Expression of diazepam-binding inhibitor peptide in human skin: an immunohistochemical and ultrastructural study.

Human diazepam binding inhibitor (DBI) was originally isolated from the brain and subsequently found to be present in several peripheral tissues. The various physiologic effects recently attributed to DBI include acting as an endogenous ligand for the central and peripheral (mitochondrial) benzodiazepine receptors. The present work provides, for the first time, evidence of DBI immunoreactivity in skin. DBI immunoreactivity was found in the epidermis, in the eccrine sweat and in sebaceous glands. Ultrastructurally, DBI was distributed throughout the cytoplasm. Although the physiologic role of DBI in skin is unknown, our results indicate that DBI may serve as an endogenous ligand for mitochondrial benzodiazepine receptors. Its activity could be related to the regulation of lipid and cholesterol synthesis in keratinocytes and sebaceous glands and to the secretion of sweat in sweat glands.

Ultrasound-amplified immunohistochemistry.

We describe a novel technique for improving the sensitivity of immunofluorescence staining by use of ultrasonic irradiation. Free-floating vibratome sections from rat cerebellum were incubated with primary antiserum and simultaneously were briefly exposed to ultrasound (US) in a conventional ultrasound bath. After the US treatment, a conventional immunohistochemical method was employed. Two different antisera and two conventional immunohistochemical detection systems were tested. In all cases a 10-20-sec US treatment strengthened immunoreactivity considerably. Irradiated samples showed a good morphology compared with non-irradiated sections. Our results demonstrate that with ultrasonic treatment the dilutions of primary antibodies can be increased and the incubation times of primary antisera can be reduced. The ultrasonic method described here requires no special equipment. It is easy, reproducible, and it can be considered a new method for the enhancement at immunohisto- and cytochemical staining of free-floating vibratome sections.

Neonatal noradrenaline depletion prevents the transition of Bergmann glia in the developing cerebellum.

The influence of neonatal administration of 6-hydroxydopamine (6-OHDA) on the cell proliferation in cerebellum was studied using 10-30 days-old rats. Compared to their littermates, treated rats had poor ability in searching, skills performance and orienting in the new environment. Elimination of noradrenergic terminals by 6-OHDA led to a delay in granular cell migration. The secondary foliation in neo-cerebellum was absent. The Bergmann glial cells were abnormally located, structurally different and did not form the intimate association with Purkinje cells. Our findings indicate that without noradrenergic influence neurones and glial cells do not proliferate normally and noradrenaline may act as an important trophic factor also for Bergmann glial cells.

Microglial response to the neurotoxicity of 6-hydroxydopamine in neonatal rat cerebellum.

Depletion of noradrenaline in newborn rats by 6-hydroxydopamine (6-OHDA) affects the postnatal development and reduces the granular cell area in the neocerebellum (lobules V-VII). During the first postnatal month, Bergmann glial fibers guide the migration of immature granule cells to the internal granule cell layer. Microglia and Bergmann glia may play an important role in this process, but the exact mechanism behind this phenomenon is not known. We studied the effect of systemic administration of 6-OHDA on the expression and localization on microglia and Bergmann glia in the neonatal cerebellum by immunohistochemistry. In the neocerebellum, 6-OHDA treatment caused a significant increase in the number of activated microglia. The increase was observed mainly in the granule cell layer and the cerebellar medulla. Bergmann glial cells in treated brains were abnormally located, did not form intimate associations with Purkinje cells, and the glial fibers were structurally different. Our findings indicate that a noradrenergic influence may be necessary for the normal maturation and migration of granule cells, and abnormal migration may be the result of Bergmann glia destruction and the activation of microglia. Activated microglia in the granule cell layer may be used as a marker for an injured cerebellar area.

Neonatal 6-hydroxydopamine treatment affects GABA(A) receptor subunit expression during postnatal development of the rat cerebellum.

Neurotoxic elimination of noradrenergic terminals by 6-hydroxydopamine (6-OHDA) leads to alteration of the granule cell layer formation. We have studied the developmental expression of GABA(A) receptor subunits in rat cerebellum after neonatal administration of 6-OHDA during the first postnatal month of life. 6-OHDA was injected subcutaneously. The expression of GABA(A) receptor subunits was studied by in situ hybridization and immunohistochemistry. The alterations were observed in the neocerebellum - the part of the cerebellum which starts development postnatally. The migration of granule cells was delayed, and the total area of the granule cell layer in the neocerebellum from 6-OHDA-treated rats was reduced to 22.6+/-5% of the corresponding area from control rats. In situ hybridization with subunit-specific antisense oligonucleotide probes was performed for alpha1, alpha2, alpha3, alpha5, alpha6, beta1, beta2, gamma1 and gamma2 subunits of the GABA(A) receptor. In neocerebellum, 6-OHDA treatment caused a significant reduction in the alpha1, alpha6 and gamma2 subunit mRNA levels. The expression of the other subunits was not changed. It has been shown that in the postnatal cerebellum alpha1 and alpha6 subunits can be detected in granule cells only when the cells had migrated to their final destination. Our findings indicate that a noradrenergic influence may be necessary for the normal maturation and migration of cerebellar granule cells.

Reduction of GABAergic transmission and alterations in behavior after 6-OHDA treatment of rats.

We studied the effects of neonatal administration of 6-hydroxydopamine (6-OHDA) upon gamma-aminobutyric acid (GABA) and noradrenergic neurotransmission in the developing rat brain. After 6-OHDA administration tyrosine hydroxylase (TH) immunolabelling revealed more than 70% loss of catecholaminergic terminals in cortex. Glutamic acid decarboxylase (GAD) immunolabelling showed that the intensity of staining and the density of labelled terminals were decreased by approximately 50% in the prefrontal cortex of 6-OHDA treated animals, but in visual and somatosensory zones there was no difference between lesioned and control cortex. The open field test revealed an altered development of the searching activity after neonatal 6-OHDA injections. A significant difference was found between 6-OHDA treated and control rats in searching, orienting and skills performance. Our results indicate that the behavioral changes observed in young rats after 6-OHDA treatment may be reflections not only of reduced catecholaminergic transmission but also of GABAergic disturbance, occurring in the frontal cortex.

Increased expression of peripheral benzodiazepine receptors and diazepam binding inhibitor in human tumors sited in the liver.

The peripheral benzodiazepine receptor system triggers intracellular metabolic events and has been associated with cell proliferation. Its endogenous ligand, the diazepam binding inhibitor, contributes to steroidogenesis by promoting cholesterol delivery to the inner mitochondrial membrane. The present study was undertaken to verify whether this system is altered in tumors sited in the liver. Peripheral benzodiazepine receptors and diazepam binding inhibitor were studied using immunocytochemistry and in situ hybridization in 9 human tumors sited in the liver, in liver hyperplasia, cirrhotic nodular regeneration, intestinal adenocarcinoma and in surrounding non-tumoral tissue. Immunocytochemical staining and in situ hybridization demonstrated that peripheral benzodiazepine receptors and diazepam binding inhibitor were more prominently expressed in neoplastic cells than in non-tumoral tissue. They were present in the same cells, suggesting that diazepam binding inhibitor may act in an intracrine manner in these cells. Higher peripheral benzodiazepine receptors and diazepam binding inhibitor expression in tumor cells suggest an implication of this system in the metabolism of neoplastic cells. Furthermore the evaluation of peripheral benzodiazepine receptor and diazepam binding inhibitor expression might be useful in evaluating malignancy and in diagnostic approaches of tumors in liver tissue.

[Damage to the catecholaminergic system causes a slowing in the formation of early behavioral reactions in rats]. / Povrezhdenie katekholaminergicheskoi sistemy obuslovlivaet zamedlenie fomirovaniia rannikh povedencheskikh reaktsii u krys.

Influence of 6-hydroxydopamine (6-OHDA) on catecholaminergic brain system and development of early behavioural patterns was studied in 1-20-days-old Wistar rats. After 6-OHDA neonatal injection the number on catecholaminergic terminals in deep layers of pons-medulla was considerably reduced while the number of catecholaminergic neurons was 2-3 times increased. Damage of catecholaminergic innervation of the forebrain of rats was shown to cause a delay in development of washing behaviour, searching activity and disturbance of orientation in new environment.

[The dynamics of intracortical interaction during thinking activities]. / Issledovanie dinamiki vnutrikorkovogo vzaimodeistviia v protsesse myslititel'noi deiatel'nosti.

A new method is described of the brain mapping, based on determination of the probability of appearance of isofrequency components in the EEG derivations allowing to evaluate functional interaction of the brain structures in the process of psychic activity. The process of mental construction of visual image from separate elements includes three stages. At the stage of image search the focus of activity is in the occipital cortical area; in the stage of construction it moves to the frontal cortical areas; completion of the task and verbalization of the image are accompanied by joining of the cortical connections in common system. Alongside with the main focus of activity secondary focuses in the temporal cortex are also revealed during the search of the visual image. The topography of interaction at the frequencies of alpha-range in mainly determined by the stage of image construction. In case of prevalence of the image and abstract thinking shift is marked of the activity focuses at the frequencies of theta-range respectively to the right and left hemispheres.

Alterations in the development of rat cerebellum and impaired behavior of juvenile rats after neonatal 6-OHDA treatment.

The effects of neonatal systemic administration of the neurotoxin 6-hydroxydopamine (6-OHDA) on cerebellum development and behavior were studied in juvenile rats. The methods employed were immunohistochemistry, in situ hybridization, ligand binding, and behavioral testing. The results revealed, for the first time, that 6-OHDA treatment alters Bergmann glial cells and reduced the expression GABAA receptor subtypes alpha1 and alpha6 especially in granule cells. The Bergmann glial cells were abnormally located and structurally different (e.g., no intimate associations with Purkinje cells). Significant microglial activation was also observed. The animals showed impairment in behavior, especially in their orientation to a novel environment. Recent data on neuron-glia interactions support the conclusion that the observed structural changes in Bergmann glia and granular neurons disrupted the normal functioning of the Purkinje cells which then in turn resulted in the impaired sensory-motor coordination at least in juvenile rats. This paper is a summary of previously published work and some recent data in this field obtained at our laboratory.

Neonatal 6-hydroxydopamine treatment affects GABA(A) receptor subunit expression in the frontal cortex but not the hippocampus of rats during postnatal development.

The influence of neonatal administration of 6-hydroxydopamine (6-OHDA) on the maturation of GABA(A) receptors in the frontal cortex and hippocampus was studied using 5- to 40-day-old rats. In situ hybridization with antisense oligonucleotide probes was performed for alpha(1), alpha(2), alpha(5), beta(2), beta(3) and gamma(2) subunit mRNAs of the GABA(A) receptor. We demonstrated that neonatal treatment with 6-OHDA temporarily delays the postnatal transcription of the alpha(1) and gamma(2) subunits in the rat prefrontal cortex, as assessed by in situ hybridization histochemistry. The effect was selective for these subunits (the alpha(2), alpha(5), beta(2), and beta(3) subunit mRNAs remained unchanged) and for this region (the mRNA levels in the hippocampus were not changed). The reduction in mRNA levels at early postnatal stages (postnatal day 5, PD5, and PD10) also affected the subunit protein levels, as shown by immunohistochemistry for the alpha(1) subunit, and the formation of GABA(A) receptor-associated picrotoxinin-insensitive TBPS binding sites, as shown by autoradiography. Our findings indicate that without a noradrenergic influence, the maturation of GABAergic interneurons in the frontal cortex is transiently delayed (from PD5 to PD40). However, it is possible that this transient reduction of the expression of certain GABA subunits - caused by depletion of noradrenergic innervation - cannot cause a lasting alteration to the GABAergic function in the prefrontal cortex.

Apoptosis in acute pancreatitis.

BACKGROUND: Apoptosis may be involved in the mechanism of acinar cell injury in acute experimental pancreatitis. AIM: This study was to investigate whether apoptosis also is involved in human acute pancreatitis. METHOD: A needle biopsy pancreatic specimen was obtained from a patient with acute oedematous pancreatitis. The specimen was stained with In Situ Cell Death Detection Kit. Similar specimen from a patient undergoing pancreatoduodenectomy for bile duct cancer served as a control. RESULTS: Extended acinar cell apoptosis was found in the pancreatitis specimen. No single apoptotic cell was found in the control pancreas. CONCLUSION: Apoptosis probably is involved not only in acute experimental pancreatitis but also in human acute oedematous pancreatitis. The induction and role of apoptosis in pancreatis is discussed based on literature.