in GFP-transfected neurons, whereas Golgi Stain is used to measure both dendrites and dendritic spines. A combination of Golgi-Cox and immunofluorescence using confocal microscopy has also been suggested for the visualisation of dendrites in brain slices derived either from rodents or non-human primates (Levine et al., 2013). 

Fluorescent markers, such as Dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) permits not only the visualisation of detailed dendritic arborizations and spines in cell culture and tissue sections but is also compatible with the quantitative analysis of dendritic spine number (Cheng et al., 2014). 

Immunostaining with specific antibodies that recognize presynaptic proteins of excitatory and inhibitory neurons (i.e., vesicular glutamate transporters, vesicular GABA proteins and transporters) and the postsynaptic density protein-95 kDa (PSD-95) can be applied to enumerate synapse number (Gatto and Broadie, 2010, Akashi et al., 2009). There are commercially available ‘synaptogenesis assay kits’ that rely on the immunostaining of cells with common synaptic marker proteins such as MAP-2, PSD-95 and synaptophysin. Some other presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) markers have been shown to correlate well with the ultrastructural studies in cultured hippocampus primary cells (Grabrucker et al., 2009).

Electron microscopy can also be applied to assess the prevalence of excitatory and inhibitory synapses amongst convergent contacts (Megias et al., 2001). Recently, a high content image analysis based on RNAi screening protocols has been suggested as a useful tool to create imaging algorithm for use in both in vitro and in vivo synaptic punctae analysis (Nieland et al., 2014). 

Some of the same techniques used in rodent studies have been applied to postmortem tissue in humans.  In addition, non-invasive, structural neuroimaging techniques in living subjects are also widely used in human studies to assess hippocampal volume using voxel-based morphometry (VBM). With this approach, volume of brain regions is measured by drawing ‘regions of interest’ on images from brain scans obtained from magnetic resonance imaging (MRI) or positron emission tomography (PET) scans calculating the volume enclosed (Mechelli et al., 2005). These imaging techniques can be applied in rodent models (Powell et al., 2009; Hasegawa et al., 2010; Pirko et al., 2005; Pirko and Johnson, 2008). 

It is recognized that most of these biochemical, molecular, cellular, histological and anatomical methods (e.g., morphometrics, protein quantification using different types of cellular staining, immunohistochemistry, and imaging procedures) can also be applied to complex in vitro test systems (Pamies et al., 2016; Hartman et al., 2023; Pomeshchik et al., 2020) Human brain spheres or brain organoids could be developed in three-dimensional cell culture, resembling hippocampus  at different stages development (Sakaguchi et al., 2015). These new methods, if assessed as robust, reliable and reproducible, would allow measurement of the KE in a human-relevant test system. 

The hippocampus is generally similar in structure function across most mammalian species (West, 1990). The vast majority of information on the structure of the hippocampus is from mice, rats and primates including humans.

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