Christine Pesold

The phenotypic characteristics of heterozygous reeler mouse

Histological and behavioral traits are associated with reelin (Reln) haplo-insufficiency in heterozygous reeler mouse (rl+/-). These phenotypic traits are an approximately 50% decrease of brain Reln mRNA and Reln protein, an accumulation of nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons in subcortical white matter, an age-dependent decrease in prepulse inhibition of startle (PPI), and neophobic behavior on the elevated plus-maze. Possible analogies between these rl+/- phenotypic traits and signs of psychosis vulnerability are discussed.

Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse

Heterozygous reeler mice (HRM) haploinsufficient for reelin express ≈50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD67)-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD67 down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD67 knockout mice (HG67M). These mice exhibited a down-regulation of GAD67 mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.

Colocalization of integrin receptors and reelin in dendritic spine postsynaptic densities of adult nonhuman primate cortex

The expression of telencephalic reelin (Reln) and glutamic acid decarboxylase mRNAs and their respective cognate proteins is down-regulated in postmortem brains of schizophrenia and bipolar disorder patients. To interpret the pathophysiological significance of this finding, immunoelectron microscopic experiments are required, but these cannot be carried out in postmortem human brains. As an alternative, we carried out such experiments in the cortex of rats and nonhuman primates. We found that Reln is expressed predominantly in layer I of both cortices and is localized to bitufted (double-bouquet), horizontal, and multipolar gamma-aminobutyric acid-ergic interneurons, which secrete Reln into extracellular matrix. Reln secretion is mediated by a constitutive mechanism that depends on the expression of a specific signal peptide present in the Reln carboxy-terminal domain. Extracellular matrix Reln is found to aggregate in proximity of postsynaptic densities expressed in apical dendrite spines, which include also the alpha(3) subunit of integrin receptors. Most pyramidal neurons of various cortical layers express the mouse-disabled 1 (Dab1) protein, which, after phosphorylation by a soluble tyrosine kinase, functions as an adapter protein, probably mediating a modulation of cytoskeleton protein expression. We hypothesize that the decrease of neuropil and dendritic spine density reported to exist in the neocortex of psychiatric patients may be related to a down-regulation of Reln-integrin interactions and the consequent decrease of cytoskeleton protein turnover.

Aging-associated up-regulation of neuronal 5-lipoxygenase expression: putative role in neuronal vulnerability

Aging is associated with neurodegenerative processes. 5‐Lipoxygenase (5‐LO), which is also expressed in neurons, is the key enzyme in the synthesis of leukotrienes, inflammatory eicosanoids that are capable of promoting neurodegeneration. We hypothesized that neuronal 5‐LO expression can be up‐regulated in aging and that this may increase the brains vulnerability to neurodegeneration. We observed differences in the distribution of 5‐LO‐like immunoreactivity in various brain areas of adult young (2‐month‐old) vs. old (24‐month‐old) male rats. Greater 5‐LO‐like immunoreactivity was found in old vs. young rats, in particular in the dendrites of pyramidal neurons in limbic structures, including the hippocampus, and in layer V pyramidal cells of the frontoparietal cortex and their apical dendrites. The aging‐increased expression of neuronal 5‐LO protein appears to be due to increased 5‐LO gene expression. Using a quantitative reverse transcription/polymer‐ase chain reaction assay and 5‐LO‐specific oligonucleotide primers and their mutated internal standards, we observed about a 2.5‐fold greater hippocampal 5‐LO mRNA content in old rats. 5‐LO‐like immunoreactivity was also observed in small, nonpyramidal cells, which were positive for glutamic acid decarboxylase or glial fibrillary acid protein. This type of 5‐LO immunostaining did not increase in the old rats. Hippocampal excitotoxic injury induced by systemic injection of kainate was greater in old rats. Neuroprotection was observed with the 5‐LO inhibitor, caffeic acid. Together, these results suggest that aging increases both neuronal 5‐LO expression and neuronal vulnerability to 5‐LO inhibitor‐sensitive ex‐citotoxicity, and indicate that the 5‐LO system might play a significant role in the pathobiology of aging‐associated neurodegenerative diseases.—Uz, T., Pesold, C., Longone, P., Manev, H. Aging‐associated up‐regulation of neuronal 5‐lipoxygenase expression: putative role in neuronal vulnerability. FASEB J. 12, 439–449 (1998)

Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines

Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72–96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABAA (γ-aminobutyric acid type A) receptor subunits (decrease in γ2 and α1; increase in α5) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD67. In contrast, dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal.

Reelin in the extracellular matrix and dendritic spines of the cortex and hippocampus: a comparison between wild type and heterozygous reeler mice by immunoelectron microscopy.

Reelin is a glycoprotein (∼400 kDa) secreted by GABAergic neurons into the extracellular matrix of the neocortex and hippocampus as well as other areas of adult rodent and nonhuman primate brains. Recent findings indicate that the heterozygote reeler mouse (haploinsufficient for the reeler gene) shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder with mania. These include (1) a downregulation of both reelin mRNA and the translated proteins, (2) a decrease in the number of dendritic spines in cortical and hippocampal neurons, (3) a concomitant increase in the packing density of cortical pyramidal neurons, and (4) an age-dependent decrease in prepulse inhibition of startle. Interestingly, the heterozygous reeler mouse does not exhibit the unstable gait or the neuroanatomy characteristic of the null mutant reeler mouse. Immunocytochemical studies of the expression of reelin in mice have been primarily limited to light microscopy. In this study we present new immunoelectron microscopy data that delineates the subcellular localization of reelin in the cortex and hippocampus of the wild-type mouse, and compares these results to reelin expression in the heterozygous reeler mouse. In discontinuous areas of cortical layers I and II and the inner blade area of the dentate gyrus of the wild type mouse, extracellular reelin is associated with dendrites and dendritic spine postsynaptic specializations. Similar associations have been detected in the CA1 stratum oriens and other areas of the hippocampus. In the hippocampus, reelin expression is more expansive and more widespread than in cortical layers I and II. In contrast, extracellular reelin immunoreactivity is greatly diminished in all areas examined in the heterozygous reeler mouse. However, some cell bodies of GABAergic neurons in the cortex and hippocampus demonstrate an increased accumulation of reelin in the Golgi and endoplasmic reticulum. We suggest that in the heterozygous reeler mouse a downregulation of reelin biosynthesis results in a decreased rate of secretion into the extracellular space. This inhibits dendritic spine maturation and plasticity and leads to dissociation of dendritic postsynaptic density integrity and atrophy of spines. We speculate that the haploinsufficient reeler mouse may provide a model for future studies of the role of reelin, as it may be related to psychosis vulnerability.

Tolerance to diazepam and changes in GABAA receptor subunit expression in rat neocortical areas

Long-term treatment with diazepam, a full allosteric modulator of the GABA(A) receptor, results in tolerance to its anticonvulsant effects, whereas an equipotent treatment with the partial allosteric modulator imidazenil does not produce tolerance. Use of subunit-specific antibodies linked to gold particles allowed an immunocytochemical estimation of the expression density of the alpha1, alpha2, alpha3, alpha5, gamma(2L&S) and beta(2/3) subunits of the GABA(A) receptor in the frontoparietal motor and frontoparietal somatosensory cortices of rats that received long-term treatment with vehicle, diazepam (three times daily for 14 days, doses increasing from 17.6 to 70.4 micromol/kg), or imidazenil (three times daily for 14 days, doses increasing from 2.5 to 10.0 micromol/kg). In this study, tolerance to diazepam was associated with a selective decrease (37%) in the expression of the alpha1 subunit in layers III-IV of the frontoparietal motor cortex, and a concomitant increase in the expression of the alpha5 (150%), gamma(2L&S) and beta(2/3) subunits (48%); an increase in alpha5 subunits was measured in all cortical layers. In the frontoparietal somatosensory cortex, diazepam-tolerant rats had a 221% increase in the expression of alpha5 subunits in all cortical layers, as well as a 35% increase in the expression of alpha3 subunits restricted to layers V-VI. Western blot analysis substantiated that these diazepam-induced changes reflected the expression of full subunit molecules. Rats that received equipotent treatment with imidazenil did not become tolerant to its anticonvulsant properties, and did not show significant changes in the expression of any of the GABA(A) receptor subunits studied, with the exception of a small decrease in alpha2 subunits in cortical layers V-VI of the frontoparietal somatosensory cortex. The results of this study suggest that tolerance to benzodiazepines may be associated with select changes in subunit abundance, leading to the expression of different GABA(A) receptor subtypes in specific brain areas. These changes might be mediated by a unique homeostatic mechanism regulating the expression of GABA(A) receptor subtypes that maintain specific functional features of GABAergic function in cortical cell layers.

New Neurochemical Markers for Psychosis: A Working Hypothesis of Their Operation

Reelin (Reln) is expressed in specific GABAergic neurons in layer I and II of neocortex, and is secreted into the extracellular matrix where it surrounds dendrites, spines and neurite arborizations, and binds to integrin receptors located on post-synaptic densities of apical dendritic spines. Experiments in rodents (including wild type or reeler heterozygous mice) and non-human primates suggest the Reln secreted in the extracellular matrix of neocortex, via integrin receptors, modulates the function of the adaptor protein DAB1(drosophila disable-gene) homologous product) thereby participating in dynamic processes associated with plasticity changes in dendrites, dendritic spines and their synapses. A local protein synthesis at dendritic spines (ie the activity regulated cytoskeleton associated protein, Arc) probably acts as a signal for plastic modulatory activities in synapses operative in neural group interactions. A research strategy directed toward identifying specific neurochemical markers operative in the etiopathology of psychotic disorders lead to the identification of a downregulation (30–50%) of Reln and glutamic acid decarboxylase 67(GAD67) expression in prefrontal cortex and other brain areas of schizoprenia and bipolar disorder patients with psychosis. These downregulations were not due to neuronal damage, postmortem interval, or antipsychotic medication. The dysfunction of GABAergic interneurons observed in psychotic brains in combination with reduced Reln expression and downregulation of Reln-integrin receptor interaction, may provide an explanation for the reported decrease in neuropile expression including dendritic spine density reduction, in neocortex of schizophrenia patients. This downregulation of neuropile plasticity may be a factor to be considered in the etiology of the disintegration of consciousness, which is one of the primary signs of psychosis.

In Patas monkey, glutamic acid decarboxylase-67 and reelin mRNA coexpression varies in a manner dependent on layers and cortical areas

In nonhuman and human primates, reelin immunoreactivity is expressed consistently in γ‐aminobutyric acid (GABA)‐ergic interneurons of the three upper cortical layers (Impagnatiello et al. [1998] Proc. Natl. Acad. Sci. U S A 95:15718–15723; Rodriguez et al. [2000] Proc. Natl. Acad. Sci. U S A 97:3550–3555). To understand in detail the pattern of reelin synthesis in GABAergic interneurons of primate neocortex, a quantitative analysis of reelin and of glutamic acid decarboxylase‐67 (GAD67) mRNA‐positive neurons as well as a quantitative analysis of total neuronal density measured by neuron‐specific nuclear protein (NeuN) immunoreactivity was carried out in Patas monkey neocortex (Brodmanns areas 2, 3, 4, 6, 9, 17, 18, and 24). Reelin mRNA is expressed in every cortical area and layer studied, but layer II of each cortical area consistently revealed the largest neuronal population expressing reelin mRNA compared with other layers. The percentages of GAD67‐positive neurons in each layer of the eight cortical areas were 83–98% in layer I, 55–64% in layer II, 37–49% in layer III, 71–89% in layer IV, 54–68% in layer V, and 71–85% in layer VI. The percentages of GABAergic neurons expressing reelin were 86–100% in layer I, 76–84% in layer II, 52–96% in layer III, 23–33% in layer IV, 33–57% in layer V, and 34–54% in layer VI. These findings suggest that there may be two classes of GABAergic neurons that can be differentiated by their ability to express reelin mRNA and reelin protein. This differentiation may have a functional significance, considering that reelin is secreted into the extracellular matrix, where it plays a putative role in the maturation of newly formed dendritic spines and binds selectively to dendritic shafts and to spine postsynaptic densities and presumably to integrin receptors, including α3 subunits (Rodriguez et al. [2000]). J. Comp. Neurol. 451:279–288, 2002.

Subcellular localization of the α7 nicotinic receptor in rat cerebellar granule cell layer

THE distribution of the α7 nicotinic receptor subunit in the rat cerebellum was studied immunohistochemically at the electron microscope level using an α7 subunitspecific antibody. The granule cell layer showed a much lower level of immunoreactivity for the α7 subunit than the Purkinje cell layer. Granule cell somata were completely devoid of labeling; this appeared to be restricted to glomeruli exclusively located in the membranes of granule cell dendrites. The α7 immunolabeling was located not at active synaptic areas but was mostly perisynaptic. This localization suggests that nicotinic receptors containing the α7 subunit could have a modulatory function and/or play a direct role in the generation of synaptic currents.