Edward G. Jones

Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data

Genome-wide expression profiling is a powerful tool for implicating novel gene ensembles in cellular mechanisms of health and disease. The most popular platform for genome-wide expression profiling is the Affymetrix GeneChip. However, its selection of probes relied on earlier genome and transcriptome annotation which is significantly different from current knowledge. The resultant informatics problems have a profound impact on analysis and interpretation the data. Here, we address these critical issues and offer a solution. We identified several classes of problems at the individual probe level in the existing annotation, under the assumption that current genome and transcriptome databases are more accurate than those used for GeneChip design. We then reorganized probes on more than a dozen popular GeneChips into gene-, transcript- and exon-specific probe sets in light of up-to-date genome, cDNA/EST clustering and single nucleotide polymorphism information. Comparing analysis results between the original and the redefined probe sets reveals ∼30–50% discrepancy in the genes previously identified as differentially expressed, regardless of analysis method. Our results demonstrate that the original Affymetrix probe set definitions are inaccurate, and many conclusions derived from past GeneChip analyses may be significantly flawed. It will be beneficial to re-analyze existing GeneChip data with updated probe set definitions.

Two classes of cortical GABA neurons defined by differential calcium binding protein immunoreactivities

SummaryCalcium ions play a key role in many aspects of neuronal behavior and certain calcium binding proteins that may influence this behavior are differentially distributed in the central nervous system. In this study it is shown that immunoreactivity for calbindin-28 and for parvalbumin is localized in separate populations of inhibitory GABA interneurons in all areas of the neocortex of Old World monkeys. Virtually all GABA neurosn show immunoreactivity for one or other calcium binding protein but, except for a few cells in layer IV, GABA cells do not show immunoreactivity for both proteins. Among the two cell populations, parvalbumin immunoreactivity characterizes basket neurons while calbindin immunoreactivity characterizes double bouquet neurons. These findings suggest that the two GABA cell types differ in their regulation of calcium homeostasis and may yield clues to their different roles in intracortical circuitry.

Altered cortical glutamatergic and GABAergic signal transmission with glial involvement in depression

Abnormalities in l-glutamic acid (glutamate) and GABA signal transmission have been postulated to play a role in depression, but little is known about the underlying molecular determinants and neural mechanisms. Microarray analysis of specific areas of cerebral cortex from individuals who had suffered from major depressive disorder demonstrated significant down-regulation of SLC1A2 and SLC1A3, two key members of the glutamate/neutral amino acid transporter protein family, SLC1. Similarly, expression of l-glutamate-ammonia ligase, the enzyme that converts glutamate to nontoxic glutamine was significantly decreased. Together, these changes could elevate levels of extracellular glutamate considerably, which is potentially neurotoxic and can affect the efficiency of glutamate signaling. The astroglial distribution of the two glutamate transporters and l-glutamate-ammonia ligase strongly links glia to the pathophysiology of depression and challenges the conventional notion that depression is solely a neuronal disorder. The same cortical areas displayed concomitant up-regulation of several glutamate and GABAA receptor subunits, of which GABAAα1 and GABAAβ3 showed selectivity for individuals who had died by suicide, indicating their potential utility as biomarkers of suicidality. These findings point to previously undiscovered molecular underpinnings of the pathophysiology of major depression and offer potentially new pharmacological targets for treating depression.

The thalamic matrix and thalamocortical synchrony

High-frequency synchronous activity of neurons in the cerebral cortex and thalamus is a concomitant of discrete conscious events. In the primate thalamus, a newly identified population of neurons provides a basis for this synchronization. A matrix of calbindin-immunoreactive neurons extends throughout the thalamus and projects to superficial layers of cortex over wide areas, unconstrained by boundaries between areas. In some nuclei, a core of parvalbumin-immunoreactive neurons is superimposed upon the matrix. Core neurons project in a topographically ordered fashion to middle layers of the cortex in an area-specific manner. Matrix neurons, recruited by corticothalamic connections, can disperse activity across cortical areas and thalamic nuclei. Their superficial terminations can synchronize specific and nonspecific elements of the thalamocortical network in coherent activity that underlies cognitive events.

Differential Calcium Binding Protein Immunoreactivity Distinguishes Classes of Relay Neurons in Monkey Thalamic Nuclei

The distributions of neurons displaying immunoreactivity for two calcium binding proteins, parvalbumin and 28Kd calbindin, were studied in the thalamus of M. fascicularis. Colocalization experiments were carried out to determine the extent to which parvalbumin‐ and calbindin‐like immunoreactivity was found in the same cells and the extent to which either was localized in GABAergic interneurons. Anterograde and retrograde tracing experiments involving the fluorescent tracer, fast blue, were also used to determine that cells expressing the calcium binding proteins projected upon the cerebral cortex.

A new parcellation of the human thalamus on the basis of histochemical staining

Serial sections of human thalami, cut in the 3 standard planes, were stained in alternating series for Nissl substance, myelin, cytochrome oxidase and acetylcholinesterase. Nissl and acetylcholinesterase-stained sections revealed a parcellation of the nuclei that could be correlated with that used in the macaque monkey thalamus. Human nuclei were accordingly re-named using the monkey nomenclature. Apart from differences of size, the nuclei of the human and monkey thalamus are remarkably similar. In the human ventral nuclear complex there is a very clear histochemical distinction between nuclei which, on the basis of comparison with the monkey, probably form the pallidal, cerebellar and lemniscal relays to premotor, motor and somatic sensory cortex, respectively. In the human somatic sensory relay nucleus there is a further clear cytoarchitectonic distinction between components that are probably equivalent to the relays for deep and cutaneous receptors in the equivalent monkey nucleus.

VIEWPOINT: THE CORE AND MATRIX OF THALAMIC ORGANIZATION

The integration of the whole cerebral cortex and thalamus during forebrain activities that underlie different states of consciousness, requires pathways for the dispersion of thalamic activity across many cortical areas. Past theories have relied on the intralaminar nuclei as the sources of diffuse thalamocortical projections that could facilitate spread of activity across the cortex. A case is made for the presence of a matrix of superficially-projecting cells, not confined to the intralaminar nuclei but extending throughout the whole thalamus. These cells are distinguished by immunoreactivity for the calcium-binding protein, D28K calbindin, are found in all thalamic nuclei of primates and have increased numbers in some nuclei. They project to superficial layers of the cerebral cortex over relatively wide areas, unconstrained by architectonic boundaries. They generally receive subcortical inputs that lack the topographic order and physiological precision of the principal sensory pathways. Superimposed upon the matrix in certain nuclei only, is a core of cells distinguished by immunoreactivity for another calcium-binding protein, parvalbumin, These project in highly ordered fashion to middle layers of the cortex in an area-specific manner. They are innervated by subcortical inputs that are topographically precise and have readily identifiable physiological properties. The parvalbumin cells form the basis for sensory and other inputs that are to be used as a basis for perception. The calbindin cells, especially when recruited by corticothalamic connections, can form a basis for the engagement of multiple cortical areas and thalamic nuclei that is essential for the binding of multiple aspects of sensory experience into a single framework of consciousness.

Effect of Agonal and Postmortem Factors on Gene Expression Profile: Quality Control in Microarray Analyses of Postmortem Human Brain

There are major concerns that specific agonal conditions, including coma and hypoxia, might affect ribonucleic acid (RNA) integrity in postmortem brain studies. We report that agonal factors significantly affect RNA integrity and have a major impact on gene expression profiles in microarrays. In contrast to agonal factors, gender, age, and postmortem factors have less effect on gene expression profiles. The Average Correlation Index is proposed as a method for evaluating RNA integrity on the basis of similarity of microarray profiles. Reducing the variance due to agonal factors is critical in investigating small but validated gene expression differences in messenger RNA levels between psychiatric patients and control subjects.

Activity-dependent regulation of GABA expression in the visual cortex of adult monkeys

Levels of the inhibitory transmitter, GABA, and its synthesizing enzyme, GAD, appear to be regulated in the visual cortex of young adult monkeys in an activity-dependent manner. In monkeys subjected to monocular deprivation by eye removal, tetrodotoxin injection, or eyelide suture, the number of GABA and GAD immunoreactive neurons in deprived-eye columns of the cortex is reduced by up to 50%. This effect is unaccompanied by cell death and is reversible. After cessation of TTX injection or reopening of the eyes, the number of immunostained cells returns to normal. The effect appears after 4-5 days of eye removal or tetrodotoxin injection, but only after 7-16 weeks of eyelid suture. In the latter case, it is more severe in the younger monkeys. The reversible reduction in GABA and GAD immunostaining extends out of layer IVC into lay IVA and to neurons around but not in cytochrome oxidase periodicities of layer III. This may indicate selective vulnerability of GABA cells sensitive to high spatial frequency.

Switching of NMDA Receptor 2A and 2B Subunits at Thalamic and Cortical Synapses during Early Postnatal Development

Switching of the NMDA receptor 2A (NR2A) and NR2B subunits at NMDA receptors is thought to underlie the functional changes that occur in NMDA receptor properties during the developmental epoch when neural plasticity is most pronounced. The cellular expression of NR2A and NR2B and the NR2 synaptic binding protein postsynaptic density-95 (PSD-95) was examined in the mouse somatosensory cortex and thalamus from postnatal day 2 (P2) to P15 using reverse transcription-PCR, in situ hybridization histochemistry, and immunocytochemistry. The localization of NR2A and NR2B subunits and PSD-95 was then studied at synapses in layer IV of somatosensory cortex and in the ventral posterior nucleus of the thalamus using high-resolution immunoelectron microscopy. At both cortical and thalamic synapses, a quantitative switch in the dominant synaptic subunit from NR2B to NR2A was accompanied by a similar change in the cellular expression of NR2A but not of NR2B. Synaptic PSD-95 developed independently, although both NR2A and NR2B colocalized with PSD-95. Displacement of NR2B subunits from synapses was not accompanied by an increase in an extrasynaptic pool of this subunit. Thus, the switch in synaptic NR2 subunit predominance does not occur by changes in expression or displacement from synapses and may reflect the formation of new synapses from which NR2B is lacking.