Jon I. Arellano

Microstructure of the neocortex: Comparative aspects

The appearance of the neocortex, its expansion, and its differentiation in mammals, represents one of the principal episodes in the evolution of the vertebrate brain. One of the fundamental questions in neuroscience is what is special about the neocortex of humans and how does it differ from that of other species? It is clear that distinct cortical areas show important differences within both the same and different species, and this has led to some researchers emphasizing the similarities whereas others focus on the differences. In general, despite of the large number of different elements that contribute to neocortical circuits, it is thought that neocortical neurons are organized into multiple, small repeating microcircuits, based around pyramidal cells and their input-output connections. These inputs originate from extrinsic afferent systems, excitatory glutamatergic spiny cells (which include other pyramidal cells and spiny stellate cells), and inhibitory GABAergic interneurons. The problem is that the neuronal elements that make up the basic microcircuit are differentiated into subtypes, some of which are lacking or highly modified in different cortical areas or species. Furthermore, the number of neurons contained in a discrete vertical cylinder of cortical tissue varies across species. Additionally, it has been shown that the neuropil in different cortical areas of the human, rat and mouse has a characteristic layer specific synaptology. These variations most likely reflect functional differences in the specific cortical circuits. The laminar specific similarities between cortical areas and between species, with respect to the percentage, length and density of excitatory and inhibitory synapses, and to the number of synapses per neuron, might be considered as the basic cortical building bricks. In turn, the differences probably indicate the evolutionary adaptation of excitatory and inhibitory circuits to particular functions.

Ultrastructure of Dendritic Spines: Correlation Between Synaptic and Spine Morphologies

Dendritic spines are critical elements of cortical circuits, since they establish most excitatory synapses. Recent studies have reported correlations between morphological and functional parameters of spines. Specifically, the spine head volume is correlated with the area of the postsynaptic density (PSD), the number of postsynaptic receptors and the ready-releasable pool of transmitter, whereas the length of the spine neck is proportional to the degree of biochemical and electrical isolation of the spine from its parent dendrite. Therefore, the morphology of a spine could determine its synaptic strength and learning rules. To better understand the natural variability of neocortical spine morphologies, we used a combination of gold-toned Golgi impregnations and serial thin-section electron microscopy and performed three-dimensional reconstructions of spines from layer 2/3 pyramidal cells from mouse visual cortex. We characterized the structure and synaptic features of 144 completed reconstructed spines, and analyzed their morphologies according to their positions. For all morphological parameters analyzed, spines exhibited a continuum of variability, without clearly distinguishable subtypes of spines or clear dependence of their morphologies on their distance to the soma. On average, the spine head volume was correlated strongly with PSD area and weakly with neck diameter, but not with neck length. The large morphological diversity suggests an equally large variability of synaptic strength and learning rules.

Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling

Primary cilia are present on mammalian neurons and glia, but their function is largely unknown. We generated conditional homozygous mutant mice for a gene we termed Stumpy. Mutants lack cilia and have conspicuous abnormalities in postnatally developing brain regions, including a hypoplasic hippocampus characterized by a primary deficiency in neural stem cells known as astrocyte-like neural precursors (ALNPs). Previous studies suggested that primary cilia mediate sonic hedgehog (Shh) signaling. Here, we find that loss of ALNP cilia leads to abrogated Shh activity, increased cell cycle exit, and morphological abnormalities in ALNPs. Processing of Gli3, a mediator of Shh signaling, is also altered in the absence of cilia. Further, key mediators of the Shh pathway localize to ALNP cilia. Thus, selective targeting of Shh machinery to primary cilia confers to ALNPs the ability to differentially respond to Shh mitogenic signals compared to neighboring cells. Our data suggest these organelles are cellular “antennae” critically required to modulate ALNP behavior.

Non-synaptic dendritic spines in neocortex.

A long-held assumption states that each dendritic spine in the cerebral cortex forms a synapse, although this issue has not been systematically investigated. We performed complete ultrastructural reconstructions of a large (n=144) population of identified spines in adult mouse neocortex finding that only 3.6% of the spines clearly lacked synapses. Nonsynaptic spines were small and had no clear head, resembling dendritic filopodia, and could represent a source of new synaptic connections in the adult cerebral cortex.

Everything that Glitters Isn't Gold: A Critical Review of Postnatal Neural Precursor Analyses

Adult neurogenesis research has made enormous strides in the last decade but has been complicated by several failures to replicate promising findings. Prevalent use of highly sensitive methods with inherent sources of error has led to extraordinary conclusions without adequate crossvalidation. Perhaps the biggest culprit is the reliance on molecules involved in DNA synthesis and genetic markers to indicate neuronal neogenesis. In this Protocol Review, we present an overview of common methodological issues in the field and suggest alternative approaches, including viral vectors, siRNA, and inducible transgenic/knockout mice. A multipronged approach will enhance the overall rigor of research on stem cell biology and related fields by allowing increased replication of findings between groups and across systems.

Pyramidal cell axons show a local specialization for GABA and 5-HT inputs in monkey and human cerebral cortex

Various mechanisms are thought to control excitation of pyramidal cells of the cerebral cortex. With immunocytochemical methods, we found that the proximal portions of numerous pyramidal cell axons (Pyr‐axons) in the human and monkey neocortex are immunoreactive for the serotonin (5‐HT) receptor 5‐HT‐1A. With double‐labeling experiments and confocal laser microscopy, we found that most (93.4%) of the 5‐HT1A–immunoreactive Pyr‐axons present in layers II and III were innervated by parvalbumin‐immunoreactive chandelier cell axon terminals. In addition, Pyr‐axons were compartmentalized: 5‐HT‐1A receptors were found proximal to inputs from chandelier cells. Although we found close appositions between GABAergic chandelier cell axon terminals and Pyr‐axons, suggesting synaptic connections, we did not observe 5‐HT–immunoreactive fibers in close proximity to the Pyr‐axons. These results suggested that Pyr‐axons are under the influence of 5‐HT in a paracrine manner (via 5‐HT‐1A receptors) and, more distally, are under the influence of γ‐aminobutyric acid (GABA) in a synaptic manner (through the axons of chandelier cells). The local axonal specialization might represent a powerful inhibitory mechanism by which the responses of large populations of pyramidal cells can be globally controlled by subcortical serotonin afferents, in addition to local inputs from GABAergic interneurons. J. Comp. Neurol. 433:148–155, 2001.

Quantitative analysis of parvalbumin-immunoreactive cells in the human epileptic hippocampus

Hippocampal sclerosis is the most frequent pathology encountered in mesial temporal structures resected from patients with intractable temporal lobe epilepsy and it mainly involves hippocampal neuronal loss and gliosis. These alterations are accompanied by changes in the expression of a variety of molecules in the surviving neurons, as well as axonal reorganization in both excitatory and inhibitory circuits. The alteration of a subpopulation of GABAergic interneurons that expresses the calcium binding protein parvalbumin (PV) is thought to be a key factor in the epileptogenic process. We investigated the distribution and density of parvalbumin-immunoreactive (PV-ir) neurons in surgically resected hippocampal tissue from epileptic patients with and without sclerosis. Using quantitative stereological methods, we show for the first time that there is no correlation between total neuronal loss and PV-ir neuronal loss in any of the hippocampal fields. We also observed higher values of the total neuronal density in the sclerotic subiculum, which is accompanied by a lower density of PV-ir when compared with non-sclerotic epileptic and autopsy hippocampi. These findings suggest that, the apparently normal subiculum from sclerotic patients also shows unexpected changes in the density and proportion of PV-ir neurons.

Species-Dependent Posttranscriptional Regulation of NOS1 by FMRP in the Developing Cerebral Cortex

Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism, results from loss of function of the RNA-binding protein FMRP. Here, we show that FMRP regulates translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is widely expressed, NOS1 protein is transiently coexpressed with FMRP during early synaptogenesis in layer- and region-specific pyramidal neurons. These include midfetal layer 5 subcortically projecting neurons arranged into alternating columns in the prospective Brocas area and orofacial motor cortex. Human NOS1 translation is activated by FMRP via interactions with coding region binding motifs absent from mouse Nos1 mRNA, which is expressed in mouse pyramidal neurons, but not efficiently translated. Correspondingly, neocortical NOS1 protein levels are severely reduced in developing human FXS cases, but not FMRP-deficient mice. Thus, alterations in FMRP posttranscriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.

CA1 Hippocampal Neuronal Loss in Familial Alzheimer's Disease Presenilin-1 E280A Mutation Is Related to Epilepsy

Summary:  Purpose: Alzheimer disease (AD) and epilepsy are brain disorders frequently associated with neuronal cell loss in mesial temporal lobe structures, but presenting different patterns of damage. Recently it was proposed that a causal relation may exist between AD pathology and the appearance of epilepsy in some cases with AD. This study aimed to determine the neuronal loss in CA1 hippocampal region from patients bearing the presenilin‐1 [E280A] mutation (PS1[E280A]) associated with seizures.

Development and Distribution of Neuronal Cilia in Mouse Neocortex

Neuronal primary cilia are not generally recognized, but they are considered to extend from most, if not all, neurons in the neocortex. However, when and how cilia develop in neurons are not known. This study used immunohistochemistry for adenylyl cyclase III (ACIII), a marker of primary cilia, and electron microscopic analysis to describe the development and maturation of cilia in mouse neocortical neurons. Our results indicate that ciliogenesis is initiated in late fetal stages after neuroblast migration, when the mother centriole docks with the plasma membrane, becomes a basal body, and grows a cilia bud that we call a procilium. This procilium consists of a membranous protrusion extending from the basal body but lacking axonemal structure and remains undifferentiated until development of the axoneme and cilia elongation starts at about postnatal day 4. Neuronal cilia elongation and final cilia length depend on layer position, and the process extends for a long time, lasting 8–12 weeks. We show that, in addition to pyramidal neurons, inhibitory interneurons also grow cilia of comparable length, suggesting that cilia are indeed present in all neocortical neuron subtypes. Furthermore, the study of mice with defective ciliogenesis suggested that failed elongation of cilia is not essential for proper neuronal migration and laminar organization or establishment of neuronal polarity. Thus, the function of this organelle in neocortical neurons remains elusive. J. Comp. Neurol., 2012.