Anatomy and three-dimensional reconstructions of the brain of the white whale (Delphinapterus leucas) from magnetic resonance images.

Magnetic resonance imaging offers a means of observing the internal structure of the brain where traditional procedures of embedding, sectioning, staining, mounting, and microscopic examination of thousands of sections are not practical. Furthermore, internal structures can be analyzed in their precise quantitative spatial interrelationships, which is difficult to accomplish after the spatial distortions often accompanying histological processing. For these reasons, magnetic resonance imaging makes specimens that were traditionally difficult to analyze, more accessible. In the present study, images of the brain of a white whale (Beluga) Delphinapterus leucas were scanned in the coronal plane at 119 antero-posterior levels. From these scans, a computer-generated three-dimensional model was constructed using the programs VoxelView and VoxelMath (Vital Images, Inc.). This model, wherein details of internal and external morphology are represented in three-dimensional space, was then resectioned in orthogonal planes to produce corresponding series of "virtual" sections in the horizontal and sagittal planes. Sections in all three planes display the sizes and positions of such structures as the corpus callosum, internal capsule, cerebral peduncles, cerebral ventricles, certain thalamic nuclear groups, caudate nucleus, ventral striatum, pontine nuclei, cerebellar cortex and white matter, and all cerebral cortical sulci and gyri.

Cyclic AMP-elevating agents induce the expression of MAP kinase phosphatase-1 in PC12 cells.

Stimulation of pheochromocytoma PC12 cells by cAMP-elevating agents caused the induction of the immediate early gene 3CH134, which encodes MAP kinase phosphatase-1 (MKP-1). Forskolin was as potent as serum in stimulating MKP-1 gene expression, whereas dibutyryl-cAMP and neuropeptide PACAP were less effective. Induction of the MKP-1 gene was accompanied by neo-synthesis of MKP-1 protein. MAP kinase activation was not involved in the cAMP-induced MKP-1 gene expression. The MAP kinase inactivation, that would result from MKP-1 induction in response to increased intracellular cAMP level, contributes to explain how hormones or neurotransmitters signaling through cAMP influence cell growth and differentiation.

Regulation of human D1 dopamine receptor function and gene expression in SK-N-MC neuroblastoma cells.

SK-N-MC human neuroblastoma cells express functional D1, but not D5, dopaminergic receptors. Stimulating cells with dopamine or the D1-selective agonist, SKF R-38393, rapidly (t(1/2) = 1 h) resulted in > 95% attenuation of dopamine-mediated accumulation of cyclic AMP, without any change in D1 dopamine receptor levels. Prolonged (> 4 h) exposure of cells to dopamine attenuated D1 receptor levels to 45-50% of control (t(1/2) = 8 h) and was accompanied by a loss of high-affinity binding sites. At the molecular level, the expression of D1 receptor messenger RNA was bimodal: an initial increase (by approximately 60%) of receptor messenger RNA within 2 h of treatment of cells with dopamine was followed by a decline to 50% below control messenger RNA levels. Low concentrations (1-10 nM) of dopamine also potentiated D1 messenger RNA levels (up to 48%), resulting in a twofold increase in receptor levels. Transfection studies with the cloned human D1 promoter construct, pGL-D1P, indicated that the up-regulation of D1 messenger RNA was due to activation of promoter by dopamine. The dopamine-mediated up-regulation of both D1 receptor messenger RNA and promoter was prevented by the D1-selective antagonist, SCH 23390. The results suggest that dopamine regulates D1 receptor gene and protein expression in a bimodal manner, partly through activation of the receptor promoter. Moreover, the effects of dopamine are independent of the second messenger, cyclic AMP.

Cocaine and fluoxetine induce the expression of the hVH-5 gene encoding a MAP kinase phosphatase.

A novel class of immediate early genes that encode enzymes of the MAP kinase phosphatase family has recently been described. These enzymes are dual-specificity protein phosphatases and some show tissue-specific distribution, like the hVH-5 gene (homologue of vaccinia virus H1 phosphatase gene clone 5), which is expressed predominantly in the adult brain. In this paper, we investigated whether the hVH-5 gene is induced by psychostimulants in rat brain, as has been demonstrated for immediate early genes encoding transcription factors. Using in situ hybridization, we found that i.p. injection of cocaine, amphetamine and caffeine induced hVH-5 mRNA expression within 40 min in the nucleus accumbens (NAc), caudate putamen, frontal cortex and hippocampus, with a maximal effect in the NAc. The cocaine-induced hVH-5 gene induction involves the serotonergic system, since it was abolished in the NAc by lesioning serotonergic raphé projections with 5,7-dihydroxytryptamine. Moreover, the effect of cocaine was fully mimicked by the selective serotonin uptake inhibitor fluoxetine. In contrast to what has been described for c-fos and egr-1 immediate early genes, we found that hVH-5 mRNA expression in the NAc and hippocampus was as significant after repeated cocaine injections for 10 days as after a single injection. The considerable and prolonged induction of the MAP kinase phosphatase hVH-5 gene by psychostimulant drugs in postmitotic brain cells, particularly in the NAc, could indicate that MAP kinase substrates are involved in the reinforcing properties of drugs of abuse.

The serotonergic system modulates the cocaine-induced expression of the immediate early genes egr-1 and c-fos in rat brain.

Transcription regulatory factors are rapidly induced in brain by a wide variety of stimuli and may be important in coordinating changes in gene expression under-lying neuronal plasticity. Using in situ hybridization, we found that acute cocaine administration (20 mg/kg, intraperitoneally (i.p.)) produced a robust induction of both c-fos and egr-1 immediate early genes. Egr-1 messenger RNA induction was highest in the caudate putamen and in the shell of the nucleus accumbens. No significant induction was noticed after injection of fluoxetine, a selective inhibitor of serotonin uptake. Cocaine-induced egr-1 and c-fos expression was substantially reduced in the brain areas from rats in which the serotonergic projections were lesioned by injection of the neurotoxin 5,7-dihydroxytryptamine and in rats that have been injected with tropisetron, an antagonist of the 5-hydroxytryptamine (5-HT3) receptor. Conversely, the 5-HT3 receptor agonist 2-methylserotonin induced the expression of these early genes in structures including the caudate putamen and nucleus accumbens.

The Serotonergic System Modulates the Cocaine-Induced Expression of the Immediate Early Genes egr-1 and c-fos in Rat Brain.

Transcription regulatory factors are rapidly induced in brain by a wide variety of stimuli and may be important in coordinating changes in gene expression underlying neuronal plasticity. Using in situ hybridization, we found that acute cocaine administration (20 mg/kg, intraperitoneally (i.p.)) produced a robust induction of both c-fos and egr-1 immediate early genes. Egr-1 messenger RNA induction was highest in the caudate putamen and in the shell of the nucleus accumbens. No significant induction was noticed after injection of fluoxetine, a selective inhibitor of serotonin uptake. Cocaine-induced egr-1 and c-fos expression was substantially reduced in the brain areas from rats in which the serotonergic projections were lesioned by injection of the neurotoxin 5,7-dihydroxytryptamine, and in rats that have been injected with tropisetron, an antagonist of the 5-hydroxytryptamine (5-HT3) receptor. Conversely, the 5-HT3 receptor agonist 2-methyl-serotonin induced the expression of these early genes in structures including the caudate putamen and nucleus accumbens.

5-Hydroxytryptamine induces TIS8/egr-1 and c-fos expression in PC12 cells. Involvement of tyrosine protein phosphorylation.

The TIS8/egr-1 gene is a member of the class of immediate early genes. Originally discovered as a mitogen-induced gene, it can also be induced by synaptic activity. We report here the induction of the TIS8/egr-1 gene by the neurotransmitter 5-hydroxytryptamine (5-HT) in cultured rat phaeochromocytoma PC12 cells. Induction was maximal 40 min after addition of 5-HT to the cells, and declined very rapidly to reach the basal level after 90 min. The electrophoretic mobility-shift assay showed that induction of the TIS8/egr-1 gene by 5-HT was accompained by increased Egr-1 protein binding to its DNA consensus sequence. We found an overall correlation of 5-HT-induced egr-1 expression with that of c-fos expression. The kinetics of the ability of both gene products to bind to their respective DNA consensus sequence was also similar. The 5-HT-induced activation in Egr-1 binding was inhibited by ketanserin and mesulergine, indicating that 5-HT exerted its action via a 5-HT2 receptor subtype. The tyrosine protein kinase inhibitor genistein abolished induction of the TIS8/egr-1 gene, suggesting that tyrosine kinase activity is required for the induction of early genes by 5-HT. Genistein also inhibited 5-HT-induced Egr-1 binding activity. The increase in phosphotyrosine content of focal adhesion kinase we noticed upon addition of 5-HT to cells suggests that this cytoplasmic tyrosine protein kinase mediates the effect of 5-HT in eliciting early gene induction.

Induction of c-fos, jun B and egr-1 expression by haloperidol in PC12 cells: involvement of calcium.

Acute injection of haloperidol, a dopamine D2 receptor antagonist, is known to increase immediate early gene expression of the fos and jun families in rodent striatal neurons. A set of gene induction, including c-fos, jun B and TIS8/egr-1, was found when haloperidol was added to PC12 cells in culture. Electrophoretic mobility-shift assays show that haloperidol-evoked gene induction was accompanied by a transient and dose-dependent increase in AP1 and EGR-1 binding activities in these cells. Gene expression is tentatively explained by the rapid and transient increase in cytosolic free Ca2+ concentration observed upon haloperidol addition. The cytosolic calcium rise and AP1 binding activation elicited by haloperidol were dependent on extracellular Ca2+, suggesting that haloperidol exerted its effects by promoting Ca2+ entry into PC12 cells. The haloperidol-induced increase in AP1 binding activity and intracellular Ca2+ was not reproduced by two other dopamine D2 receptor antagonists, sulpiride and (+)-butaclamol.