Increased striatal proenkephalin mRNA subsequent to production of spreading depression in rat cerebral cortex: activation of corticostriatal pathways?

Cortical Spreading Depression (CSD) is a slowly propagating wave of depolarization and negative interstitial DC potential, that when induced in the rat brain extends across the entire homolateral hemisphere. Despite evidence that CSD does not penetrate into subcortical regions, neurochemical changes in areas anatomically connected to cortex have been reported. In this study in situ hybridization histochemistry was used to examine the levels of cholecystokinin (CCK), proenkephalin (ENK) and prodynorphin (DYN) mRNA in cortex and forebrain basal ganglia following KCl-induced CSD. Unilateral CSD was induced by topical application of 3 M KCl ( approximately 10 microliter) onto the right parietal cortex for 10 min and rats were then killed 1-6 h and 1-28 days later. CCK mRNA levels were increased (P<0.01) in the ipsilateral neocortex 3 h after CSD (13% above levels in contralateral side), reached a peak at 2 days ( approximately 70%) and were still elevated at 7 (30%) but not, 14 or 28 days later. Unilateral CSD also produced a rapid and sustained increase (P<0.05) in ENK mRNA in ipsilateral piriform cortex (from 3 h to 2 days; 70-250% above contralateral), and a delayed increase in caudate putamen and olfactory tubercle at 1 and 2 days ( approximately 25% in both regions), but levels were again equivalent to control at 7 days and beyond. In contrast, no marked changes in neocortical ENK mRNA, or DYN mRNA in both cortex and basal ganglia, were observed under these conditions. These findings demonstrate that CSD has specific, rapid and long-lasting effects on neuropeptide expression in neocortex and subcortical areas. CSD-induced changes in mesostriatal ENK mRNA are proposed to reflect synaptic activation of local neurons via cortical afferent projections.     

Pilocarpine-induced seizures are accompanied by a transient elevation in the messenger RNA expression of the prohormone convertase PC1 in rat hippocampus: comparison with nerve growth factor and brain-derived neurotrophic factor expression

Several prohormone convertases that are involved in the posttranslational processing of precursor proteins, including neuropetides, hormones and neurotrophic factors, are produced in the central nervous system. These include enzymes named furin, PC1, PC2, PC5 and PACE4. To understand better the potential role played by prohormone convertases in the central nervous system we studied the expression of their messenger RNAs in the hippocampus of rats with pilocarpine-induced seizures. Moreover, we compared their expression patterns with those of neurotrophins such as nerve growth factor and brain-derived neurotrophic factor, which are up-regulated in the hippocampus during seizures. Pilocarpine (380 mg/kg, i.p.) induced seizure activity that appeared within the first hour and persisted for approximately 8 h. In situ hybridization showed transient increases in messenger RNA for nerve growth factor and brain-derived neurotrophic factor that peaked at 120 min in the hippocampus. Among the convertases studied, only PC1 messenger RNA displayed up-regulation, with temporal and topographic features comparable to those of nerve growth factor and brain-derived neurotrophic factor messenger RNA. The expression of furin, PC2 and PC5 messenger RNA changed little, while PACE4 was not expressed at all, both before and after pilocarpine administration. The highest increase in PC1 messenger RNA expression was found in granule cells of the dentate gyrus and, to a lesser extent, in the pyramidal layer of CA1 and CA3 subfields. Thus, in the rat hippocampus, the epileptiform activity induced by pilocarpine mediates a co-ordinated expression of messenger RNAs for PC1, nerve growth factor and brain-derived neurotrophic factor. Our findings suggest the involvement of PC1 in the processing of precursor proteins during seizure activity.     

The induction of megakaryocyte differentiation is accompanied by selective Ser133 phosphorylation of the transcription factor CREB in both HEL cell line and primary CD34+ cells

Alteration of the nuclear matrix protein composition during active cell death was investigated by high resolution 2-dimensional gel electrophoresis and computer-assisted image analysis. Nuclear matrices were isolated from purified nuclei of a rat embryo cell line showing an immediate apoptotic response to serum reduction. While cell shrinkage and cytoplasmic compaction, characteristic features of apoptosis, were induced, the nuclear matrix protein pattern was not altered 1 h after induction of apoptosis. However, two sets of novel nuclear matrix protein spots appeared with differing kinetics within the following 5 h of apoptosis. They consisted of five and six protein spots, respectively. In addition, the intensity of five nuclear matrix protein spots that had already been present in the uninduced cells increased continuously within an observation period of 12 h. These coincidences point to a potential involvement of the described nuclear matrix proteins in the apoptotic process.     

Relationships between the prefrontal cortex and the basal ganglia in the rat: physiology of the corticosubthalamic circuits

The prelimbic-medial orbital areas (PL/MO) of the prefrontal cortex are connected to the medial part of the subthalamic nucleus (STN) through a direct projection and an indirect circuit that involves the core of the nucleus accumbens (NAcc) and the ventral pallidum (VP). In the present study, the influence of the PL/MO on the discharge of STN cells has been characterized. The major pattern of the responses observed after stimulation of PL/MO consisted of two excitatory peaks often separated by a brief inhibitory period. The early excitation was most likely to be caused by the activation of direct cortical inputs because its latency matches the conduction time of the prefrontal STN projections. The late excitation resulted from the activation of the indirect PL/MO-STN pathway that operates through a disinhibitory process. Indeed, the late excitation was no longer observed after acute blockade of the glutamatergic corticostriatal transmission by CNQX application into the NAcc. A similar effect was obtained after the blockade of the GABAergic striatopallidal transmission by bicuculline application into the VP. Finally, the brief inhibition that followed the early excitation was likely to result from the activation of a feedback inhibitory loop through VP because this inhibition was no longer observed after the blockade of STN inputs by CNQX application into the VP. This study further indicates the implication of STN in prefrontal basal ganglia circuits and underlines that in addition to a direct excitatory input, medial STN receives an indirect excitatory influence from PL/MO through an NAcc-VP-STN disinhibitory circuit.     

Induction of astroglial gene expression by experimental seizures in the rat: spatio-temporal patterns of the early stages

The levels of glial fibrillary acidic protein mRNA were analysed by in situ hybridization during the first 6 h in experimental models of status epilepticus in the rat. Two different models of status epilepticus were studied: one is produced by the administration of pilocarpine to lithium-treated rats and the other by the intracerebroventricular administration of kainate. Results obtained in the present study showed a very rapid (as early as 1.5 h in periventricular zones of hypothalamus, cerebral cortex, and hippocampal area) up-regulation of GFAP mRNA levels following the pharmacological induction of seizures. Several other areas showed a GFAP activation starting at 3 h such as septum, habenular nuclei, corpus callosum, and cingulum. The comparison of the results obtained in the two models of status epilepticus revealed interesting differences in some brain areas, such as cerebellum and striatum, which can be related to the specific neurotransmitter receptors and neurochemical pathways stimulated by the drugs. Interestingly, some brain areas whose neurons are strongly activated by pilocarpine and kainate (amygdala and CA3 hippocampal field) and that undergo neuronal degeneration did not show the early GFAP response. An interesting spatial feature was observed in several brain regions examined (striatum, septum, and hypothalamus): the response first appeared in the periventricular zones and then diffused to the rest of the brain area. In general GFAP responses in the periventricular zones were early and intense.     

Nerve gas-induced seizures: role of acetylcholine in the rapid induction of Fos and glial fibrillary acidic protein in piriform cortex

Soman (pinacolymethylphosphonofluoridate), a highly potent irreversible inhibitor of acetylcholinesterase (AChE), causes seizures and rapidly increases Fos and glial fibrillary acidic protein (GFAP) staining in piriform cortex (PC). This suggests that the inhibition of AChE by soman leads to increased acetylcholine (ACh) and neuronal excitability in PC. The sole source of cholinergic input to PC is from the nucleus of the diagonal band (NDB). To investigate the role of ACh in soman-induced seizures, we lesioned cholinergic neurons in NDB unilaterally with 192-IgG-saporin. By 10 d, saporin eliminated staining for choline acetyltransferase (ChAT), the synthetic enzyme for ACh, in NDB ipsilateral to the lesion. Staining for AChE, the degradative enzyme for ACh, was eliminated in PC ipsilateral to the lesioned NDB. By 45-60 min after soman, increased Fos and GFAP staining in PC was evident only ipsilateral to the unlesioned NDB. By 90-120 min after soman, Fos and GFAP staining increased bilaterally in PC. In a second experiment, electrical stimulation electrodes were implanted unilaterally in the NDB to activate focally the projections to PC in unanesthetized rats. Within 5 min of NDB stimulation, there were clear behavioral and EEG signs of convulsions. After 45-60 min of NDB stimulation, there was increased Fos and GFAP staining in layer II of PC ipsilateral to the stimulation site. Pretreatment with the selective muscarinic receptor antagonist scopolamine blocked the convulsions and prevented increased Fos and GFAP staining in PC. These results suggest that ACh release in PC triggers the initiation of seizures and gliosis after soman administration, predominantly by the activation of muscarinic receptors.     

Identification of sequences involved in both basal expression and phenobarbital induction of a CYP2B2 gene using in vitro transcription system

Physical and chemical agents that promote DNA damage can induce high levels of mitotic crossing-over in eukaryotic diploid cells. Similarly, foreign DNA segments introduced by transformation processes, in the cell genome, can also induce mitotic crossing-over as an outcome of the reactions leading to chromosomic balance or due to the mechanisms aiming at the integration of the exogenous DNA. Zucchi et al. have described a system showing that RNA treatments are capable of inducing changes in the genome of haploid receptor strains of Aspergillus nidulans. To verify the genetic consequences of this process in diploid cells, conidia from two strains of this fungus were protoplastized, treated with homologous RNA and analyzed. Alterations in the gene expression and in the mitotic crossing-over frequencies between linked markers were detected. Among the main observed effects there was a generalized alteration in gene expression which was very likely caused by a reversible gene inactivation mechanism due to the methylation of cytosine residues. This was confirmed by treating the haploid segregants with the hypomethylating agent 5-azacytidine, that restored the original gene activity. The presence of a duplicated segment in the chromosome I of one of the treated diploids, interfered with the RNA general effects on its genome.     

Transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor expression in normal and diseased human adrenal cortex by immunohistochemistry and in situ hybridization

Because epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGFR) have been implicated in the regulation of adrenocortical function, we used immunohistochemistry and in situ hybridization of EGF and TGF-alpha to study 41 specimens of human adrenal cortex, including 10 normal specimens, 15 aldosteronomas, five Cushing's adenomas, six adrenocortical incidentalomas, and five carcinomas to determine what role these growth factors play in controlling human adrenocortical function. Neither immunoreactivity nor mRNA hybridization signals to EGF was detected in any specimens, and EGF therefore may exert its effects on adrenal function as an endocrine hormone. TGF-alpha expression was detected at both protein and mRNA levels in normal and neoplastic adrenal cortex, demonstrating that TGF-alpha is synthesized locally in human adrenal cortex. TGF-alpha expression was observed in the cells with increased steroidogenesis, including compact tumor cells and zona fasciculata cells with lipid depletion, but did not necessarily correlate with production sites of any specific steroid hormone. EGFR immunoreactivity was more widely distributed than TGF-alpha immunoreactivity. Both TGF-alpha and EGFR expression were markedly elevated in adrenocortical carcinomas. TGF-alpha and EGFR thus appear to be involved in biological function in both normal and neoplastic human adrenal cortex. In addition, TGF-alpha and EGFR may play important roles in some biological features of adrenocortical malignancy.     

Behavioural sensitization in 6-hydroxydopamine-lesioned rats is related to compositional changes of the AP-1 transcription factor: evidence for induction of FosB- and JunD-related proteins

Rats with unilateral dopamine denervation exhibit turning behaviour in response to the selective D1 agonist SKF 38393 only after a previous exposure to dopamine agonists. We demonstrate here that this 'priming' phenomenon is related to both an increased expression of the pre-existing AP-1 complex and the occurrence of novel AP-1 complexes which are formed by FosB- and JunD-related proteins. While the former protein is expressed as a consequence of the dopamine denervation, the latter is related to the first exposure to a dopamine agonist. Pre-treatment with MK-801, an antagonist for glutamatergic receptors, prevents both the priming development and the AP-1 compositional changes. Rotational behaviour induced by SKF 38393 closely correlates with the presence of the priming AP-1 complexes, regardless of the capability of the D1 agonist to induce the immediate-early gene cFos.     

Tissue factor expression in mesothelial cells: induction both in vivo and in vitro

Docking algorithms play an important role in the process of rational drug design and in understanding the mechanism of molecular recognition. An important determinant for successful docking is the extent to which the configurational space (including conformational changes) of the ligand/receptor system is searched. Here we describe a new, combinatorial method for flexible docking of peptides to proteins that allows full rotation around all single bonds of the peptide ligand and around those of a large set of receptor side chains. We have simulated the binding of several viral peptides to murine major histocompatibility complex class I H-2Kb. In addition, we have explored the limits of our method by simulating a complex between calmodulin and an 18-residue long helical peptide from calmodulin-dependent protein kinase IIalpha. The calculated peptide conformations generally matched well with the X-ray structures. Essential information about local flexibility and about residues that are responsible for strong binding was obtained. We have frequently observed considerable side-chain flexibility during the simulations, showing the need for a flexible treatment of the receptor. Our method may also be useful whenever the receptor side-chain conformation is not available or uncertain, as illustrated by the docking of an H-2Kb binding nonapeptide to the receptor structure taken from an octapeptide/H-2Kb complex.     

Involvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells

The EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have used in situ hybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8. Three of the receptors examined correspond to the novel chicken homologs of EphA2, EphA6, and EphA7. Unexpectedly, we found that many Eph receptors are expressed not only in retinal ganglion cells, but also in tectal cells, In particular, EphA3 mRNA is prominently expressed in the anterior tectum, with a pattern reciprocal to that of ephrin-A2 and ephrin-A5. Similarly, ephrin-A5 is expressed not only in tectal cells but also in the nasal retina, with a pattern reciprocal to that of its receptor EphA3 and partially overlapping with that of its other receptor EphA4. Consistent with the even distribution of EphA4 and the polarized distribution of EphA4 ligands in the retina, probing EphA4 immunoprecipitates from different sectors of the retina with anti-phosphotyrosine antibodies revealed spatial differences in receptor phosphorylation. These complex patterns of expression and tyrosine phosphorylation suggest that Eph receptors and ephrins contribute to establishing topography of retinal axons through multiple mechanisms, in addition to playing a role in intraretinal and intratectal organization.