Ozonized autohaemotransfusion and fibrinolytic balance in peripheral arterial occlusive disease.

The acute effects of a major ozonized autohaemotransfusion on blood fibrinolytic capacity were evaluated in 20 subjects affected by peripheral arterial occlusive disease (PAOD). The parameters examined were tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1). In subjects not previously submitted to autohaemotransfusion ('unaccustomed' subjects), whether they were PAOD patients or healthy volunteers, the PAI-1/t-PA ratio in the blood samples taken 15 min before the autohaemotransfusion was higher (P < or = 0.05) than at baseline. These changes were independent of the presence of ozone in the autohaemotransfusion blood. Values in both healthy and PAOD-affected individuals were again at baseline 120 min after the end of autohaemotransfusion. In PAOD patients and in healthy subjects previously submitted to several autohaemotransfusions ('accustomed' subjects), the PAI-1/t-PA ratio did not significantly change before, during and after an additional autohaemotransfusion. The results (the increased heart rate and epinephrine and norepinephrine urinary excretion only in non-accustomed subjects) suggest that the acute fibrinolytic imbalance is caused by the apprehensive state produced by the procedure in unaccustomed subjects. Autohaemotransfusion with ozonized blood per se does not significantly influence the fibrinolytic balance.

Heterogeneous immunoreactivity of glial cells in the mesencephalon of a lizard: a double labeling immunohistochemical study.

Astrocytes and radial glia coexist in the adult mesencephalon of the lizard Gallotia galloti. Radial glia and star-shaped astrocytes express glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The same cell markers are also expressed by round or pear-shaped cells that are therefore astrocytes with unusual morphology. Other round or pear-shaped cells, also scattered in the tegmentum and the tectum, display only GS. Electron microscopy reveals that these cells may be oligodendrocytes. In this lizard, the GS is expressed in some oligodendrocytes while this does not occur in the central nervous system of mammals in situ. These results confirm that the cellular specificity of GS is different in various species and suggest that ependymal cells are also immunoreactive for GS but they do not contain GFAP.

Distribution of metabotropic glutamate receptor type 1a in Purkinje cell dendritic spines is independent of the presence of presynaptic parallel fibers.

The metabotropic glutamate receptor type 1a (mGluR1a) is expressed at a high level in the molecular layer of the cerebellar cortex, where it is localized mostly in dendritic spines of Purkinje cells, innervated by parallel fibers. Treatment with methylazoxymethanol (MAM) of mouse pups at postnatal days (PND) 0 + 1 or 5 + 6 results in the partial loss of granule cells, the extent of which depends on the age of the animal at the time of injection. As a consequence of hypogranularity, the number of parallel fibers is decreased to such an amount that many of the postsynaptic Purkinje cell dendritic spines are devoid of axonal input, and only a limited number of spines participate in the formation of parallel fiber synapses, or, infrequently, in heterologous or heterotopic synapses with other presynaptic partners. At PND 30, 50% of the spines in the cerebella of mice treated with MAM at PND 0 + 1 was not contacted by any presynaptic element, compared to 5% in controls or 15% in the cerebella of mice treated with MAM at PND 5 + 6. The localization of mGluR1a was visualized by immunocytochemistry on ultrathin sections: approximately 80% of all Purkinje cell dendritic spines were immunopositive in controls and in both groups of MAM-treated mice, indicating that mGluR1a was present in Purkinje dendritic spines even when the corresponding synaptic input was absent. This observation indicates that the expression and subcellular distribution of mGluR1a are inherent, genetically determined properties of Purkinje cells.

Involvement of a cyclic-AMP pathway in group I metabotropic glutamate receptor responses in neonatal rat cortex.

3,5-Dihydroxyphenylglycine (DHPG), (S)-3-hydroxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG) stimulated phosphoinositide hydrolysis in neonatal rat cortical slices, but with lower maximal effect, in comparison with 2S,1'S,2'S-2-(2'-carboxycyclopropyl)glycine (L-CCG I) or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD). DHPG, 1S,3R-ACPD, and S-4C3HPG also evoked a rapidly desensitizing increase in [Ca2+]i in cortical layers of neonatal brain slices. (R,S)-alpha-methyl-4-tetrazolyl-phenylglycine (MTPG), and (R,S)-alpha-methyl-4-phosphono-phenylglycine (MPPG) inhibited the increase of phosphoinositide hydrolysis elicited by 1S,3R-ACPD but not that by R,S-DHPG. In contrast, the selective group II receptor agonist (1S,2S,5R,6S)-2-amino-bicyclo-[3.1.0]-hexane-2,6-dicarboxylate (LY 354740) potentiated the response of R,S-DHPG. Finally, 8-(4-chlorophenylthio)-cAMP, a membrane permeant analogue of cAMP, reversed the stimulatory effect of 1S,3R-ACPD and S-4C3HPG on phosphoinositide hydrolysis and [Ca2+]i mobilization, without affecting the response induced by R,S-DHPG. These data suggest that, in neonatal rat cortex, the activation of group II metabotropic glutamate receptors potentiates the phosphoinositide hydrolysis and [Ca2+]i responses mediated by group I metabotropic glutamate receptors.

Kainic acid, AMPA, and dihydrokainic acid effect on uptake and efflux of D-[3H] aspartic acid in cerebellar slices.

In this study we show that the glutamate ionotropic agonist kainate (KA) stimulates the efflux of preloaded D-[3H]aspartate (D-[3H]Asp) and inhibits the uptake of this amino acid in cerebellar slices. The effect of this agonist on the efflux of D-[3H]Asp is sensitive to (i) 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2-3-dione (NBQX), indicating the involvement of KA/(RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and is (ii) partially tetrodotoxin (TTX)-sensitive, indicating that pre-(TTX-insensitive) and post-synaptic (TTX-sensitive) KA/AMPA receptors are involved. In contrast, the effect on uptake is NBQX- and TTX-insensitive indicating a direct interaction with glutamate transporters. AMPA inhibited D-[3H]Asp uptake and had no effect on D-[3H]Asp efflux. In the same system, the uptake but not the efflux of D-[3H]Asp was affected by dihydrokainate (DHK). The DHK-induced uptake inhibition occurred in the presence of TTX. NBQX inhibited DHK-induced effect at 5 mM but not at 1 mM DHK concentrations.

Differential ontogenic pattern of metabotropic [3H]-L-glutamate receptors in normal and granule cell-deficient mouse cerebellum.

[3H]-L-glutamate binding site distribution corresponding to metabotropic receptors was studied by autoradiography during normal and altered cerebellar ontogeny in mice treated on postnatal days (PND) 5 and 6 with the antimitotic methylazoxymethanol (MAM). Quisqualate (QA)-induced and (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I)-induced [3H]-L-glutamate binding inhibition allowed us to distinguish between group I and group II metabotropic receptor binding sites. In control cerebellar cortex, the QA-sensitive binding site density increases during development, while the L-CCG-I-sensitive binding site density decreases. In the deep cerebellar nuclei (DCN), both populations of binding sites decrease during ontogeny. The antimitotic treatment induces: (1) a slight but significant increase in the QA-sensitive binding sites in the DCN at PND 10 and in the cerebellar cortex beginning from PND 20; (2) a retarded decrease in the L-CCG-I-sensitive metabotropic receptor binding site density. These differences could be due to a retarded cell maturation and/or an over-expression of some postsynaptic receptors in the adult cerebellum in response to the afference deficiency.

Changes of pharmacological properties of (1S,3R)-ACPD-sensitive glutamate binding sites in developing mouse cerebellum.

Autoradiography of [3H]glutamate binding to mouse cerebellar sections was used to study the distribution of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-((1S,3R)-ACPD) sensitive [3H]glutamate binding sites and the sensitivity of these sites to drugs preferentially acting on one or few types of the metabotropic receptor family. The inhibitory effect of (1S,3R)-ACPD on [3H]glutamate binding and its estimated inhibition constant showed the presence of a different global metabotropic receptor population according to the region considered. During ontogeny, the (1S,3R)-ACPD binding site density increased in the molecular layer (ML), in contrast it decreased in the internal granular layer (IGL) and the deep cerebellar nuclei (DCN). In addition, different sensitivities to (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), (S)-4-carboxyphenylglycine (4-CPG), (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I) and L-2-amino-4-phosphonobutyric acid (L-AP4) were demonstrated according to the region and the age. In the DCN, the high (1S,3R)-ACPD binding site density at PND 10 seems to be also sensitive to L-CCG-I but not to MCPG, 4-CPG or L-AP4. In the ML, the MCPG-, the 4-CPG- and the L-AP4-sensitive [3H]glutamate binding sites appeared during ontogeny and the L-CCG-I-sensitive [3H]glutamate binding sites were already present at PND 10. In the IGL, L-CCG-I-sensitive binding sites disappeared in contrast to the L-AP4-sensitive binding sites which appeared during development even if the total (1S,3R)-ACPD binding site density was relatively weak in the adults. These results all reflected the multiplicity of the receptor subtypes included in the cerebellar metabotropic component.

Autoradiographic localization of [3H]-L-glutamate binding sites in a model of cerebellar granule cell ectopia generated by methylazoxymethanol treatment.

The distribution of [3H]glutamate binding sites was studied in a model of altered cerebellar development obtained by injecting methylazoxymethanol (MAM) in 5-day-old mice. In these mice, at the 25th postnatal day, cerebella were smaller than normal, stratification was normal except for the presence in some lobes of a thin ectopic granule cell layer in the middle of the molecular layer, the proportion of the distribution of [3H]glutamate binding sites between molecular and internal granule cell layers was maintained but site density of both quisqualate- and NMDA-sensitive types was increased in the two layers. In the molecular layer, this increase was uniform in spite of the presence of the ectopic cell layer. In the internal granular layer, the increase of quisqualate-sensitive and NMDA-sensitive [3H]glutamate binding sites is topographically segregated and the first corresponds to areas of lesser cellular density. These results show that MAM treatment induces persistent alterations of the cerebellar glutamatergic system, which consist of receptor over-expression, possibly due to deficit of innervation, reactive gliosis and immaturity of surviving granule cells.

Regional distribution of transient [3H]kainic acid-binding sites in the central nervous system of the developing mouse: an autoradiographic study.

[3H]kainate-binding site distribution in mouse brain was studied by in vitro autoradiography during postnatal development. Sites, highly concentrated at early postnatal ages and undetectable at adult ages, were observed in deep cerebellar nuclei, inferior olive, pontine nuclei, inferior colliculus and stratum lacunosum moleculare of the area CA1 in the hippocampus as well as in previously described rat brain areas. It is suggested that the molecules carrying these sites play a role in the development of the regions where they are transiently expressed.

Developmental changes of EAA metabotropic receptor activity in rat cerebellum.

The potency but not the efficacy of t-ACPD stimulation of phosphatydil inositide hydrolysis changes in developing rat cerebellum. This suggests that the excitatory amino-acid-stimulated metabotropic receptors and/or their coupling are ontogenically regulated. In this, cerebellum differs from other CNS regions where only efficacy changes were described. Differently from hippocampus, the t-ACPD effect, at all ages, is independent of the activation of the NMDA receptor.

Ectopic granule cells in mouse cerebellum molecular layer express high affinity GABAA receptor.

The antimitotic/mutagenic agent methylazoxymethanol when injected in mice pups at postnatal days 3 and 4 produces hypogranular adult cerebella with subpial cells clusters and with a supplementary, ectopic granule cells layer in the molecular layer. The internal granular layer of these treated mice displayed a much lower density of [3H]muscimol binding sites than in controls. At the same time these binding sites are expressed in the ectopic granule cells in the middle of the molecular layer, in spite of the unusual localization of the cells and of the alteration of their nerve inputs. The subpial cells do not express these sites. Our autoradiographic data confirm the suggestion that during ontogeny the GABAA receptors in the granule cells appear when these cells leave the subpial region and indicate that the expressed receptor subtype is the same whether granule cells are in the internal granular layer or in the middle of the molecular layer.

Muscimol and flunitrazepam binding sites in a subcellular fraction enriched in rat cerebellar glomeruli.

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of GABAA and of benzodiazepine binding sites between membranes derived from this fraction (fraction G) and from a total cerebellar homogenate (fraction T) was studied. The benzodiazepine and GABA binding sites were measured by the binding of agonists [3H]flunitrazepam and [3H]muscimol, respectively. The results indicate that both binding sites are present, but only slightly enriched, in the glomerular synapses. We found a muscimol/flunitrazepam binding site ratio of two, which is consistent with the enrichment of muscimol binding sites in the granular layer shown by both autoradiographic with radioactive glutamatergic ligands and in situ hybridization experiments respectively.

[3H]muscimol and [3H]flunitrazepam binding sites in the developing cerebellum of mice treated with methylazoxymethanol at different postnatal ages.

Two models of perturbed cerebellar ontogenesis were obtained by a single administration of methylazoxymethanol (MAM), a potent antimitotic agent, to mouse pups either on the day of birth (MAM0 mice) or at postnatal day 5 (MAM5 mice). The alterations of the cerebellar GABAergic system were studied by measuring glutamic acid decarboxylase activity, [3H]muscimol binding sites, which are known to be concentrated in the GABAA receptors in the internal granular layer, and [3H]flunitrazepam binding sites, which are more abundant in the molecular layer. The primary target of the antimitotic agent are the precursors of the glutamatergic and GABAceptive granule cells. In both models GABAergic structures, as revealed by GAD activity measurements, appear to be relatively spared, and recovery of granule cell numbers occurs during development in MAM5 mice. In MAM treated mice the number of [3H]muscimol binding sites (on a per cerebellum basis) decrease as the number of granule cells decrease, although some recovery occurred in MAM5 mice, but not in MAM0 mice. In MAM5 mice, [3H]flunitrazepam binding sites (on a per cerebellum basis) were relatively unaffected, while they were decreased significantly, but to a lesser extent than [3H]muscimol binding sites, in MAM0 animals. The more significant reduction of granule cell numbers and the cytoarchitectural disruption resultant from the more precocious application of the antimitotic appear responsible for the significant alteration and lack of recovery in MAM0 mice.

Myelin and myelinization in the telencephalon and mesencephalon of the lizard Gallotia galloti as revealed by the immunohistochemical localization of myelin basic protein.

We have studied in the telencephalon and mesencephalon of the lizard Gallotia galloti the localization and the chronology of appearance of the immunoreactivity due to the presence of a myelin-specific protein: the Myelin Basic Protein (MBP). MBP-like immunoreactivity was present with different degrees of intensity in many nerve fibers (isolated, in tracts and in commissurae) and it was apparently more abundant in mesencephalon. During ontogeny the earliest MBP-like immunoreactivity was detected at E.36 in few tracts in mesencephalon and appeared at E.40 in telencephalon, proceeding caudo-rostrally and from the ventral (basal) to the dorsal (alar) regions. Accumulation of MBP continued after hatching. Oligodendrocyte cell bodies were not immunopositive, not even at the youngest ages studied.

An improved method for the preparation of rat cerebellar glomeruli.

Cerebellar glomeruli consist of large portions of the mossy fiber giant terminal, granule cell dendrites and Golgi neuron terminals. By modifying previously reported procedures we have developed a new method for bulk preparation of this polysynaptic complex from rat cerebellum. We obtained well preserved isolated glomeruli of satisfactory purity and homogeneity as indicated by electron microscopy and by determination of appropriate biochemical markers. The method is fast and simple, and it provides a glomerular fraction suitable for investigation of neurotransmitter receptors.

Stoichiometry of muscimol and benzodiazepine binding sites in developing mouse cerebellum.

The ontogeny of high affinity GABAA and central benzodiazepine receptors in the mouse cerebellum was investigated by measuring [3H]muscimol and [3H]flunitrazepam binding to membrane preparations during postnatal development. In the P2 fraction, [3H]muscimol binding was much more abundant than [3H]flunitrazepam binding at all ages. [3H]muscimol Bmax exhibited a peak around postnatal day 25 while [3H]flunitrazepam binding did not follow a parallel course. These results can be explained by the preferential presence in cerebellum of certain variants of the different subunits of the GABAA receptor complex and with different topographical distributions of the different receptor subtypes. Development dependent changes of organelle distribution during subcellular fractionation also contributed to the described developmental pattern.