Astroglial in vivo response to cocaine in mouse dentate gyrus: a quantitative and qualitative analysis by confocal microscopy.

Astrocytes have been proved to play a critical role in neuromodulation, neuroprotection, pH maintenance, axon guidance control during development, homeostasis preservation and blood brain barrier maintenance in the CNS (Kimmelberg and Norenberg, 1989). Quantitative changes in the expression of glial fibrillary acidic protein (GFAP), a cytoskeletal intermediate filament protein exclusively expressed in astrocytes (Bignami et al, 1972), have been observed after administration of alcohol (Framke, 1995), morphine (Beitner-Johnson et al., 1993), amphetamine and its derivates (Aguirre et al., 1999), cannabinoids (Suarez et al., 2000), nicotine (Janson and Moller, 1993), caffeine (Marret et al., 1993) and prenatal exposure to cocaine (Clarke et al., 1996; Nassogne et al., 1998). However, the general astrocytic response to drugs of abuse is still far from being defined. In the present study we examined the in vivo astroglial response to cocaine in mouse dentate gyrus, the hippocampus being a common target of neurotoxic agents (Walsh and Emerich, 1988) which has a prominent effect on learning and memory processes (Eichenbaum et al., 1992). Quantitative changes in immunoreactivity of GFAP were investigated 24 h after acute and repeated daily administration of intraperitoneal cocaine (20 mg/kg). Drug-induced morphological alterations and spatial distribution of astrocytes were evaluated by means of confocal microscope. The results show that, compared to control animals, GFAP expression is two-fold enhanced after a single cocaine injection, still significantly higher after seven consecutive daily administrations, but not statistically different after prolonged (14 days) drug treatment. Moreover, morphological and morphometric analyses reveal significant modifications in astrocytic numbers, cell size and shape complexity. These data demonstrate that in mouse dentate gyrus, cocaine exposure differently affects the expression of GFAP and induces strong changes in astrocytes proliferation rate and cell morphology. Taken together, our findings provide the first in vivo quantitative and qualitative evaluation of astrocytic response to several regimens of cocaine in adult animals brain.

Application of the Cajal-De Castro method to the laser scanning confocal microscope in studying the peripheral nervous system.

We tested the Cajal-De Castro method with the Laser Scanning Confocal Microscope (LSCM). This method is a valid investigatory tool for anatomical-morphological study and for the tridimensional reconstruction of the structures of the peripheral nervous system. The combination technique makes it possible to exploit the properties of reflection and fluorescence of the deposited reduced silver. The quantity of which is dependent on the different tissue affinity. On the other hand, since scanning in fluorescence is not selective, both the nerve structures and the surrounding tissue are evidenced and the structures that demonstrate more specific affinity for silver salt are more prominent. Being able to study the preparations using both methods contemporaneously makes it possible to obtain very significant tridimensional images that abound in detail. The Cajal-De Castro method also offers interesting possibilities in the morphometric field and in image analysis.

Kinetics of tert-[35S]butylbicyclophosphorothionate binding in the cerebral cortex of newborn and adult rats: effects of GABA and receptor desensitization.

The effects of GABA on the kinetics of tert-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to the convulsant site of GABAA receptors were studied in membrane suspensions from the cerebral cortex of newborn (1-day-old) and adult (90-day-old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [35S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast (t1/2, 11 min) and slow (t1/2, 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 microM GABA, however, was more robust in neonates than in adults (six- to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 microM GABA (dissociation started by adding 40 microM picrotoxin) were two- to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 microM picrotoxin plus 2 microM GABA). Taken together, these results suggest that (1) the closed state of GABAA receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS.

Modulation of [35S]TBPS binding by ligands with preferential affinity for benzodiazepine BZ1 sites in the cerebral cortex of newborn and adult rats.

The present study was designed to compare the allosteric coupling between the Cl- channel of the GABAA receptor and the different benzodiazepine recognition site subtypes (BZ sites) in the cerebral cortex of newborn (5-day-old) and adult rats (90-day-old). To this aim, we reexamined the heterogeneity of cortical GABAA receptors in self- and cross-competition binding experiments using [3H]flunitrazepam and two ligands with higher affinity for benzodiazepine BZ1 sites relative to benzodiazepine BZ2 sites, the triazolopyridazine 3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo [4,3-b] pyridazine (CL 218,872) and the imidazopyridine N,N,6-trimethyl-2-(4-methylphenyl)-imidazo[1,2-a]-pyridine-3-acetamide hemitartrate (zolpidem). Benzodiazepine BZ1 sites accounted for 52% of the total number of binding sites in adult rats, but were not detected in newborn rats. On the other hand, two classes of benzodiazepine BZ2 sites with high and low affinity for zolpidem were present in newborn and adult rats. These sites were designated as benzodiazepine BZ2H (high affinity for zolpidem, Kd approximately 150 nM) and benzodiazepine BZ2L (low affinity for zolpidem, Kd approximately 3000 nM). High densities of benzodiazepine BZ2H sites were measured in both newborn and adult rats (75% and 41% of the total number of [3H]flunitrazepam binding sites, respectively), whereas benzodiazepine BZ2L sites accounted for 25% and 7% of the total number of cortical sites in neonates and adults, respectively. Flunitrazepam, CL 218,872 and zolpidem inhibited in a concentration-dependent manner the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to the convulsant site of cortical GABAA receptors in newborn and adult rats. The IC50 for flunitrazepam was about 3-fold greater in adults than in neonates. This rightward shift in the concentration-response curve may be due to a decrease with age in the intrinsic efficacy of flunitrazepam. In contrast, CL 218,872 and zolpidem were 4-fold more potent at inhibiting [35S]TBPS binding in adult rats relative to neonates. The different affinities of CL 218,872 and zolpidem for benzodiazepine BZ1 and BZ2 receptors may account, at least in part, for the age-related changes in their inhibitory potencies. These results demonstrate that benzodiazepine BZ2 sites mediate the modulation of [35S]TBPS binding by benzodiazepine recognition site ligands in the cerebral cortex of newborn rats. Further, benzodiazepine BZ2 sites may be involved in the inhibition of [35S]TBPS binding by flunitrazepam, CL 218,872 and zolpidem in the cerebral cortex of adult rats.

The myoepithelial and basal cells of ducts of human major salivary glands: a SEM study.

The cytoarchitecture and distribution of myoepithelial (mecs) and basal (bc) cells of intralobular (intercalated, striated) and interlobular (excretory) ducts of human major salivary glands were studied by SEM through a variety of maceration and microdissection techniques. Intercalated ducts are covered by mecs which, unlike the large stellate cells of acini, are spindle shaped. Small star shaped mecs are rarely observed even in the most distal striated ducts, while no such cells are seen in excretory ducts. Basal cells form a more or less continuous row of small, basally placed cells in excretory ducts. Sparse bc are occasionally present in proximal striated ducts as well. Following microdissection, bc exhibit a cup-shaped apex which embraces the convex base of principal cells. This configuration may explain why, in TEM, bc seem to possess lateral processes which may be mistaken for mecs processes. Moreover, the lateral surfaces of bc do not exhibit the complex system of plasmalemma folds typical of principal cells. The present study demonstrates that fusate mecs are present in intercalated ducts and that basal cells are distinct from myoepithelial cells. These results may have some relevance in histogenetic studies of salivary gland neoplasms.

[An atrial hematopoietic locus in the heart of the cave salamander (urodele amphibian)]. / Un locus emopoietico atriale nel cuore del geotritone (anfibi urodeli).

Lungless Salamanders of the family Plethodontidae have a reduced interatrial septum. The pulmonary vein is lacking. In these species, the septum as a membranous thin sheet attaches near the dorsal lip of the sino-atrial valve where a connective and muscular column, supporting the valve, extends its branches over the upper wall of the undivided atrial cavity where a sponge-like structure is formed. The meshes of this structure are the site of a erythropoietic activity as shown in the plates. Early stages in active reproduction are found in the external acid layer while in the basic inner layer the red cells undergo differentiation. This locus may be correlated to the particular anatomy of the heart concerning the lacking of the pulmonary vein, the position of the sino-arterial aperture shifted to the left side and the reduced interatrial septum. In the large upper cavity of the atrium a certain degree of blood stagnation could be possible which could allow the settlement of this locus. No ventricular erythropoiesis nor epicardial granulopoiesis have been found. This hemopoietic locus is lacking in the family Salamandridae and Anura.

Differential effect of aging on 3H-SCH 23390 binding sites in the retina and in distinct areas of the rat brain.

The effects of age on the binding parameters of the selective D-1 dopamine (DA) receptor antagonist 3H-SCH 23390 were studied in membrane preparations from rat striatum, substantia nigra, olfactory tubercle, prefrontal cortex and retina. When compared with 3-month-old animals, there was a significant decrease in the density of 3H-SCH 23390 binding sites in the striatum (-25%), substantia nigra (-24%), and olfactory tubercle (-23%), but not in the prefrontal cortex of senescent (23-month-old) rats. The affinity of 3H-SCH 23390 for D-1 DA receptors did not change with age in any of the brain areas analyzed. In contrast, the density of 3H-SCH 23390 binding sites was higher (+53%) in the retina of aged rats that in 3-month-old controls. Confirming previous studies, we observed that light deprivation induced a significant increment in the density of 3H-SCH 23390 binding sites in the retina of adult rats (+31%) but not in the retina of aged animals. The ability of light exposure to activate DAergic neurons in the rat retina was not altered by normal aging. In fact, a similar increase in the concentration of DOPAC was observed in the retina of light-adapted adult and senescent rats when compared to their respective dark-adapted controls (+94% and +95%, respectively). The results indicate that aging has a differential effect on D-1 DA receptors in the retina and different areas of the rat brain. Finally, the age-related increment in the density of retinal D-1 DA receptors does not appear to depend on presynaptic mechanisms, since DA metabolism is increased by light to the same extent in young and aged rats.

[The epithelial bell]. / La campana epiteliale.

The epithelial bell which surrounds the clitoris in most mammals arises by fusion of visceral and parietal layers of the prepuce. Adhesion of the epithelia occurs between the squamous cells and a normal connection is formed by desmosomes and other junctions. There is total absence of cornifications. The squamous epithelial double lamina splits occasionally in some patches where the free surfaces are getting back to a normal process of keratinization as in the wall of the vestibule. In the closed pits the keratin scales form squamous granules without sebaceous secretion. Sometimes a similar process occurs in the human prepuce with the same results as described in cat and rabbit. The granules of keratin are different from the smegmaliths as they do not contain sebaceous secretion.