Reduced nerve growth factor levels in stress-related brain regions of folate-deficient mice.

Folate deficiency has been linked to neurodegenerative and stress-related diseases such as stroke, dementia and depression. The role of the neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT-3) in stress-related disorders and neurodegeneration has garnered increasing attention in recent years. Uracil misincorporation is involved in the neuropsychiatric dysfunction induced by experimental folate deprivation. However, the effects of folate deficiency on the expression of NGF and NT-3 in brain tissue have not yet been investigated. In a 2×2 design, aged mice lacking uracil-DNA N-glycosylase (Ung(-/-)) versus wild-type (Ung(+/+)) controls were subjected to a folate-deficient diet versus a regular diet for three months. Independent of genotype, folate deficiency led to decreased NGF protein levels in the frontal cortex and amygdala. In the hippocampus, NGF levels were increased in UNG(-/-) mice on the normal diet, but not under folate deficiency, while in UNG(+/+) mice, folate deprivation did not affect hippocampal NGF content. NT-3 protein concentrations were neither affected by genotype nor by folate deficiency. Altogether, the results of our study show that folate deficiency affects NGF levels in the frontal cortex, amygdala and hippocampus. The decrease in NGF content in the hippocampus in response to folate deficiency in Ung(-/-) mice may contribute to their phenotype of enhanced anxiety and despair-like behavior as well as to selective hippocampal neurodegeneration.

Patterns of mRNA and protein expression for 12 GABAA receptor subunits in the mouse brain.

The GABAA receptor is the main inhibitory receptor in the brain and its subunits originate from different genes or gene families (α1-α6, ß1-ß3, γ1-γ3, δ, ε, θ, π, or ρ1-3). In the mouse brain the anatomical distribution of GABAA receptor subunit mRNAs so far investigated is restricted to subunits forming benzodiazepine-sensitive receptor complexes (α1-α3, α5, ß2, ß3 and γ2) in the forebrain and midbrain as assessed by in situ hybridization (ISH). In the present study the anatomical distribution of the GABAA receptor subunits α1-α6, ß1-ß3, γ1-γ2 and δ was analyzed in the mouse brain (excluding brain stem) by ISH and immunohistochemistry (IHC). In several brain areas such as hippocampus, cerebellum, bulbus olfactorius and habenula we observed that mRNA levels did not reflect protein levels, indicating that the protein is located far distantly from the cell body. We also compared the distribution of these 12 subunit mRNAs and proteins with that reported in the rat brain. Although in general there is a considerable correspondence in the distribution between mouse and rat brains, several species-specific differences were observed.

The impact of environmental enrichment on sex-specific neurochemical circuitries - effects on brain-derived neurotrophic factor and the serotonergic system.

Experimental evidence in mice indicates that environmental conditions affect females and males differently. However, in a recent study analyzing the heterozygous mutation of brain-derived neurotrophic factor (BDNF), both sexes presented a similar emotional phenotype, which became obvious only under impoverished, but not in enriched conditions suggesting an "enrichment-induced" rescue. To investigate the basis of this behavioral "rescue" effect, we analyzed neurochemical changes (BDNF expression, serotonergic changes, and corticosterone) in the hippocampus, frontal cortex and hypothalamus of animals housed under respective conditions. In male mice, enrichment induced an increase of BDNF expression in the hippocampus of both BDNF heterozygous (BDNF(+/-)) and wild-types. Notably, in enriched-reared BDNF(+/-) mice BDNF mRNA and protein increased to levels comparable to those of wild-types in impoverished environment. In the frontal cortex of males, only wild-types presented an enrichment-induced increase of BDNF mRNA, while no effect of environment could be detected in BDNF protein levels of the male hypothalamus. A further male-specific effect of "environment" is the significant reduction of hypothalamic 5-hydroxyindoleacetic acid in enriched-housed wild-types. In female mice, environmental enrichment did not affect BDNF expression in the hippocampus and hypothalamus. However, comparable to males, an enrichment-induced increase of BDNF mRNA was detected in the frontal cortex of wild-types only. In contrast to males, no influence of environment on serotonergic parameters was observed. Male and female corticosterone levels were neither affected by "genotype" nor by "environment". In conclusion, we propose that the rescue of the emotional phenotype by environmental enrichment in BDNF(+/-) mice is directed by distinct mechanisms in males and females. Only in male BDNF(+/-) mice the rescue is related to an increase in hippocampal BDNF expression suggesting that enrichment triggers different neuronal systems in a gender-specific manner.

AMPA receptor subunit 1 (GluR-A) knockout mice model the glutamate hypothesis of depression.

Recent evidence indicates that glutamate homeostasis and neurotransmission are altered in major depressive disorder, but the nature of the disruption and the mechanisms by which it contributes to the syndrome are unclear. Glutamate can act via AMPA, NMDA, or metabotropic receptors. Using targeted mutagenesis, we demonstrate here that mice with deletion of the main AMPA receptor subunit GluR-A represent a depression model with good face and construct validity, showing behavioral and neurochemical features of depression also postulated for human patients. GluR-A(-/-) mice display increased learned helplessness, decreased serotonin and norepinephrine levels, and disturbed glutamate homeostasis with increased glutamate levels and increased NMDA receptor expression. These results correspond well with current concepts regarding the role of AMPA and NMDA receptors in depression, postulating that compounds that augment AMPA receptor signaling or decrease NMDA receptor functions have antidepressant effects. GluR-A(-/-) mice represent a model to investigate the pathophysiology underlying the depressive phenotype and to identify changes in neural plasticity and resilience evoked by the genetic alterations in glutamatergic function. Furthermore, GluR-A(-/-) mice may be a valuable tool to study biological mechanisms of AMPA receptor modulators and the efficacy of NMDA antagonists in reducing behavioral or biochemical changes that correlate with increased helplessness.

Changes in the brain serotonin satiety system in transgenic rats lacking brain angiotensinogen.

In transgenic rats, TGR(ASrAOGEN)680, with reduced glial expression of angiotensinogen, changes in brain angiotensinogen are associated with reductions in serotonin (5-HT) content and/or 5-HT metabolism as determined in various brain regions, including the hypothalamus. These rats showed an anxious phenotype upon a first behavioural screen. The present study aimed to extend the search for functional consequences of changes in brain 5-HT with respect to feeding behaviour in these transgenic rats. In feeding experiments, rats were treated with the anorectic drug fenfluramine to probe for functional changes in the serotonergic satiety system. Fenfluramine (0.3 mg/kg, i.p.) reduced food intake in TGR(ASrAOGEN)680 rats whereas the minimal effective dose in wild-type rats was 3 mg/kg, i.p. Although, in the cortex, no differences were apparent in the expression of serotonin 5-HT(1A), 5-HT(1B), 5-HT(2C) receptor and 5-HT transporter mRNAs between TGR(ASrAOGEN)680 and wild-type rats, the expression of mRNAs for the 5-HT(2C) receptor and 5-HT transporter mRNA were significantly higher in the hypothalamus of TGR(ASrAOGEN)680 rats compared to wild-type rats. No differences were found in the mRNA levels for hypothalamic 5-HT(1A) and 5-HT(1B) receptors between TGR(ASrAOGEN)680 and wild-type rats. Taken together, these findings suggest that the transgenic effect on the brain 5-HT system is paralleled by functional changes of the serotonergic feeding system.

Embryonic and postnatal development of the serotonergic raphe system and its target regions in 5-HT1A receptor deletion or overexpressing mouse mutants.

The neurotransmitter 5-HT regulates early developmental processes in the CNS. In the present study we followed the embryonic and postnatal development of serotonergic raphe neurons and catecholaminergic target systems in the brain of 5-HT1A receptor knockout (KO) and overexpressing (OE) in comparison with wild-type (WT) mice from embryonic day (E) 12.5 to postnatal day (P) 15.5. Up to P15.5 no differences were apparent in the differentiation and distribution of serotonergic neurons in the raphe area as revealed by the equal number of serotonergic neurons in the dorsal raphe in all three genotypes. However, the establishment of serotonergic projections to the mesencephalic tegmentum and hypothalamus was delayed at E12.5 in KO and OE animals and projections to the cerebral cortex between E16.5 and E18.5 were delayed in OE mice. This delay was only transient and did not occur in other brain areas including septum, hippocampus and striatum. Moreover, OE mice caught up with WT and KO animals postnatally such that at P1.5 serotonergic innervation of the cortex was more extensive in the OE than in KO and WT mice. Tissue levels of 5-HT and of its main metabolite 5-hydroxyindoleacetic acid as well as 5-HT turnover were considerably higher in brains of OE mice and slightly elevated in KO mice in comparison with the WT, starting at E16.5 through P15.5. The initial differentiation of dopaminergic neurons and fibers in the substantia nigra at E12.5 was transiently delayed in KO and OE mice as compared with WT mice, but no abnormalities in noradrenergic development were apparent in later stages. The present data indicate that 5-HT1A receptor deficiency or overexpression is associated with increased 5-HT synthesis and turnover in the early postnatal period. However, they also show that effects of 5-HT1A KO or OE on the structural development of the serotonergic system are at best subtle and transient. They may nonetheless contribute to the establishment of increased or reduced anxiety-like behavior, respectively, in adult mice.

Effects of brain-derived neurotrophic factor (BDNF) on glial cells and serotonergic neurones during development.

Serotonergic neurones are among the first to develop in the central nervous system. Their survival and maturation is promoted by a variety of factors, including serotonin itself, brain-derived neurotrophic factor (BDNF) and S100beta, an astrocyte-specific Ca(2+) binding protein. Here, we used BDNF-deficient mice and cell cultures of embryonic raphe neurones to determine whether or not BDNF effects on developing serotonergic raphe neurones are influenced by its action on glial cells. In BDNF-/- mice, the number of serotonin-immunoreactive neuronal somata, the amount of the serotonin transporter, the serotonin content in the striatum and the hippocampus, and the content of 5-hydroxyindoleacetic acid in all brain regions analysed were increased. By contrast, reduced immunoreactivity was found for myelin basic protein (MBP) in all brain areas including the raphe and its target region, the hippocampus. Exogenously applied BDNF increased the number of MBP-immunopositive cells in the respective culture systems. The raphe area displayed selectively reduced immunoreactivity for S100beta. Accordingly, S100beta was increased in primary cultures of pure astrocytes by exogenous BDNF. In glia-free neuronal cultures prepared from the embryonic mouse raphe, addition of BDNF supported the survival of serotonergic neurones and increased the number of axon collaterals and primary dendrites. The latter effect was inhibited by the simultaneous addition of S100beta. These results suggest that the presence of BDNF is not a requirement for the survival and maturation of serotonergic neurones in vivo. BDNF is, however, required for the local expression of S100beta and production of MBP. Therefore BDNF might indirectly influence the development of the serotonergic system by stimulating the expression of S100beta in astrocytes and the production MBP in oligodendrocytes.

Time gated fluorescence spectroscopy in Barrett's oesophagus.

BACKGROUND AND AIMS: Specialised intestinal metaplasia and its dysplastic transformation, which precedes cancer in Barrett's oesophagus cannot be differentiated in standard gastroscopy. The aim of this study was to investigate whether laser induced protoporphyrin IX fluorescence permits the detection of specialised intestinal metaplasia and dysplasia during endoscopy and to take biopsy specimens in a guided rather than random manner. METHODS: In 53 patients with Barrett's oesophagus 5-aminolaevulinic acid was sprayed on the mucosa. Approximately 60 to 120 minutes later, biopsy specimens were taken based on point-like measurements of delayed fluorescence intensity ratios of protoporphyrin IX in vivo. Two independent pathologists examined the 596 biopsy specimens taken, 168 of which were selected to be investigated by a third pathologist. Among these specimens only those (n=141) with a consensus diagnosis by at least two pathologists and p53 expression as additional marker were included in the analysis. RESULTS: The median of normalised fluorescence intensity (ratio of delayed PpIX fluorescence intensity to immediate autofluorescence intensity) in non-dysplastic specialised intestinal metaplasia (0.51, 68% CI 0.09 to 1.92) and low grade dysplasia (1.89, 68% CI 0.55 to 3.92) differed significantly (p<0.005). Dysplasia was detected at a rate 2.8-fold higher compared with screening endoscopy despite taking fewer specimens. In addition, three early cancers were detected for the first time. Moreover, this method permitted differentiation of specialised intestinal metaplasia from junctional or gastric-fundic type epithelium (p<0.013). CONCLUSIONS: For the first time it was possible to differentiate low grade dysplasia from non-dysplastic Barrett's mucosa during endoscopy based on delayed laser induced fluorescence endoscopy of PpIX. Furthermore, the method helps to detect specialised intestinal metaplasia in short Barrett's oesophagus.

Role of 5-HT1A receptors in the control of food intake in obese Zucker rats of different ages.

The present study describes the role of 5-HT1A receptors in the serotonergic control of food intake in obese Zucker rats of different ages. In addition, serotonin (5-HT) and cholecystokinin (CCK) content and 5-HT turnover were determined in various brain regions. The 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 100 microg/kg) stimulated food intake in 3-month-old lean control rats but inhibited feeding in obese Zucker rats (300 microg/kg). This pattern remained the same in 6-month-old rats. At 10 months of age, 8-OH-DPAT lost its inhibitory activity in the obese rats but still stimulated feeding in lean controls (300 microg/kg). 5-HT levels were higher in the hypothalamus and in the frontal and parietal cortices of 3-month-old obese Zucker rats and were associated with a lower cortical turnover. In the parietal cortex and the hypothalamus of 6-month-old rats, 5-HT levels were still higher, linked with a lower hypothalamic turnover. No differences were observed in 10-month-old rats. CCK content was not different between obese Zucker rats and lean rats. The persistently different feeding responses to 8-OH-DPAT in obese Zucker rats and lean controls may be related to changes in brain 5-HT metabolism in the obese Zucker rats.

Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death.

After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of the cell cycle machinery is thought to contribute to apoptosis in various conditions including ischemia. We demonstrate that loss of endogenous cyclin-dependent kinase (Cdk) inhibitor p16(INK4a) is an early and reliable indicator of delayed neuronal death in striatal neurons after mild cerebral ischemia in vivo. Loss of p27(Kip1), another Cdk inhibitor, precedes cell death in neocortical neurons subjected to oxygen-glucose deprivation in vitro. The loss of Cdk inhibitors is followed by upregulation of cyclin D1, activation of Cdk2, and subsequent cytoskeletal disintegration. Most neurons undergo cell death before entering S-phase, albeit a small number ( approximately 1%) do progress to the S-phase before their death. Treatment with Cdk inhibitors significantly reduces cell death in vitro. These results show that alteration of cell cycle regulatory mechanisms is a prelude to delayed neuronal death in focal cerebral ischemia and that pharmacological interventions aimed at neuroprotection may be usefully directed at cell cycle regulatory mechanisms.

Serotonergic lesion of median raphe nucleus alters nerve growth factor content and vulnerability of cholinergic septohippocampal neurons in rat.

About 45% of the serotonergic raphe neurons are reported to express nerve growth factor (NGF) receptors. We therefore investigated whether selective serotonergic lesions of the median or dorsal raphe nuclei are associated with changes in NGF protein levels of the brain and whether the loss of serotonergic function alters the vulnerability of cholinergic septohippocampal neurons. In adult rats the hippocampal NGF content changed in a biphasic way after lesion of the median raphe nucleus by 5,7-dihydroxytryptamine (5,7-DHT), with a significant increase after 2-3 weeks of up to 35%, followed by a significant reduction of 22% below control levels after 7 weeks, and a return to control levels within the following 4 weeks. By contrast, the decrease in hippocampal serotonin and 5-hydroxyindoleacetic acid remained throughout the observation period of 11 weeks, being still reduced to 15 and 30% of the control levels, respectively. In the frontal cortex the partial loss of the serotonergic innervation projecting from the median raphe was associated 5 weeks after 5,7-DHT injection with an increase in NGF protein of 39.7+/-9.6% (P<0.05), which remained elevated up to 11 weeks. At 9 weeks after 5,7-DHT, the lesion of the septohippocampal cholinergic neurons induced by the cholinotoxin ethylcholine aziridinium (AF64A) was exaggerated (P<0.05) as compared to AF64A-treated rats with intact serotonergic innervation. The present data indicate that a serotonergic lesion of the median raphe nucleus results in biphasic changes of NGF protein content and in a delayed increase in the vulnerability of septohippocampal cholinergic neurons.

Differential mechanisms of neuroprotection by 17 beta-estradiol in apoptotic versus necrotic neurodegeneration.

The major goal of this study was to compare mechanisms of the neuroprotective potential of 17 beta-estradiol in two models for oxidative stress-independent apoptotic neuronal cell death with that in necrotic neuronal cell death in primary neuronal cultures derived from rat hippocampus, septum, or cortex. Neuronal apoptosis was induced either by staurosporine or ethylcholine aziridinium (AF64A), as models for necrotic cell death glutamate exposure or oxygen-glucose deprivation (OGD) were applied. Long-term (20 hr) pretreatment (0.1 microm 17 beta-estradiol) was neuroprotective in apoptotic neuronal cell death induced by AF64A (40 microm) only in hippocampal and septal neuronal cultures and not in cortical cultures. The neuroprotective effect was blocked by the estrogen antagonists ICI 182,780 and tamoxifen and the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. In glutamate and OGD-induced neuronal damage, long-term pretreatment was not effective. In contrast, short-term (1 hr) pretreatment with 17 beta-estradiol in the dose range of 0.5-1.0 microm significantly reduced the release of lactate dehydrogenase and improved morphology of cortical cultures exposed to glutamate or OGD but was not effective in the AF64A model. Staurosporine-induced apoptosis was not prevented by either long- or short-term pretreatment. The strong expression of the estrogen receptor-alpha and the modulation of Bcl proteins by 17 beta-estradiol in hippocampal and septal but not in cortical cultures indicates that the prevention of apoptotic, but not of necrotic, neuronal cell death by 17 beta-estradiol possibly depends on the induction of Bcl proteins and the density of estrogen receptor-alpha.

Serotonin uptake and release mechanisms in developing cultures of rat embryonic raphe neurons: age- and region-specific differences.

The development of serotonergic neurons of the rat raphe was followed in primary neuronal cell cultures taken at embryonic days embryonic day 13 and embryonic day 14 from three different raphe sub-groups, topographically defined with respect to their position to the isthmus as rostral (R1), intermediate (R2) and caudal (R3). In neurons cultivated from embryonic day 13 raphe serotonin, immunoreactivity was detected after only two days in vitro in the rostral R1 and the intermediate R2 sub-groups. Within two weeks of cultivation the number of serotonergic neurons as well as the dendritic branching continuously increased in all three sub-groups. In cultures obtained from embryonic day 13 raphe a specific uptake of [3H]serotonin could not be detected during the first days in vitro. Specific uptake as well as regulated serotonin release, however, was clearly discernible in these cultures after nine days in vitro, indicating developmental differentiation of the initially immature serotonergic neurons in culture. In contrast, serotonergic neurons obtained from the three raphe sub-groups at embryonic day 14 took up and released [3H]serotonin, as early as after two days in culture. Basal as well as stimulated serotonin release was diminished when preincubating the cells with tetanus toxin, which cleaves synaptobrevin thereby blocking exocytosis. Our data indicate that the differential development of serotonergic neurons in the various raphe sub-groups in vivo is also sustained in culture. The differences observed when comparing neurons from embryonic days 13 and 14 suggest that a short time-period of about 24h appears to be crucial for the developmental upregulation of serotonin uptake, storage and release.

Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures.

To assess the neuroprotective potential of melatonin in apoptotic neuronal cell death, we investigated the efficacy of melatonin in serum-free primary neuronal cultures of rat cortex by using three different models of caspase-dependent apoptotic, excitotoxin-independent neurodegeneration and compared it to that in necrotic neuronal damage. Neuronal apoptosis was induced by either staurosporine or the neurotoxin ethylcholine aziridinium (AF64A) with a delayed occurrence of apoptotic cell death (within 72 h). The apoptotic component of oxygen-glucose deprivation (OGD) unmasked by glutamate antagonists served as a third model. As a model for necrotic cell death, OGD was applied. Neuronal injury was quantified by LDH release and loss of metabolic activity. Although melatonin (0.5 mM) partly protected cortical neurons from OGD-induced necrosis, as measured by a significant reduction in LDH release, it was not effective in all three models of apoptotic cell death. In contrast, exaggeration of neuronal damage by melatonin was observed in native cultures as well as after induction of apoptosis. The present data suggest that the neuroprotectiveness of melatonin strongly depends on the model of neuronal cell death applied. As demonstrated in three different models of neuronal apoptosis, the progression of the apoptotic type of neuronal cell death cannot be withhold or is even exaggerated by melatonin, in contrast to its beneficial effect in the necrotic type of cell death.

Clonidine modulates BAY K 8644-induced rat behavior and neurotransmitter changes in the brain.

BAY K 8644 (methyl-1,4-dihydro-2, 6-dimethyl-3-nitro-4[2-trifluoromethyl-phenyl]-pyridine-5-carboxylate), an activator of dihydropyridine-sensitive Ca(2+) channels, injected in rats [2 mg/kg intraperitoneally (i.p.)], induces behavioral changes including ataxia, increased sensitivity to auditory stimulation, stiff tail, arched back, limb tonus and clonus, and rolling over. Neurochemical changes in the brain 45 min after application of 2 mg/kg were characterized by a significant decrease of noradrenaline in the amygdala (-27.8%, P<0.02) and piriform cortex (-16.3%, P<0.02). No significant changes of catecholamines were found in the hippocampal subregions CA1, CA3 and dentate gyrus or in the septum as compared to controls. The dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the amygdala were elevated by 60% (P<0.02) and 66.7% (P<0.02), respectively. In the septum, a 52.6% (P<0.02) increase of HVA was observed. Analysis of amino acids revealed a marked increase of gamma-aminobutyric acid (GABA) content (+50.4%, P<0.001) in the septum. Pretreatment of the rats with the alpha(2)-adrenoceptor agonist, clonidine (0.1 mg/kg i.p.), 30 min before BAY K 8644 (2 mg/kg i.p.) injection completely abolished the behavioral and neurochemical changes. The data suggest that the Ca(2+)-dependent neurotransmitter release provoked by BAY K 8644 can be modulated by stimulation of presynaptic alpha(2)-adrenoceptors. The effect of clonidine on the GABAergic system may represent an important mechanism involved in the prevention of BAY K 8644-induced behavior.

Role of nitric oxide in the ethylcholine aziridinium model of delayed apoptotic neurodegeneration in vivo and in vitro.

The involvement of nitric oxide in neurodegenerative processes still remains incompletely characterized. Although nitric oxide has been reported to be an important mediator in neuronal degeneration in different models of cell death involving NMDA-receptor activation, increasing evidence for protective mechanisms has been obtained. In this study the role of nitric oxide was investigated in a model of NMDA-independent, delayed apoptotic cell death, induced by the neurotoxin ethylcholine aziridinium ethylcholine aziridinium both in vivo and in vitro. For the in vivo evaluation rats received bilateral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ventricle) or vehicle. In the hippocampus a transient decrease in nitric oxide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7+/-9.8% of controls). The decrease coincided with the maximal reduction in choline acetyltransferase activity as marker for the extent of cholinergic lesion. The effect of pharmacological inhibition of nitric oxide synthase was tested by application of various nitric oxide synthase inhibitors with different selectivity for the nitric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition resulted in a significant potentiation of the loss of choline acetyltransferase activity in the hippocampus measured seven days after ethylcholine aziridinium application, whereas the specific inhibition of neuronal or inducible nitric oxide synthase was ineffective. These pharmacological data are suggestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-free primary neuronal cell cultures from hippocampus, cortex and septum of E15-17 Wistar rat embryos. Ethylcholine aziridinium-application in a range of 5-80microM resulted in delayed apoptotic neurodegeneration with a maximum after three days as confirmed by morphological criteria, life-death assays and DNA laddering. Nitric oxide synthase activity in harvested cells decreased in a dose- and time-dependent manner. Nitric oxide production as determined by measurement of the accumulated metabolite nitrite in the medium was equally low in controls and in ethylcholine aziridinium treated cells (range 0.77-1.86microM nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13h after ethylcholine aziridinium application. The present data indicate that in a model of delayed apoptotic neurodegeneration as induced by ethylcholine aziridinium neuronal cell death in vitro and in vivo is independent of the cytotoxic potential of nitric oxide. This is confirmed by a decrease in nitric oxide synthase activity, absence of nitric oxide production and absence of inducible nitric oxide synthase expression. In contrast, evidence for a neuroprotective role of nitric oxide was obtained in vivo as indicated by the exaggeration of the cholinergic lesion after unspecific nitric oxide synthase inhibition by N-nitro-L-arginine methylester.

The neuronal monoamine transporter VMAT2 is regulated by the trimeric GTPase Go(2).

Monoamines such as noradrenaline and serotonin are stored in secretory vesicles and released by exocytosis. Two related monoamine transporters, VMAT1 and VMAT2, mediate vesicular transmitter uptake. Previously we have reported that in the rat pheochromocytoma cell line PC 12 VMAT1, localized to peptide-containing secretory granules, is controlled by the heterotrimeric G-protein Go(2). We now show that in BON cells, a human serotonergic neuroendocrine cell line derived from a pancreatic tumor expressing both transporters on large, dense-core vesicles, VMAT2 is even more sensitive to G-protein regulation than VMAT1. The activity of both transporters is only downregulated by Galphao(2), whereas comparable concentrations of Galphao(1) are without effect. In serotonergic raphe neurons in primary culture VMAT2 is also downregulated by pertussis toxin-sensitive Go(2). By electron microscopic analysis from prefrontal cortex we show that VMAT2 and Galphao(2) associate preferentially to locally recycling small synaptic vesicles in serotonergic terminals. In addition, Go(2)-dependent modulation of VMAT2 also works when using a crude synaptic vesicle preparation from this brain area. We conclude that regulation of monoamine uptake by the heterotrimeric G proteins is a general feature of monoaminergic neurons that controls the content of both large, dense-core and small synaptic vesicles.

Role of tryptophan in the elevated serotonin-turnover in hepatic encephalopathy.

The increase of the brain levels of 5-hydroxyindoleacetic acid (5-HIAA) in hepatic encephalopathy (HE) suggests an increased turnover of serotonin (5-HT). To study the role of tryptophan on the increased brain 5-HT metabolism in HE, we attempted to monitor brain levels of tryptophan in rats with thioacetamide-induced acute liver failure by intravenous infusion of branched-chain amino acids (BCAA). The effect of this treatment on 5-HT synthesis and metabolism was investigated in five brain areas. BCAA-infusions (1 and 2 gm/kg/24 h) increased the ratio BCAA/aromatic amino acids in plasma two- and fourfold, respectively, and lowered both plasma and brain levels of tryptophan. At the higher BCAA-dose all parameters suggesting an altered brain 5-HT metabolism (increased brain levels of 5-HT and 5-HIAA, increased 5-HIAA/5-HT ratio) were almost completely normalized. These results provide further evidence for the role of tryptophan in the elevation of brain 5-HT metabolism and for a potential role of BCAA in the treatment of HE.

Photodynamic therapy of nonresectable cholangiocarcinoma.

BACKGROUND & AIMS: Successful treatment in nonresectable Bismuth type III and IV cholangiocarcinoma is seldom achieved. The aim of this study was to evaluate the effect of photodynamic therapy on cholestasis, quality of life, and survival in these patients. METHODS: Nine patients with advanced nonresectable cholangiocarcinomas Bismuth type III and IV, who showed no sufficient drainage (bilirubin decrease <50%) after endoscopic stent insertion, underwent photodynamic therapy. Two days after intravenous application of a hematoporphyrin derivate, intraluminal photoactivation was performed cholangioscopically. Serum bilirubin, quality of life, and survival time were assessed in two monthly intervals after photodynamic therapy. RESULTS: After photodynamic therapy, bilirubin serum levels declined from 318 +/- 72 to 103 +/- 35 micromol/L (P = 0.0039) with no significant increase during the two monthly follow-ups. Quality of life indices improved dramatically and remained stable (e.g., Karnofsky index from 32.2% +/- 8.13% to 68.9% +/- 6.1%; P = 0.0078). Thirty-day mortality was 0%, and median survival time was 439 days. CONCLUSIONS: This study provides clear evidence that photodynamic therapy is effective in restoring biliary drainage and improving quality of life in patients with nonresectable disseminated cholangiocarcinomas Bismuth type III and IV. Compared with published data, survival time seems to be prolonged.