Anda Cornea

Determination of Axonal and Dendritic Orientation Distributions Within the Developing Cerebral Cortex by Diffusion Tensor Imaging

As neurons of the developing brain form functional circuits, they undergo morphological differentiation. In immature cerebral cortex, radially-oriented cellular processes of undifferentiated neurons impede water diffusion parallel, but not perpendicular, to the pial surface, as measured via diffusion-weighted magnetic resonance imaging, and give rise to water diffusion anisotropy. As the cerebral cortex matures, the loss of water diffusion anisotropy accompanies cellular morphological differentiation. A quantitative relationship is proposed here to relate water diffusion anisotropy measurements directly to characteristics of neuronal morphology. This expression incorporates the effects of local diffusion anisotropy within cellular processes, as well as the effects of anisotropy in the orientations of cellular processes. To obtain experimental support for the proposed relationship, tissue from 13 and 31 day-old ferrets was stained using the rapid Golgi technique, and the 3-D orientation distribution of neuronal proceses was characterized using confocal microscopic examination of reflected visible light images. Coregistration of the MRI and Golgi data enables a quantitative evaluation of the proposed theory, and excellent agreement with the theoretical results, as well as agreement with previously published values for locally-induced water diffusion anisotropy and volume fraction of the neuropil, is observed.

Sexual differentiation of projections from the principal nucleus of the bed nuclei of the stria terminalis

The principal nucleus of the bed nuclei of the stria terminalis (BSTp) is sexually dimorphic and participates in several aspects of reproduction. A detailed analysis of its projections revealed that the BSTp provides major inputs to forebrain regions that are sexually dimorphic and contain high densities of neurons that express receptors for sex steroid hormones in a pattern that is remarkably similar to that of the medial amygdaloid nucleus. The BSTp sends its strongest outputs to the periventricular zone of the hypothalamus and innervates structures thought to play important roles in regulating hormone secretion from the anterior pituitary, but it also provides strong inputs to the medial preoptic and ventromedial nuclei of the hypothalamus. The BSTp also sends a strong return projection to the medial nucleus of the amygdala. The projections of the BSTp appear to be more robust in males with striking sex differences observed in most, but not all, major terminal fields. Moreover, various terminal fields appeared to differ in their developmental sensitivity to manipulation of circulating levels of sex steroids during the neonatal period. Thus, the organization of projections from the BSTp suggests that it plays a particularly important role in regulating neuroendocrine function and that neurons in this nucleus may relay olfactory information to the hypothalamus differently in male and female rats. Furthermore, the differential action of sex steroids on the density of afferents from the BSTp in various regions indicates that these hormones exert a target‐specific influence on the development of BSTp projections. J. Comp. Neurol. 460:542–562, 2003.

Vascular Endothelial Cells Promote Acute Plasticity in Ependymoglial Cells of the Neuroendocrine Brain

Glial and endothelial cells interact throughout the brain to define specific functional domains. Whether endothelial cells convey signals to glia in the mature brain is unknown but is amenable to examination in circumventricular organs. Here we report that purified endothelial cells of one of these organs, the median eminence of the hypothalamus, induce acute actin cytoskeleton remodeling in isolated ependymoglial cells and show that this plasticity is mediated by nitric oxide (NO), a diffusible factor. We found that both soluble guanylyl cyclase and cyclooxygenase products are involved in this endothelial-mediated control of ependymoglia cytoarchitecture. We also demonstrate by electron microscopy that activation of endogenous NO release in the median eminence induces rapid structural changes, allowing a direct access of neurosecretory axons containing gonadotropin-releasing hormone (GnRH) (the neuropeptide controlling reproductive function) to the portal vasculature. Local in vivo inhibition of NO synthesis disrupts reproductive cyclicity, a process that requires a pulsatile, coordinated delivery of GnRH into the hypothalamic-adenohypophyseal portal system. Our results identify a previously unknown function for endothelial cells in inducing neuroglial plasticity and raise the intriguing possibility that endothelial cells throughout the brain may use a similar signaling mechanism to regulate glial-neuronal interactions.

REFOLDING OF MISFOLDED MUTANT GPCR: POST-TRANSLATIONAL PHARMACOPERONE ACTION IN VITRO

All reported GnRH receptor mutants (causing human hypogonadotropic hypogonadism) are misfolded proteins that cannot traffic to the plasma membrane. Pharmacoperones correct misfolding and rescue mutants, routing them to the plasma membrane where they regain function. Because pharmacoperones are often peptidomimetic antagonists, these must be removed for receptor function after rescue; in vivo this necessitates pulsatile pharmacoperone administration. As an antecedent to in vivo studies, we determined whether pharmacoperones need to be present at the time of synthesis or whether previously misfolded proteins could be refolded and rescued. Accordingly, we blocked either protein synthesis or intra-cellular transport. Biochemical and morphological studies using 12 mutants and 10 pharmacoperones representing three different chemical classes show that previously synthesized mutant proteins, retained by the quality control system (QCS), are rescued by pharmacoperones, showing that pharmacoperone administration in vivo likely need not consider whether the target protein is being synthesized at the time of drug administration.

Molecular mechanism of action of pharmacoperone rescue of misrouted GPCR mutants: the GnRH receptor.

The human GnRH receptor (hGnRHR), a G protein-coupled receptor, is a useful model for studying pharmacological chaperones (pharmacoperones), drugs that rescue misfolded and misrouted protein mutants and restore them to function. This technique forms the basis of a therapeutic approach of rescuing mutants associated with human disease and restoring them to function. The present study relies on computational modeling, followed by site-directed mutagenesis, assessment of ligand binding, effector activation, and confocal microscopy. Our results show that two different chemical classes of pharmacoperones act to stabilize hGnRHR mutants by bridging residues D(98) and K(121). This ligand-mediated bridge serves as a surrogate for a naturally occurring and highly conserved salt bridge (E(90)-K(121)) that stabilizes the relation between transmembranes 2 and 3, which is required for passage of the receptor through the cellular quality control system and to the plasma membrane. Our model was used to reveal important pharmacophoric features, and then identify a novel chemical ligand, which was able to rescue a D(98) mutant of the hGnRHR that could not be rescued as effectively by previously known pharmacoperones.

Single-cell analysis of insulin-regulated fatty acid uptake in adipocytes

Increased body fat correlates with the enlargement of average fat cell size and reduced adipose tissue insulin sensitivity. It is currently unclear whether adipocytes, as they accumulate more triglycerides and grow in size, gradually become less insulin sensitive or whether obesity-related factors independently cause both the enlargement of adipocyte size and reduced adipose tissue insulin sensitivity. In the first instance, large and small adipocytes in the same tissue would exhibit differences in insulin sensitivity, whereas, in the second instance, adipocyte size per se would not necessarily correlate with insulin response. To analyze the effect of adipocyte size on insulin sensitivity, we employed a new single-cell imaging assay that resolves fatty acid uptake and insulin response in single adipocytes in subcutaneous adipose tissue explants. Here, we report that subcutaneous adipocytes are heterogeneous in size and intrinsic insulin sensitivity. Whereas smaller adipocytes respond to insulin by increasing lipid uptake, adipocytes with cell diameters larger than 80-100 microm are insulin resistant. We propose that, when cell size approaches a critical boundary, adipocytes lose insulin-dependent fatty acid transport. This negative feedback mechanism may protect adipocytes from lipid overload and restrict further expansion of adipose tissue, which leads to obesity and metabolic complications.

Simultaneous and independent visualization of the gonadotropin-releasing hormone receptor and its ligand: Evidence for independent processing and recycling in living cells

The first step in GnRH signaling is binding by the peptide to its plasma membrane receptor (GnRHR). The receptor is a member of the seven transmembrane G protein-coupled class but lacks the characteristic C-terminal cytoplasmic tail, making it among the smallest receptors in this superfamily. It has been known since 1980 that agonist occupancy of the GnRHR results in patching, capping, and internalization, although it has not been possible to localize the unoccupied GnRHR, because elaboration of receptor antisera has not been easy to achieve. The recent production of a green fluorescent protein (GFP) conjugate of the GnRHR (“rGnRHR-C-tail-GFP”) that is expressed in cells, targeted to the plasma membrane, binds GnRH analogs and couples to G proteins has made it possible to monitor movement of the unoccupied receptor by confocal microscopy. In the present study, we used this probe, along with Texas Red conjugates of a GnRH agonist, to examine simultaneous processing of the receptor and its ligands. The prep...

Hypothalamic Tumor Necrosis Factor-α Converting Enzyme Mediates Excitatory Amino Acid-Dependent Neuron-to-Glia Signaling in the Neuroendocrine Brain

Glial erbB1 receptors play a significant role in the hypothalamic control of female puberty. Activation of these receptors by transforming growth factor α (TGFα) results in production of prostaglandin E2, which then stimulates luteinizing hormone releasing hormone (LHRH) neurons to secrete LHRH, the neuropeptide controlling sexual development. Glutamatergic neurons set in motion this glia-to-neuron signaling pathway by transactivating erbB1 receptors via coactivation of AMPA receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Because the metalloproteinase tumor necrosis factor α converting enzyme (TACE) releases TGFα from its transmembrane precursor before TGFα can bind to erbB1 receptors, we sought to determine whether TACE is required for excitatory amino acids to activate the TGFα–erbB1 signaling module in hypothalamic astrocytes, and thus facilitate the advent of puberty. Coactivation of astrocytic AMPARs and mGluRs caused extracellular Ca2+ influx, a Ca2+/protein kinase C-dependent increase in TACE-like activity, and enhanced release of TGFα. Within the hypothalamus, TACE is most abundantly expressed in astrocytes of the median eminence (ME), and its enzymatic activity increases selectively in this region at the time of the first preovulatory surge of gonadotropins. ME explants respond to stimulation of AMPARs and mGluRs with LHRH release, and this response is prevented by blocking TACE activity. In vivo inhibition of TACE activity targeted to the ME delayed the age at first ovulation, indicating that ME-specific changes in TACE activity are required for the normal timing of puberty. These results suggest that TACE is a component of the neuron-to-glia signaling process used by glutamatergic neurons to control female sexual development.

Diffusion MRI of the developing cerebral cortical gray matter can be used to detect abnormalities in tissue microstructure associated with fetal ethanol exposure.

Fetal alcohol spectrum disorders (FASDs) comprise a wide range of neurological deficits that result from fetal exposure to ethanol (EtOH), and are the leading cause of environmentally related birth defects and mental retardation in the western world. One aspect of diagnostic and therapeutic intervention strategies that could substantially improve our ability to combat this significant problem would be to facilitate earlier detection of the disorders within individuals. Light microscopy-based investigations performed by several laboratories have previously shown that morphological development of neurons within the early-developing cerebral cortex is abnormal within the brains of animals exposed to EtOH during fetal development. We and others have recently demonstrated that diffusion MRI can be of utility for detecting abnormal cellular morphological development in the developing cerebral cortex. We therefore assessed whether diffusion tensor imaging (DTI) could be used to distinguish the developing cerebral cortices of ex vivo rat pup brains born from dams treated with EtOH (EtOH; 4.5 g/kg, 25%) or calorie-matched quantities of maltose/dextrin (M/D) throughout gestation. Water diffusion and tissue microstructure were investigated using DTI (fractional anisotropy, FA) and histology (anisotropy index, AI), respectively. Both FA and AI decreased with age, and were higher in the EtOH than the M/D group at postnatal ages (P)0, P3, and P6. Additionally, there was a significant correlation between FA and AI measurements. These findings provide evidence that disruptions in cerebral cortical development induced by EtOH exposure can be revealed by water diffusion anisotropy patterns, and that these disruptions are directly related to cerebral cortical differentiation.

Redistribution of Gq/11α in the Pituitary Gonadotrope in Response to a Gonadotropin-Releasing Hormone Agonist1

In the present study, we took advantage of high-resolution multilaser confocal microscopy to examine the distribution of the α-subunit of the guanyl nucleotide binding protein subfamily Gq/11 (Gq/11α). Dispersed cultures of pituitary cells were prepared from female weanling rats, fixed, permeabilized, and then stained with monoclonal antiserum (mouse) to the gonadotrope-specific form of secretogranin (SIIp), which was then tagged with Texas Red. Accordingly, the subpopulation of gonadotropes (∼15% of total cells) could be identified against a background of other pituitary cell types. Gq/11α was localized with antiserum made in rabbit, then tagged with fluorescein. Hoechst 33258 nuclear stain was also used in some experiments for topological reference. The data indicate localization of the Gq/11α in a cellular region near the plasma membrane and external to the border of the layer occupied by secretory granules. In the absence of activation, there were an average of six clusters of Gq/11α in a section 1 μm...