Giuliana P. Serra

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.

Morphine withdrawal‐induced abnormalities in the VTA: confocal laser scanning microscopy

Morphine withdrawal is characterized by functional alterations at the level of the ventrotegmental area. We investigated the effects of chronic morphine administration and withdrawal on the morphological properties of immuno-labelled tyrosine hydroxylase-positive neurons of the rat ventrotegmental area with a confocal laser scanning microscope. Morphological evaluation revealed a reduction in the area and perimeter of tyrosine hydroxylase-positive somata in morphine-withdrawn rats. Conversely, the number of cells per field was found to have increased in the naloxone group. Collectively, the present results indicate that withdrawal from a chronic morphine treatment, and not chronic morphine per se, modifies cellular morphology of tyrosine hydroxylase-positive, presumably dopamine-containing, neurons of the rat VTA. This is consistent with the idea that withdrawal from morphine alters functioning of the mesolimbic dopamine system and provides a direct morphological correlate for the functional abnormalities typical of morphine withdrawal.

Effect of Dopamine Agonists and Antagonists on DOPA Formation in the Substantia Nigra

Abstract: The effect of different psychotropic drugs on the rate of DOPA accumulation after administration of a decarboxylase inhibitor (NSD 1015) was compared in the substantia nigra (SN) and caudate nucleus (CN) by a new radioenzymatic method. Inhibition of monoamine oxidase with pargyline or stimulation of dopamine (DA) receptors with apomorphine, N‐n‐propyl‐norapomorphine or d‐amphetamine reduced DOPA formation in the CN and SN to the same extent. Vice versa, both inhibition of DA receptors with haloperidol or (‐)sulpiride and depletion of DA concentration with reserpine enhanced DOPA formation to a greater extent in the CN than in the SN. Apomorphine antagonized not only the effect of haloperidol and (‐)sulpiride, but also, and even more effectively, that of reserpine. The results indicate that DA synthesis in the SN is controlled by both end‐product inhibition and DA receptor‐mediated mechanisms.

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.

TRH activates mesolimbic dopamine system: behavioural evidence.

The effect of TRH pretreatment on locomotor activity and stereotypy induced by d-amphetamine was examined in rats. The results show that TRH potentiates d-amphetamine-induced locomotor activity, but not d-amphetamine-induced stereotypy, suggesting that this neuropeptide selectively activates the mesolimbic dopamine system.

Somatostatin in the brain of the cave salamander, Hydromantes genei (Amphibia, Plethodontidae): Immunohistochemical localization and biochemical characterization

The distribution of somatostatin‐like immunoreactivity in the brain of the cave salamander Hydromantes genei (Amphibia, Plethodontidae) was investigated by using two distinct antisera raised against somatostatin‐14. Most somatostatin‐positive cells were detected in the ependymal cell layer surrounding the ventricles. These cells possessed the typical morphological characteristics of tanycytes or radial glial cells. Double‐labeling with an antiserum against somatostatin and a monoclonal antibody against glial fibrillary acidic protein showed that somatostatin‐immunoreactive cells lining the ventricles also exhibited GFAP‐like immunoreactivity. Injection of the neurotracer biocytin into the lateral ventricle revealed that neurons lining the ventricles did not contain somatostatin‐like immunoreactivity. In the telencephalon, somatostatin‐like immunoreactivity was confined to radial glial cells. In the diencephalon, in addition to somatostatin‐immunoreactive cells in the ependyma, positive cell bodies were also found in the periventricular preoptic nucleus, the infundibular nucleus, the epiphysis, and the subcommissural organ. In the metencephalon, positive cell bodies were found in the auricula cerebelli, whereas in the rhombencephalon numerous somatostatin‐immunoreactive cells were seen lining the ventricular cavity. Immunoreactive nerve fibers were observed in the hypothalamus‐median eminence complex. In the pituitary, a discrete group of somatostatin‐positive cells was found in the pars distalis. High‐performance liquid chromatography analysis of brain extracts revealed that the immunoreactive material coeluted with somatostatin‐14. The present results show that the somatostatin peptidergic system in the brain of the cave salamander has a more simple organization than those described in the brain of frog and other vertebrates. This feature is probably related to the expression of high pedomorphic characters in plethodontids. The distribution of somatostatin‐like immunoreactivity suggests that, in the cave salamander, somatostatin may act as a neurotransmitter and/or neuromodulator, a central regulator of fluid homeostasis, and a hypophysiotropic neurohormone. J. Comp. Neurol. 475:163–176, 2004.

Ultrastructural study of the mental body of Hydromantes genei (Amphibia: Plethodontidae)

The mental glands of Hydromantes genei are considered a specialized form of the urodele serous cutaneous glands. Use of a variety of techniques of maceration and digestion as well as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) has shown the three‐dimensional morphology of secretory and myoepithelial cells. Secretory cells are pyramidal and rest on an almost continuous layer of myoepithelial cells. The latter have a long ribbon‐like body from which branch off transversal and longitudinal processes with swallow‐tailed ends. Cytoplasmic processes of secretory cells, containing irregular dense vesicles, squeeze through clefts between myoepithelial cells and may reach, at some points, the basal lamina. The interstices between myoepithelium and secretory cells are extraordinarily rich in nerve endings with clear vesicles. The glandular outlets appear as elliptical stomata in the superficial layer of the epidermis and are lined by horny cells, which invaginate to circumscribe the excretory duct. The morphological results indicate that the myoepithelium of Plethodontidae mental glands differ in some respects from that of amphibian serous cutaneous glands. A double polarity for the secretory cells is also suggested.

Immunohistochemical localization of atrial natriuretic factor and autoradiographic distribution of atrial natriuretic factor‐binding sites in the brain of the cave salamander Hydromantes genei (Amphibia, Plethodontidae)

The distribution of atrial natriuretic factor (ANF)‐like immunoreactivity in the central nervous system of the cave salamander Hydromantes genei (Amphibia, Plethodontidae) was investigated by using antisera raised against rat and human ANF(1–28). Concurrently, the location of ANF‐binding sites was determined by autoradiography, using radioiodinated human ANF(1–28) as a tracer. In several regions of the brain, including the olfactory bulb, the preoptic area, the ventral thalamus, the tectum of the mesencephalon, and the choroid plexuses inside the ventricles, a good correlation was observed between the distribution of ANF‐immunoreactive elements and the location of ANF‐binding sites. Mismatching was found in the habenular nucleus, the commissura habenularis, the fasciculum retroflexus, and the interpeduncular nucleus, which contained high levels of binding sites but were devoid of ANF‐immunoreactive structures. In contrast, a few other regions, such as the pineal gland and the subcommissural organ, showed a high concentration of ANF‐like immunoreactivity but did not contain ANF‐binding sites. This study provides the first localization of ANF‐like immunoreactivity and ANF‐binding sites in the brain of an urodele amphibian. The results show that the ANF peptidergic system in the cave salamander has an organization more simple than the organizations described for the brain of frog or other vertebrates. This feature is probably related to the expression of highly pedomorphic characters in plethodontids. The anatomical distribution of ANF‐immunoreactive elements and ANF‐binding sites suggests that ANF‐related peptides may act as hypophysiotropic hormones as well as neurotransmitters and/or neuromodulators in the salamander brain. J. Comp. Neurol. 437:240–258, 2001.

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

Abstract: 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 µM 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 µM GABA (dissociation started by adding 40 µM picrotoxin) were two‐ to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 µM picrotoxin plus 2 µM 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.

Evidence of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) in Pancreatic Islet Cells by Confocal Microscopy

Several studies have shown that pituitary adenylate cyclase activating polypeptide (PACAP) stimulates at very low concentration insulin release from pancreatic beta cells. In addition, PACAP has been evidenced in pancreatic nervous fibers surrounding the islets, the core of the islet, and the capillaries. The aim of the present study was to demonstrate internalization of PACAP in pancreatic islet cells. Pancreatic islets were obtained from Wistar rat pancreata by modified Lacys isolation method. The isolated islets were incubated in the presence of Fluo-PACAP 27, a fluorescent ligand specific for PACAP receptors. At the end of incubation the islets were fixed in paraformaldehyde and then observed by confocal microscope. Fluo-PACAP 27 was internalized into pancreatic islet cells, and this process was time-and temperature-dependent (37°C). The fluorescent molecules converged toward the nucleus where an intense fluorescence was evidenced after 60 minutes. Incubation with phenyl arsine oxide as well as with PACAP 6–38, a receptor antagonist, prevented the internalization process. Further studies are required to explain the internalization process of PACAP 27 into the nucleus of pancreatic islet cells.