Stereology and neuronal connectivity of the rat hippocampus: from 2d images to 3d modeling

Synaptic micro-circuit properties in neuronal networks are fundamental to information processing in brain systems. These properties, intrinsic to brain structure, are directly related to neural activity and function. Characterization and mapping the micro-circuitry has been a long standing challenge in mammalian hippocampal research. One of the stumbling blocks in this endeavor is the absence of a construct to integrate the available anatomical knowledge. Thus, integrating hippocampal anatomy from neuronal dendrites to whole-system level may help explain its relation to spatial navigation and episodic memory. This dissertation describes a novel approach to map existing morphological data onto an in-silico based template of the rat hippocampus and address important scientific questions on macroscopic stereology and potential connectivity between various cell types in the hippocampus. Towards this aim, we digitally traced the cytoarchitectural boundaries of the dentate gyrus (DG) and areas CA3/CA1 throughout their entire longitudinal extent from high-resolution images of thin cryostatic sections of adult rat brain. A custom developed computational framework further extends the functionality of this model by transforming the digital trace stack into volumetric representations with arbitrary voxel size. Next, virtually embedding 1.8 million neuronal morphologies stochastically resampled from 244 digital reconstructions, emulated the dense packing of granular and pyramidal layers, and orienting the principal dendritic axes according to local curvature. Utilizing this unique systems level digital representation, the first part of this research study reports and discusses the macroscopic stereological properties such as volumes, and neuropil occupancy ratios across the various cytoarchitectonic layers of DG & CA regions in the rat hippocampus. The neuropil occupancy reproduced recent electron microscopy data specifically measured in a restricted location. Extension of this analysis across each layer and sub-region throughout the whole longitudinal extent of the hippocampus revealed highly non-homogeneous dendritic density. In CA1, dendritic occupancy was >60% higher temporally than septally (0.46 vs. 0.28). CA3 values varied both across subfields (from 0.35 in CA3b/CA3c to 0.50 in CA3a) and layers (0.48, 0.34, and 0.27 in oriens, radiatum, and lacunosum-moleculare, respectively). Dendritic occupancy was substantially lower in DG, especially in the supra-pyramidal blade (0.18). The computed probability of dendro-dendritic collision significantly correlated with expression of the membrane repulsion signal DSCAM. These heterogeneous stereological properties reflect and complement the non-uniform molecular composition, circuit connectivity, and computational function of the hippocampus across its hippocampal-transverse, longitudinal, and laminar organization. The second part of this dissertation reports and discusses the potential synaptic connectivity computed by mapping and orienting digital axonal reconstructions of five principal and two CA3 interneuron classes across the CA pyramidal dendritic network within this 3D model. In the mammalian cortex, structural plasticity of spines and boutons makes 'potential synapses' functionally relevant to learning capability and memory capacity. To date, however, potential synapses have only been mapped in the surrounding of a neuron and relative to its local orientation rather than in a system-level anatomical reference. Analyzing connectivity in terms of close spatial appositions between axons and dendrites could thus bridge the opposite scales, from synaptic level to whole systems. We report the potential connectivity onto pyramidal cell dendrites from the axons of a dentate granule cell, three CA3 pyramidal cells, one CA2 pyramidal cell, and 13 CA3b interneurons. The numbers, densities, and distributions of potential synapses were analyzed in each sub-region (e.g. CA3 vs. CA1), layer (e.g. oriens vs. radiatum), and septo-temporal location (e.g. dorsal vs. ventral). The overall ratio between the numbers of actual and potential synapses was ∼0.20 for the granule and CA3 pyramidal cells. All potential connectivity patterns are strikingly dependent on the anatomical location of both pre-synaptic and post-synaptic neurons.