Damijana Mojca Jurič

Monoaminergic neuronal activity up-regulates BDNF synthesis in cultured neonatal rat astrocytes.

Astrocytes as an active part of the tripartite synapse can respond to the synaptically released neurotransmitters. Because brain-derived neurotrophic factor (BDNF) is produced by astrocytes, in addition to neurons, we focused our present study on the regulatory effects of monoamines noradrenaline (NA), serotonin (5-HT), and dopamine (DA) on the synthesis of BDNF protein in rat neonatal astrocytes from specific brain regions (cortex, cerebellum). All tested neurotransmitters are able to potently and transiently increase BDNF cellular contents; their maximal effects are dose and time dependent and differ between the two brain regions. In cultured cortical astrocytes, NA (1 microM; 6 h) elevates BDNF levels by a 4-fold, 5-HT (1 microM; 4 h) by a 2.3-fold, and DA (150 microM; 4 h) by a 2.2-fold. In cerebellar astrocytes, NA (1 microM; 4 h) increases BDNF content by a 4.7-fold, 5-HT (1 microM; 4 h) by a 1.7-fold, and DA (150 microM; 4 h) by a 1.4-fold. The initial increase in the BDNF levels return to basal levels when incubation with monoamines is extended beyond 12 h (for 5-HT) or 24 h (for NA and DA). Our results confirm the involvement of monoaminergic systems in the regulation of BDNF production in astrocytes and suggest the existence of a positive reciprocal interaction between monoaminergic neuronal activity and astrocytic neurotrophic support in neuron-astrocyte crosstalk, which has a dynamic role in mediating neuronal plasticity and trophic functions in the brain.

Differences in the regulation of BDNF and NGF synthesis in cultured neonatal rat astrocytes.

Using a new brain‐derived neurotrophic factor (BDNF) specific enzyme‐immunoassay, we determined the basal cellular content of BDNF protein in neonatal rat astrocytes in primary culture, thus confirming the ability of astrocytes to synthesize BDNF in addition to nerve growth factor (NGF). We subsequently monitored the influence of different pharmacological agents: neurotransmitter receptor agonists, cytokines, and second messenger up‐regulators, on the synthesis of BDNF and NGF. Marked differences in the regulation of their synthesis by the above pharmacological agents were observed in our study.

Interleukin-1β, but not IL-1α, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin‐1β (IL‐1β). In the present study, the role of IL‐1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL‐1 receptors, and, by comparing the effects of both the agonists (IL‐1α and IL‐1β) and the antagonist (IL‐1ra)—members of the IL‐1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor‐binding studies, binding of [125I] IL‐1β to cultured astrocytes was saturable and of high affinity. Mean values for the KD and Bmax were calculated to be 60.7±7.4 pM and 2.5±0.1 fmol mg‐1 protein, respectively. The binding was rapid and readily reversible. IL‐1 receptor agonists IL‐1α (Ki of 341.1 pM) and IL‐1β (Ki 59.9 pM), as well as the antagonist IL‐1ra (Ki 257.6 pM), displaced specific [125I] IL‐1β binding from cultured astrocytes in a monophasic manner. Anti‐IL‐1RI antibody completely blocked specific [125I] IL‐1β binding while anti‐IL‐1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL‐1α and IL‐1β revealed the functional difference between the agonists in influencing NGF release. In contrast to IL‐1β (10 U/ml), which caused a 3‐fold increase in NGF secretion compared to control cells, IL‐1α by itself had no stimulatory action on NGF release. The simultaneous application of IL‐1α and IL‐1β elicited no additive response. IL‐1ra had no effect on basal NGF release but dose‐dependently inhibited the stimulatory response induced by IL‐1β. We concluded that IL‐1β‐induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL‐1 receptors, whereas IL‐1α and IL‐1ra, in spite of their affinity for IL‐1RI, have no effect on NGF secretion from these cells. Type II IL‐1R is not present on rat neonatal cortical astrocytes.

Regulatory role of monoamine neurotransmitters in astrocytic NT-3 synthesis

Astrocytes actively control neuronal activity and synaptic transmission and by producing various neurotrophic factors represent an important local cellular source of trophic support in the normal and diseased brain. Our present study showed the ability of astrocytes to synthesize neurotrophin‐3 (NT‐3) and the active involvement of the monoamine neurotransmitters noradrenaline, adrenaline, dopamine, and serotonin, as well as basic intracellular second messenger systems, in the regulation of NT‐3 production in neonatal rat cortical astrocytes.

Identification and pharmacological characterization of the histamine H3 receptor in cultured rat astrocytes

Recently we reported that cultured rat cortical astrocytes express histamine H3 receptor that is functionally coupled to Gi/o proteins and participates to the stimulatory effect of histamine. Due to the lack of data on the distribution of histamine H3 receptors on glial cells we further investigated their presence in cultured astrocytes from different brain regions. Real-time PCR was performed to examine the expression of native histamine H3 receptor in cultured rat astrocytes from cortex, cerebellum, hippocampus and striatum. Double-antigen immunofluorescence staining and [3H]N-α-methylhistamine ([3H]NαMH) binding studies were utilized to specifically identify and characterize receptor binding sites in astrocytes. Histamine H3 receptor mRNA was detected in rat astrocytes from all the regions under investigation with the highest levels in striatal astrocytes followed by hippocampal astrocytes and approximately equal levels in cerebellar and cortical astrocytes. Double-antigen immunofluorescence confirmed the presence of histamine H3 receptors on the membrane of all examined astroglial populations. [3H]NαMH bound with high affinity and specificity to an apparently single class of saturable sites on cortical astrocytic membranes (KD=4.55±0.46nM; Bmax=5.63±0.21fmol/mg protein) and competition assays with selective agonists and antagonists were consistent with labeling of histamine H3 receptor (range of pKi values 7.50-8.87). Our study confirmed the ability of cultured astrocytes from different rat brain regions to express histamine H3 receptors. The observed diverse distribution of the receptors within various astrocytic populations possibly mirrors their heterogeneity in the brain and indicates their active involvement in histamine-mediated effects.

Histamine and astrocyte function

Astrocytes support the brain through numerous functional interactions in health and disease. The recent advances in our knowledge of astrocyte involvement in various neurological disorders raised up several questions about their role and functioning in the central nervous system. From the evidence discussed in this review, we show that histamine importantly influences the main astrocytic activities such as ion homeostasis, energy metabolism, neurotransmitter clearance, neurotrophic activity and immune response. These processes are mediated through at least three histamine receptor subtypes, H1, H2 and H3, expressed on the astrocyte surface. Thus, we recognize histamine as an important player in the modulation of astrocytic functions that deserves further considerations in exploring involvement of astrocytes in neurological disorders.

The effectiveness of oxygen therapy in carbon monoxide poisoning is pressure- and time-dependent: A study on cultured astrocytes

Carbon monoxide (CO) poisoning causes neuronal and glial apoptosis that can result in delayed neurological symptoms. The damage of brain cells can be prevented by oxygen therapy. Based on the central role of astrocytes in maintaining neuronal function and viability we investigated the toxic effects of 3000ppm CO in air followed by 24h of normoxia and evaluated the possible protective influence of 100% normobaric oxygen or 100% oxygen at a pressure of 3bar (hyperbaric) against CO poisoning in these cells. CO/normoxia caused a progressive decline of viability, increase in reactive oxygen species and decline of mitochondrial membrane potential and intracellular ATP levels in cultured rat astrocytes. Increased caspase-9, caspase-8 and calpain activity converged in activation of caspase-3/7. 1h treatment with oxygen disclosed pressure- and time-dependent efficacy in restoring astrocytic mitochondrial function and the prevention of apoptosis. The protective effect was most evident when the astrocytes were exposed to hyperbaric oxygen, but not normobaric oxygen, 1-5h after exposure to CO.

Selective cytotoxicity of microcystins LR, LW and LF in rat astrocytes.

Microcystins (MCs) comprise a group of cyanobacterial toxins with hepatotoxic, nephrotoxic and, possibly, neurotoxic activity in mammals. In order to understand the development of their neurotoxicity we investigated the toxic effects of MC variants, MC-LR, MC-LW and MC-LF, in astrocytes that play a central role in maintaining brain homeostasis. 24h exposure of cultured rat cortical astrocytes to MCs revealed dose-dependent toxicity of MC-LF and MC-LW, but not of MC-LR, observed by significant reduction in cell number, declined viability monitored by MTT test and an increased percentage of apoptotic cells, confirmed by Annexin-V labelling. The cultured astrocytes expressed organic anion-transporting polypeptides (Oatp) Oatp1a4, Oatp1c1 and Oatp1a5, but not Oatp1b2. Intracellular localisation of MC-LF and MC-LW, proven by anti-Adda primary antibody, demonstrated transport of tested MCs into cultured astrocytes. Acute MC-LW and MC-LF intoxication induced cytoskeletal disruption as seen by the degradation of glial fibrillary acid protein (GFAP), actin and the tubulin network. In this in vitro study, MC-LF and MC-LW, but not MC-LR, are shown to cause the dysfunction of astrocytic homeostatic capabilities, already at low concentrations, suggesting that astrocyte atrophy, with loss of function, could be expected in the brain response to the toxic insult.

Hyperbaric oxygen preserves neurotrophic activity of carbon monoxide-exposed astrocytes

In astrocytes, carbon monoxide (CO) poisoning causes oxidative stress and mitochondrial dysfunction accompanied by caspase and calpain activation. Impairment in astrocyte function can be time-dependently reduced by hyperbaric (3bar) oxygen (HBO). Due to the central role of astrocytes in maintaining neuronal function by offering neurotrophic support we investigated the hypothesis that HBO therapy may exert beneficial effect on acute CO poisoning-induced impairment in intrinsic neurotrophic activity. Exposure to 3000ppm CO in air followed by 24-72h of normoxia caused a progressive decline of gene expression, synthesis and secretion of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) to different extent. 1h treatment with 100% oxygen disclosed a pressure- and time-dependent efficacy in preserving astrocytic neurotrophic support. The beneficial effect was most evident when the astrocytes were exposed to HBO 1-5h after exposure to CO. The results further support an active role of hyperbaric, not normobaric, oxygenation in reducing dysfunction of astrocytes after acute CO poisoning. By preserving endogenous neurotrophic activity HBO therapy might promote neuronal protection and thus prevent the occurrence of late neuropsychological sequelae.

Monoamine neurotransmitters modulate NT-3 levels in astrocytes

Background Neurotrophin-3 (NT-3), a member of the neurotrophin family of neurotrophic factors, displays profound neuromodulatory functions in the normal and in the diseased brain. Under physiological conditions, NT-3 is produced by neuronal cells and also by local glial cells. We focused our investigation on the ability of astrocytes to synthesize NT-3 and, additionally, on the active involvement of the neurotransmitters noradrenaline, adrenaline, dopamine, histamine and serotonin (5-HT) in the regulation of NT-3 production in neonatal rat cortical astrocytes.