Uneven distribution of NG2 cells in the rat cerebellar vermis and changes in aging.

We describe by NG2 (neuron-glia chondroitin sulphate proteoglycan 2) immunocytochemistry an uneven distribution of NG2 glial cells in the rat cerebellum, being them more represented in the central lobules of the cerebellar vermis, belonging to the cerebrocerebellum. The cerebellar distribution of NG2 cells changes in aging rats, in which the area where the cells appear to be densely scattered throughout all cerebellar layers involves also more rostral and caudal lobules. In addition, in aging rats, in the most rostral and caudal lobules belonging to the spinocerebellum, punctate reaction product is present at the apical pole of Purkinje cells, i.e. in the area where the majority of synapses between olivary climbing fibers and Purkinje cells occur. Data suggest that the different distribution of NG2 cells is correlated to differences in physiology among cerebellar areas and reflects changes during aging.

Increase of the ornithine decarboxylase/polyamine system and transglutaminase upregulation in the spinal cord of aged rats.

We have investigated changes in ornithine decarboxylase (ODC) activity and in polyamine levels in the central nervous system of aged rats. We measured a significant increase of ODC catalytic activity in the spinal cord from 30 month-old rats (+105%) as compared to 4 month-old rats. No changes were noticed in the cerebellum, cortex and hippocampus from the same animals. A related putrescine increase was measured in the spinal cord of 30 month-old rats (+168%), together with a smaller increase of spermidine (+33%). A parallel increase (+78%) of the Ca2+-dependent transglutaminase activity was detected in the spinal cord of 30 month-old rats, while no changes were apparent in the cortex and cerebellum. Our observations indicate a possible role of the ODC/polyamine system during the normal process of ageing in rats and point to the spinal cord as the most sensitive area for this kind of modification. A possible role of protein polyamination by transglutaminase is discussed.

Effect of insemination time of frozen semen on incidence of uterine fluid in mares.

Ninety five mares were inseminated with frozen semen either within 12 h before ovulation or within 8 h after ovulation. The effect of preovulatory versus postovulatory insemination (AI) on the subsequent detection of uterine fluid was studied. The overall pregnancy rate was 43% and this was not significantly influenced by preovulatory or postovulatory insemination. When mares were first examined 12 h after AI, 18 of 52 mares (35%) had accumulated uterine fluid. However, when mares were first examined 18 to 24 h after AI, only 6 of 43 mares (14%) had uterine fluid. Presence of intrauterine fluid significantly lowered pregnancy rates. Timing of insemination did not affect incidence of uterine fluid. Serum concentrations of estrogen and progesterone at time of insemination did not influence uterine clearance or pregnancy rates, but both hormones were higher at preovulatory than at postovulatory inseminations. We concluded that there was no evidence that postovulatory inseminations would predispose mares to persistence of uterine fluid after AI.

Abnormal vestibular control of gaze and posture in a strain of a waltzing rat.

The waltzing behavior is usually attributed to vestibular dysfunction. However, the vestibular control of gaze and posture has not yet been measured quantitatively in any waltzing mutant. Therefore, this study was aimed at investigating the relationship between inner-ear morphology, the circling behavior, and the vestibular control of gaze and posture in a new strain of waltzing rats. Light- and electron-microscopy studies of these mutants did not reveal any structural abnormalities of the vestibular neural epithelia. In addition, the expression of Calretinin and 200-kD phosphorylated and non-phosphorylated neurofilaments was also found to be normal in the vestibular neural epithelia and ganglion cells. In contrast, the mutants showed severe dysfunctions of the vestibular control of gaze and posture. The skeletal geometry of the alert unrestrained animals was studied using cineradiography. At rest, waltzing rats held their heads tilted down: the horizontal semicircular-canal's plane was near the earth-horizontal's plane, instead of being tilted up as in Long Evans control rats. In addition, their cervical column was pitched more forward (33.6 degrees) than in the control group (6.9 degrees). The circling behavior was observed frequently, and the rats had episodes of circling in both directions. The episodes of circling amounted to an average of 17 turns, and the average angular velocity of the circling was 645 degrees/s. Unilateral labyrinthectomy induced the same postural and oculomotor syndromes in the waltzing and control groups. This indicates that the mutant vestibular nerve had a significant resting discharge before the lesion. Eye movements were recorded using acutely implanted search coils. Although waltzing rats were able to perform normal spontaneous eye movements, they showed a complete deficit of the horizontal vestibulo-ocular reflex (HVOR) and an impairment of the maculo-ocular reflex (MOR) during constant velocity, off-vertical axis rotation (OVAR). These results show, for the first time, that deficient transduction and/or processing of the horizontal-canal- and macular-related information can be causally related to the circling behavior and abnormal posture, respectively.

Development of the anatomical alteration of the cerebellar fissura prima.

The development of the naturally occurring malformation of the cerebellar fissura prima was monitored in rats starting from 4 days of life to the adulthood. The first sign of the malformation was evident at 10 days of life and consisted of an interruption of the pia mater and the fusion of the external granular layers on the two sides of the fissura. Later, nests of apparently mature granule cells could be seen to be encircled by cells of the external granular layer and to be connected to the granule cell layer by thin bridges of cells. Calretinin immunoreactive fibers followed the bridges of cells to reach the ectopic masses of cells. Towards the end of histogenesis and in adult animals, brush cells and Golgi cells were present in the ectopic masses of granule cells. The latter appeared to contribute to the formation of normal glomeruli, as in the orthotopic granule cell layer. In addition, bundles of parallel fibers crossed the boundary between the molecular layers on the two side of the fissure, thus suggesting that parallel fibers can contact Purkinje cells of the opposite folium.

Distribution of calretinin-like immunoreactivity in the brain of Rana esculenta.

The distribution of calretinin-like immunoreactivity has been analyzed in the brain of Rana esculenta. Several neurons of nuclei belonging to sensory pathways, subhabenular area and left habenula were immunopositive. Immunoreactivity was present in fibers of motor and sensory pathways, thalamus, tegmentum and isthmus. The immunolabeling pattern partially overlapped that previously described in the rat. However, in comparison with the rat, fewer cells and fibers were immunoreactive and there were less positive brain nuclei. especially in the pallium, septum and striatum, that were totally negative. Taking into consideration that these regions are rather simple in the frog, the presence of calretinin seems to be consistent with the degree of complexity of brain areas and segregation of different nuclei.

Nitric oxide-containing neurons in the nervous ganglia of Helix aspersa during rest and activity: immunocytochemical and enzyme histochemical detection.

Nitric oxide synthase (NOS) immunoreactivity and staining for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) activity are two cytochemical markers for nitric oxide (NO)-containing neurons. The authors examined the changes in the distribution of NOS immunolabeling and NADPH-diaphorase reactivity in the cerebral and buccal ganglia of the terrestrial snail Helix aspersa during resting and active phases. During inactivity and after 1 day of activity, in the mesocerebrum and metacerebrum of the snails, there were several reactive neurons for both markers; after 7 days of activity, the number of reactive neurons was lower. Opposite results were obtained in the buccal ganglia, in which increased staining and numbers of reactive neurons were present in the active snails (after 1 day and 7 days of activity). Although the staining patterns for the two reactions were similar, colocalization was not always observed. The comparison between inactive and active animals provided a more precise survey of NOS-containing neurons in the snail cerebral ganglia than previously described. Moreover, it suggested that not only is NO involved in distinct nervous circuits, but, as a ubiquitous molecule, it also plays a role in neuroprotection and neuropeptide release.

Bioactive peptides and serotonin immunocytochemistry in the cerebral ganglia of hibernating Helix aspersa.

The role of some neuromodulators and neurotransmitters in the functioning of molluskan cerebral neurons and in their metabolic changes during hibernation has been considered. The cerebral ganglion of mollusks is a center for the integration of different inputs from the sensory areas of the head and for the generation of motor command impulses. During hibernation, animals are deprived of many external sensory stimuli and do not have locomotion and feeding. Immunocytochemistry for bioactive peptides (BAPs), such as SP (Substance P), CCK8 (Cholecystokinin 8/Gastrin), CGRP (Calcitonin-Gene-Related Peptide) and ET (Endothelin), and serotonin was performed on cerebral ganglia of active and hibernating Helix aspersa. The distribution of the immunopositivity was analyzed in different cell-containing areas (procerebrum, mesocerebrum, metacerebrum) and in the neuropiles. With all the antibodies raised against peptides, we observed that only a few neurons, mainly of small and medium size, had immunopositivity during the period of activity, the patterns of distribution being quite similar to those previously described in Helix or other gastropods. Fibers and varicosities with BAP immunopositivity were found in the procerebral and central neuropiles and sometimes around neurons. Serotonin-immunopositive neurons, including the giant neuron, were observed in the metacerebrum; numerous fibers and varicosities immunopositive for serotonin were present in the neuropile areas. In hibernating snails, the number of fibers with BAP and serotonin immunopositivity decreased in several areas of the neuropiles. Moreover, an increased number of neurons of the metacerebrum (two-to four-fold) and mesocerebrum (8- to 28-fold) had BAP-like immunopositivity, and the intensity of the immunoreaction for serotonin of the metacerebral neurons was also higher than in the active snails. These results are discussed, taking into account two hypotheses. The first hypothesis assumes that the increased immunocytochemical staining was really linked to accumulation of BAPs and serotonin. The second hypothesis considers that the antibodies for BAPs recognized a preprotein, the synthesis of BAPs being completed during the active period only. Both the hypotheses account for the co-occurrence and co-localization of two or ore peptides and serotonin and stress that the hibernation condition is of interest for studies on the actual function of single neurons in the cerebral ganglia. Finally, the data are consistent with the changes recently found in other markers of the morphological and functional activity of neurons, demonstrating that the neuromodulation and the neurotransmission are slowed during hibernation.

Cytochemical demonstration of nitric oxide synthase and 5' nucleotidase in human glioblastoma.

Both nitric oxide (NO), formed from L-arginine by the enzyme nitric oxide synthase (NOS), and adenosine, which is produced by 5' nucleotidase (5' N) acting on adenosine 5' monophosphate (5' AMP) are implicated in several neurophysiological processes. In addition, 5' N is a linker protein involved in cell motility. Alterations of both enzyme activities seem to be responsible for some pathological states of the central nervous system (CNS). In the present report, we have studied the cytochemical demonstration of NOS and 5' N activities in human glioblastoma cells. Enzyme activity of both was observed in tumor cells; moreover, the coincidence of enzyme histochemistry and immunohistochemistry for NOS was noted in most cases. The findings were interpreted on the basis of the cytotoxic effects due to NO production by tumor cells, and on the non-catalytic role of membrane 5' N which acting as an adhesive molecule can favour tumor invasiveness.

Glial cell reaction to cis-dichlorodiammine platinum treatment in the immature rat cerebellum.

In this study we have investigated changes in glial cells of the cerebellum of cis-dichlorodiammine platinum (cisDDP)-treated rats. The expression of S-100 protein and glial fibrillary acidic protein (GFAP), taken as markers of glial cell function, was evaluated using immunocytochemical methods. In parallel, immunoreactivity for calbindin, parvalbumin, and phosphorylated 200-kDa neurofilament protein was observed in Purkinje cells as markers for neuronal integrity and activity. Results showed that, although no difference in the immunostaining of S-100 protein between control and treated animals could be observed, an increase in the frequency of GFAP immunoreactive cells was present in cisDDP-treated rats. In Purkinje cells, immunocytochemical expression of calbindin and parvalbumin was decreased after drug treatment. In addition, following immunoreaction for phosphorylated 200-kDa neurofilament protein, the somata of Purkinje cells, which were negative in control animals, were stained in treated rats. These findings suggest that cisDDP does not significantly interfere with pathways of glial cell activity and that the increased number of GFAP positive astrocytes may be due to an activation of glial cells consequent upon neuronal death.

Perineuronal glial system in the cerebral ganglion of active and hibernating Helix aspersa.

The electron microscopical changes in the glial lacunar network that surrounds the large neurons of meso- and metacerebrum of land snail cerebral ganglia were considered, in order to get an insight into the functional role of this peculiar structure in invertebrates. Compared with snails during the active period, in the hibernating animals the extension of glial processes was reduced and the glial processes appeared more regular and stacked around neurons. Moreover, they did not form deep, long interdigitations with neuronal infoldings as during the active period. In particular, data on the ultracytochemical detection of alkaline phosphatase and Ca(2+)/Mg(2+)-ATPase enzyme activities, point to a correlation between the extension of the glial system and its function in the regulation of the extracellular environment. In fact, in hibernating snails, lower reactivity was found on the glial membranes, including those of the trophospongium.

Effects of cis-dichlorodiammineplatinum on the molecular layer interneurons of the immature rat cerebellum: a putative role for calcium.

The administration of cis-dichlorodiammineplatinum to rats at 10 days of postnatal life (PD 10) alters the immunoreactivity for parvalbumin of the interneurons of the cerebellar molecular layer at late post-treatment intervals. This can result in, but may be also a consequence of the elevation in the cytosolic calcium concentration. Changes in the intracellular calcium homeostasis may induce damage of the cytoskeletal apparatus and impair growth of the cell processes leading to alteration of the ultrastructure of the cell.

Cis-dichlorodiammineplatinum alters GABAergic structures in the immature rat cerebellum.

It has been reported that injection of the antitumoral drug cis-dichlorodiammineplatinum at 10 days of life affects cerebellar development in rats. After a single dose of 5 micrograms/g of body weight, the formation of granule cells is decreased and the maturation of postmitotic neurons is slowed down. A substantial time after treatment, reduced cell packing density of the internal granule layer and atrophy of the molecular layer can be observed. In addition, there is degeneration of some Purkinje cells and Golgi neurons. In spite of all these alterations, the regular architecture of the cerebellar folia is retained in many places. In the present study, we used immunocytochemistry with an immune serum raised against glutamic acid decarboxylase to further characterize the cis-dichlorodiammineplatinum-induced alterations of GABAergic neurons. The aim was to examine cerebellar development and to test for factors controlling the settling of GABAergic circuits. At all post-treatment intervals, most of the Purkinje and Golgi neurons and molecular layer interneurons showed stronger anti-glutamic acid decarboxylase immunoreactivity than in controls; this may have been due to altered fixation because of cis-dichlorodiammineplatinum-induced damages to the blood vessels; but could also reflect cellular retention of the enzyme, maybe due to cis-dichlorodiammineplatinum-induced damage of the microtubular apparatus. After seven days, large roundish immunoreactive varicosities were present in the molecular layer adjacent to the Purkinje cell dendritic poles. These varicosities, which were not observed in control animals, may be terminals of Purkinje cell axon recurrent collaterals contributing to the supraganglionic plexus, whose abnormal development would compensate for the reduced inhibitory inputs from inhibitory interneurons and/or Purkinje cells, which degenerated at early post-treatment intervals. At later post-treatment intervals (15 and 21 days), there were also alterations in the pericellular basket at the Purkinje cell axon hillock, which was poorly developed in or absent from the majority of cells. The finding was confirmed by morphological observation of basket cells in Golgi-Cox preparation and immunocytochemistry with an antibody raised against 200,000 mol. wt phosphorylated neurofilaments. It is concluded that early changes in anti-glutamic acid decarboxylase immunoreactivity of neurons may be due to a direct interference of the drug with the cellular metabolic pathways. The late anomalies in the anti-glutamic acid decarboxylase immunoreactivity appear to be secondary to changes in the tissue cytoarchitecture rather than being primary cis-dichlorodiammineplatinum-induced lesions of the cells.