Neurochemical alterations but not nerve cell loss in aged rat neostriatum.

Numerical changes in the overall neostriatal neuronal population have been investigated by morphometric analysis of Nissl-stained and glucocorticoid receptor-immunoreactive neurons. Number and staining intensity of various chemically-identified nerve cell populations were analysed by means of immunocytochemistry coupled with computer-assisted image analysis. Three- and 24-month-old male Sprague-Dawley rats were used. No change in the number of Nissl-stained, glucocorticoid receptor-, dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein- and enkephalin-immunoreactive neurons and a 50% decrease of neuropeptide Y-immunoreactive neurons were observed in the aged rat. In our preparations, the glucocorticoid receptor antibody stains around 90% of the neostriatal neurons, the dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein and enkephalin antibodies label 25-35% and the neuropeptide Y antibody stains only 1% of neostriatal neurons. In the same preparations a significant decrease in the intensity of immunostaining was observed for enkephalin-, dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein- and neuropeptide Y-immunoreactive neuronal cell bodies and tyrosine hydroxylase-immunoreactive nerve terminals in the aged rat. In the case of neuropeptide Y- and dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein-immunoreactive neurons, the changes in the intensity of immunostaining were differentially compartmentalized within neostriatum, suggesting selective vulnerability of striatal subregions to ageing processes. In conclusion, these data indicate that no significant age-related neuronal cell loss occurs in neostriatum. On the other hand, a generalized decrease in the levels of peptide transmitters and molecules related to dopamine transmission is observed in aged rat neostriatum, possibly resulting in the known age-related deficits of neostriatally-controlled behaviours.

Melanin biosynthesis from dopamine. II. A mass spectrometric and collisional spectroscopic investigation.

Electron impact (EI) and fast atom bombardment (FAB) mass spectrometry together with collisional activation (CA) experiments were applied to the study of the oxidation pathway of dopamine by tyrosinase. In order to prevent attachment of the protein to the highly reactive intermediates, ultrafiltration was employed to remove the enzyme at different reaction times. FAB, privileging molecular species formation, was successfully used for identification of transient intermediates and their relative concentrations with respect to time, directly in the reaction mixture. The presence of isobaric molecular species made chromatographic separation necessary. Further EI mass spectrometry and collision spectroscopy led to structural identification of pure components. Of these, dopamine-o-quinone, leucoaminochrome, and aminochrome semiquinone were characterized for the first time as real intermediates in dopamine melanogenesis, in agreement with previous hypotheses. This approach elucidated the pathway of dopamine melanogenesis.

Disposition of exogenous tritium-labelled GM1lactone in the rat.

The disposition of labelled [3H]GM1lactone, the inner ester of ganglioside GM1, was studied in the rat. After i.v. administration [3H]GM1lactone was quickly converted to its corresponding open form most likely by plasma esterases, and then displayed a pharmacokinetic profile identical to [3H]GM1. Following intramuscular administration of [3H]GM1lactone [3H]GM1 levels in plasma and in tissues were higher than those obtained after the administration of an equivalent dose of [3H]GM1. This increased bioavailability means that GM1lactone can be considered as a potential prodrug of GM1.

Differential turnover rates of D1 dopamine receptors in the brain and retina of adult and senescent rats.

The present study was designed to investigate the turnover rates of D1 dopamine receptors in the brain and retina of adult and aged rats. To this aim, we monitored the increase in the density of 3H-SCH 23390 binding sites after the administration of the irreversible antagonist N-ethoxycarbonyl-2-ethoxy-1,2 dihydroquinoline (EEDQ). The results indicate that, in aged rats, the production rate of D1 receptors decreases by 41% to 61% in the striatum, nucleus accumbens and substantia nigra, whereas a smaller reduction in the degradation rate of these receptors is observed (-21% to -40%). In contrast, the production rate of D1 receptors in the retina of aged rats remains unchanged, whilst the degradation rate decreases by 25%. These alterations in the rates of receptor production and degradation may account for the age-related decrease in the density of D1 dopamine receptors in the striatum, nucleus accumbens and substantia nigra as well as for the increase in the density of these receptors in the retina of aged rats.

Siagoside selectively attenuates morphological and functional striatal impairments induced by transient forebrain ischemia in rats.

BACKGROUND AND PURPOSE: Transient forebrain ischemia induced in rats by the four-vessel occlusion method is known to produce severe neural damage in the hippocampus and striatum and a behavioral syndrome the major symptom of which is a working memory deficit. Recent evidence suggests that monosialogangliosides can ameliorate postischemic symptoms. Our purpose was to study the effect of siagoside, the inner ester of GM1 ganglioside, on some behavioral and morphological impairments induced by four-vessel occlusion in rats. METHODS: Rats were injected daily with 5 mg/kg i.p. siagoside starting 4 hours after the cerebral ischemia. After 14 days the rats were tested for working memory in a water T maze or scored for apomorphine-induced stereotypy. The rats were killed 21 days after the cerebral ischemia. Histological and computer-assisted morphometric analyses were performed on cresyl violet-stained brain sections, which were graded according to a neuropathologic score, and on sections stained with a monoclonal antiserum against dopamine and cyclic adenosine-3',5'-monophosphate-regulated phosphoprotein, a marker for striatal dopaminoceptive neurons. RESULTS: Siagoside treatment reduced the stereotypy score induced by low doses of apomorphine and the extent of striatal lesions but did not affect the working memory deficit or the extent of hippocampal lesions. CONCLUSION: Daily siagoside treatment after acute cerebral ischemia attenuates some morphological and functional deficits related to striatal damage. These effects can be interpreted as a selective protective action on striatal neural populations or as a modulatory action on neural systems involved in striatal control. These data are consistent with preliminary clinical reports showing that monosialogangliosides enhance motor recovery after acute ischemic stroke.

Regulatory aspects of nigrostriatal dopaminergic neurons.

In the urethane-anesthetized rat, electrical stimulation (10 Hz, 30 s, 250 microA) of the medial forebrain bundle (MFB), at 20-min intervals over an 8-h period, combined with intracerebral microdialysis in the striatum caused: an undiminished increase in the release of dopamine (DA) with each stimulation episode; a decreased efflux of 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA) after the first stimulation only; a delayed increased efflux of DOPAC with no change in HVA; and a poststimulation depression of firing of dopaminergic neurons in the substantia nigra (before, 3.1 +/- 0.7 Hz; after, 1.9 +/- 1.0 Hz; P < 0.05). After the last stimulation episode, the release of DA declined to prestimulation values, while the increased efflux of DOPAC persisted for three more hours. After the infusion of tetrodotoxin (4.0 x 10(-7) M, 1.5 microliters, 1.0 microliters/min) into the MFB, the basal release of DA was reduced (P < 0.05), while the efflux of DOPAC and HVA was increased (P < 0.05). A model is proposed suggesting that: (1) during increased release of DA in the striatum, the metabolism of DA is decreased; (2) inhibition of nigrostriatal dopaminergic neurons is the usual cause of increased synthesis and metabolism of DA in the striatum; and (3) increased release of DA, and increased synthesis and metabolism of DA in the striatum are not causally linked and are noncoupled processes.

Epidermal growth factor exerts neuronotrophic effects on dopaminergic and GABAergic CNS neurons: comparison with basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have been described to exert neuronotrophic effects on central nervous system neurons in culture. To study the selectivity of trophic actions of these growth factors, neurotransmitter-identified populations of embryonic rat mesencephalon were used. At 20 days in vitro, EGF (3 ng/ml) promoted survival and neurite outgrowth from these neurons. The neuritogenic effect of bFGF (3 ng/ml) was, however, more robust. Quantitative analysis with the neurofilament monoclonal antibody RR97 and ELISA confirmed the differential response, bFGF being 2-2.5 times more effective at all concentrations tested (ED100: 3-10 ng/ml for both EGF and bFGF). At 10 days in vitro, EGF displayed no trophic activity--even at 30 ng/ml. Treatment of mesencephalic cultures with EGF (3 ng/ml) for 20 days stimulated [3H]dopamine and [14C]GABA uptakes about 4-fold. While bFGF (3 ng/ml) also stimulated GABA uptake some 4-fold, dopamine uptake was increased almost 20-fold. Thus, EGF is also capable of enhancing the transmitter traits of selected central neuronal populations; however, the actions of bFGF appear to preferentially address dopaminergic cells.

Monosialoganglioside GM1 reduces NMDA neurotoxicity in neonatal rat brain.

Monosialoganglioside GM1 prevents excitatory amino acid (EAA)-related neuronal death in cultured central nervous system (CNS) neurons and reduces the severity of acute brain damage in different experimental models of cerebral ischemia. Using a model of brain damage induced by intracerebroventricular administration of N-methyl-D-aspartate (NMDA) in neonate rats, we evaluated whether GM1 is capable of exerting antiexcitotoxic effects following its systemic administration in vivo. Newborn rats subjected to brain damage by NMDA and contemporaneously treated subcutaneously with GM1 showed significantly reduced (i) loss in hemispheric weight, (ii) loss in tissue choline acetyltransferase activity, and (iii) morphological damage in various brain areas. These results indicate that systemic GM1 treatment is efficacious in reducing EAA-related neuronal damage in vivo and suggest that such a phenomenon may underlie its capability to ameliorate neurological outcome following cerebral ischemia.

Recovery of function in spinalized, neonatal rats.

Neonatal rats, when spinalized on the fourteenth postnatal day, showed minimal recovery of function in their hindlimbs. Bridging the cut spinal cord with E16 fetal spinal cord tissue did not improve functional recovery. Bridging, plus treatment with GM1 ganglioside, caused a significant (p less than 0.05) improvement in function, versus the bridged animals treated with saline. The E16 spinal cord transplants survived poorly, or not at all. Contact of the hindlimbs with a surface is necessary to elicit function. Regrowth of descending fibers into the caudal region of the cord is probably not involved in functional recovery. It is suggested that functional recovery is mediated by hindlimb proprioceptive afferents, which activate the lumbosacral motor central pattern generator.

A new model of focal brain ischemia based on the intracerebral injection of endothelin-1.

Endothelin-1 and its receptors are widely distributed in the brain of rodents and humans. In view of its potent and long-lasting vasoconstrictor activity, a role of endothelin-1 has been proposed in brain ischemia. In the present paper, the local injection of endothelin-1 was utilized to induce ischemia in rat striatum. An evaluation of the rostrocaudal extension of the lesion is reported. By using intracerebral microdialysis, a marked increase of lactate and dopamine, but not glutamate, was observed in this region upon endothelin-1 administration. Moreover, preliminary data reported show a protective effect of ganglioside treatment on endothelin-1 lesion of rat striatum. The characteristics of the present model of brain ischemia are discussed in comparison with well characterized models, such as the Pulsinelli's four vessel occlusion and the middle cerebral artery occlusion.

Selective reduction of glucocorticoid receptor immunoreactivity in the hippocampal formation and central amygdaloid nucleus of the aged rat.

Hippocampal cell loss during aging has been related to the toxic effects of corticosterone on this cell population. It is not known which receptor mediates corticosterone cytotoxicity. At least two types of receptors for corticosterone have been recognized in the rat brain, type I (corticosterone preferring receptor, CR) and type II (glucocorticoid receptor, GR). In the present study the possible changes in GR immunoreactivity (IR) in various tel- and diencephalic regions of the aged rat have been investigated using immunocytochemistry coupled with computer-assisted image analysis. Male Sprague-Dawley rats of 3, 12 and 24 months of age were used (n = 5/group). A selective decrease of GR-IR was observed in the CA1 hippocampal field and central amygdaloid nucleus of the 24-month-old with respect to both 3- and 12-month-old rats. While in the former region GR-IR decrease was paralleled by a decrease of IR field area, no age-related decrease of GR-IR profile number was detected in central amygdaloid nucleus. A significant decrease of GR-IR and IR field area was also observed in the dentate gyrus of 24- vs 12-month-old rats but not vs 3-month-old rats. The analysis of adjacent sections stained with Cresyl violet showed a pattern of age-related changes (decrease of neuronal profiles in CA1 field pyramidal layer and dentate gyrus granular layer, and no change in the central amygdaloid nucleus of aged rats) which paralleled the observed changes in GR-IR in the same areas. This study provides evidence that GR are selectively decreased in the hippocampal formation and in the central amygdaloid nucleus of the aged rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Monosialoganglioside GM1 in cerebral ischemia.

In vitro studies have shown that monosialoganglioside GM1 reduces excitatory amino acid-related neurotoxicity by limiting the downstream consequences of abusive excitatory amino acid receptor stimulation, while enhancing neuronotrophic factor action in a variety of neuronal cell types. Systemic administration of GM1 appears to be efficacious in reducing acute nerve cell damage and in facilitating medium- and long-term functional recovery after brain injury. Although the mechanism of action remains unclear, it appears likely that GM1 protective effects in the acute injury phase are at least in part due to the attenuation of excitotoxicity, while the long-term functional recovery might reflect GM1 potentiation of neuronotrophic factors. The potential therapeutic efficacy of GM1 administration in different conditions in humans, as suggested by pioneer clinical studies, is reviewed. Further larger, randomized, double-blind clinical studies are necessary to define the therapeutic efficacy.

Lysophosphatidylserine-induced activation of mast cells in mice.

Intravenous injection of lysophosphatidylserine (2.5-25 mg/kg) increases the blood histamine level in mice. Lysophosphatidyl-D-serine, alkyl-lysophosphatidylserine and glycerophosphorylserine show little or no activity. As shown by the similar efficacy of the analogue lacking the OH group in the C-2 position of glycerol conversion into phosphatidylserine is not required. The age of mice influences the activity of lysophosphatidylserine. Thus, the increase in blood histamine is greater in adult mice (8-10 weeks) than in young mice (4-6 weeks). In old mice (50-60 weeks) the tolerance to lysophosphatidylserine is reduced. Repeated parenteral administrations induce depletion of histamine stores with concomitant desensitization to lysophosphatidylserine. Well-perfused organs containing connective tissue mast cells (tongue) are more affected. When [3H] histidine is injected into lysophosphatidylserine-treated mice, the highly radioactive histamine detected in the tongue indicates the preservation of histidine decarboxylase activity after degranulation. The data suggest that lysophosphatidylserine specifically activates connective tissue mast cells in mice.

Hypoxic-ischemic damage and the neuroprotective effects of GM1 ganglioside.

In vitro studies have shown that monosialoganglioside GM1 reduces excitatory amino acid-related neurotoxicity by limiting the downstream consequences of abusive excitatory amino acid receptor stimulation. Systemic administration of GM1 appears to be efficacious in reducing acute neuronal damage and in facilitating medium- and long-term functional recovery after brain injury. We propose that GM1 protective effects in the acute injury phase results from attenuation of excitotoxicity, whereas the functional recovery seen at longer term could reflect GM1 potentiation of neuronotrophic factors. The potential therapeutic efficacy of GM1 administration in humans is suggested by clinical studies demonstrating improved neurologic outcome in stroke patients.

TSH circadian secretions in aged men and effect of phosphatidylserine treatment.

In 20 euthyroid aged men (from 65 to 85 years of age) no significant circadian periodicity of thyrotropin (TSH) secretion has been shown by the population mean cosinor method. At the end of a period of 30 days of hospitalization the cosinor evaluation of TSH secretion showed a restored highly significant (p less than 0.001) circadian rhythmicity in phosphatidylserine (PS) (400 mg/daily) treated group (10 aged subjects). By contrast, hospitalization seems to further deteriorate the periodicity of the hormone secretion in 10 placebo-treated subjects.

GM1 ganglioside reduces edema and monoaminergic neuronal changes following experimental focal ischemia in rat brain.

Seventy-two hours following a middle cerebral artery occlusion, the associated increase in water content on the ischemic side was significantly reduced by the exogenous administration of monosialoganglioside GM1 (30 mg/kg, i.p.). The levels of dopamine and serotonin on the ischemic side were approximately 50% and 80% of those on the contralateral non-ischemic side, respectively. Treatment with GM1 (5 times during the first 48 h after occlusion) produced a significant reduction in the levels of dopamine and serotonin loss. The present findings are compatible with the observed protective action of the exogenously administered GM1 following ischemic brain injury.