Regulation of glycinergic and GABAergic synaptogenesis by brain-derived neurotrophic factor in developing spinal neurons.

Brain-derived neurotrophic factor (BDNF) effects on the establishment of glycinergic and GABAergic transmissions in mouse spinal neurons were examined using combined electrophysiological and calcium imaging techniques. BDNF (10 ng/ml) caused a significant acceleration in the onset of synaptogenesis without large effects on the survival of these neurons. Amplitude and frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) associated to activation of glycine and GABA(A) receptors were augmented in neurons cultured with BDNF. The neurotrophin effect was blocked by long term tetrodotoxin (TTX) addition suggesting a dependence on neuronal activity. In addition, BDNF caused a significant increase in glycine- and GABA-evoked current densities that partly explains the increase in synaptic transmission. Presynaptic mechanisms were also involved in BDNF effects since triethylammonium(propyl)-4-(2-(4-dibutylamino-phenyl)vinyl)pyridinium (FM1-43) destaining with high K(+) was augmented in neurons incubated with the neurotrophin. The effects of BDNF were mediated by receptor tyrosine kinase B (TrkB) and mitogen-activated protein kinase kinase (MEK) activation since culturing neurons with either (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'- kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (K252a) or 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) blocked the augmentation in synaptic activity induced by the neurotrophin.

Modulation of retinal endothelial cell behaviour by insulin-like growth factor I and somatostatin analogues: implications for diabetic retinopathy.

Evidence suggests the involvement of growth hormone (GH), insulin-like growth factor I (IGF-I) and somatostatin in the pathology associated with diabetic retinopathy. We examined the effect of IGF-I on human retinal endothelial cell (HREC) survival following high glucose exposure and serum starvation, examined the signalling pathways mediating the protective effect of IGF-I on HREC, and characterized somatostatin receptor-induced retinal endothelial cell death. IGF-I (10 ng/ml) protected HREC from apoptosis induced by high glucose and serum starvation. Wortmannin, a specific inhibitor of phosphotidylinositol-3-kinase, blocks the ability of IGF-I to protect HREC from apoptosis. Incubation of HREC in serum-free medium caused a time-dependent increase in c-Jun N-terminal kinase (JNK) activity, and continuous culture of HREC in the presence of IGF-I or vascular endothelial growth factor (VEGF) prevented JNK activation and arrested apoptosis. Activation of tyrosine kinase receptors results in extracellular signal-related kinase (ERK) activation and activation of ERK is required for proliferation. Both IGF-I and VEGF produced a time- and concentration-dependent increase in the activation of ERK. Type 2 and type 3 somatostatin receptors have been implicated in cell-cycle arrest and apoptosis. Activation of the type 3 receptor in HREC resulted in cell death. These studies suggest that IGF-I is critical for HREC survival, and that somatostatin analogues acting through the type 3 receptor have direct effects on retinal endothelial cells. Furthermore, it appears that the therapeutic efficacy of somatostatin analogues lies not only in systemic inhibition of GH, but also in modulating local growth factor effects.

Proliferation, migration, and ERK activation in human retinal endothelial cells through A(2B) adenosine receptor stimulation.

PURPOSE: The nucleoside adenosine has been implicated in angiogenesis. A previous study demonstrated that activation of the A(2B) adenosine receptor (AdoR) increases cAMP accumulation, cell proliferation, and VEGF expression in human retinal endothelial cells (HRECs). In the present study, the role of this receptor was further characterized by examination of the effects of the selective A(2B) AdoR antagonists 3-N-propylxanthine (enprofylline) and 3-isobutyl-8-pyrrolidinoxanthine (IPDX) on AdoR-mediated HREC proliferation, capillary tube formation, and signal-transduction pathways. METHODS: HRECs were exposed to the adenosine analogue 5'-N-ethylcarboxamido-adenosine (NECA) in the absence or presence of AdoR antagonists. Migration was measured using Boyden chambers. Proliferation was assessed by counting cells. Western analysis was used to assess extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) in cell lysates. The effect of AdoR activation on tube formation was studied using cells grown on a synthetic basement membrane matrix. RESULTS: NECA induced proliferation in a concentration-dependent manner that was inhibited by enprofylline and IPDX. NECA stimulated chemotaxis in a concentration-dependent manner that was also blocked by both A(2B) AdoR antagonists. NECA activated ERK and CREB in HRECs. Both A(2B) AdoR antagonists diminished activation of ERK by NECA exposure. ERK activation was also blocked by the ERK-mitogen-activated protein kinase (MAPK) inhibitor PD98059, but not by the protein kinase A (PKA) inhibitor H-89. CREB activation was blocked by H-89, but not by PD98059, suggesting that ERK activation is independent of PKA. NECA enhanced tube formation on the matrix, whereas both A(2B) AdoR antagonists attenuated this effect. CONCLUSIONS: The selective A(2B) AdoR antagonists, enprofylline and IPDX, inhibited NECA-stimulated proliferation, ERK activation, cell migration, and capillary tube formation. A(2B) AdoR inhibition may offer a way to inhibit retinal angiogenesis and provide a novel therapeutic approach to treatment of diseases associated with aberrant neovascularization, such as diabetic retinopathy and retinopathy of prematurity.

The therapeutic problem of proliferative diabetic retinopathy: targeting somatostatin receptors.

Clinical management of proliferative diabetic retinopathy has changed very little in the last 5 decades, relying primarily on laser ablation of the retinal vasculature. Several lines of clinical and experimental evidence suggest that somatostatin analogues may be efficacious in inhibiting neovascularization associated with proliferative retinopathy but the mechanism of action for these compounds is unclear. Inhibition of growth hormone secretion and the subsequent suppression of insulin-like growth factor 1 (IGF-1) production by somatostatin has been suggested as the mechanism of action, however, in vitro studies suggest that somatostatin analogues suppress endothelial cell growth through a direct, somatostatin receptor-mediated inhibition of pro-survival signaling pathways. The advent of a new generation of modified peptide and peptidomimetic somatostatin analogues has allowed investigators to more carefully define the receptor subtypes responsible for somatostatin-induced endothelial cell death and may eventually lead to the clinical development of somatostatin analogues that can reduce endothelial cell proliferation, independent of suppression of circulating hormone levels.

Oxford house: a review of research and implications for substance abuse recovery and community research.

After treatment for substance abuse, whether it is in hospital-based treatment programs, therapeutic communities, or recovery homes, many patients return to former high-risk environments or stressful family situations. Returning to these settings without a network of people to support abstinence increases chances of a relapse. As a consequence, substance abuse recidivism following treatment is high for both men and women. Alternative approaches need to be explored, and there are some promising types of recovery homes. From a public health perspective, a series of studies conducted at DePaul University suggests that one type of recovery home for alcohol abuse recovery has much potential. For example, within this self-help communal living setting, recovering alcoholics were able to maintain employment, thereby reducing their need for government subsidies. Maintaining employment for recovering alcoholics may promote increased personal responsibility, which may impact self-efficacy beliefs. These pilot studies, then, raised both theoretical and practical issues needing further evaluation.

Isentropic compressibilities--experimental origin and the quest for their rigorous estimation in thermodynamically ideal liquid mixtures.

In this review, attention is initially focused upon the evolution of the Newton-Laplace Equation, that links the measured speed of sound in a fluid in conjunction with its density, to a reliable estimate of its isentropic compressibility κS. Definitions of ideal and excess isentropic quantities are formulated on the premise that the thermodynamic properties of an ideal mixture are mutually related in the same manner as are those of a real mixture or a pure substance. It is shown that both intensive and extensive properties can be derived from the ideal Gibbs energy. Different approaches previously used to calculate ideal isentropic quantities are examined and some subtle errors are identified. The consequences of using conflicting definitions are pointed out. Isentropic pressure derivatives obtained under different conditions and empirical models for estimating the differences between ultrasonic speeds in real and ideal liquid mixtures are discussed.

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.

Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein-coupled receptors, termed A(1), A(2A), A(2B), and A(3). We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A(1) agonist N(6)-cyclopentyladenosine nor the A(2A) agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A(2A) antagonist SCH58261 or the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A(2B) adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A(2B) adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A(2B) adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.

In vivo activation and in situ BDNF-stimulated nuclear translocation of mitogen-activated/extracellular signal-regulated protein kinase is inhibited by ethanol in the developing rat hippocampus.

In order to test the hypothesis that ethanol (EtOH)-induced changes in growth factor signal transduction contribute to the teratogenic effects of EtOH in the developing brain, neonatal rat pups were administered a single dose of EtOH during the brain growth spurt (5 days of age, PN5). Hippocampal mitogen-activated/extracellular signal-regulated protein kinase (MAPK/ERK) activation was analyzed one to 6 h after exposure by electrophoretic-mobility shift assay combined with western blot. Brain-Derived Neurotrophic Factor (BDNF) was used to stimulate ERK in hippocampal slices prepared from PN5 pups and activation and cellular localization was determined with immunofluorescence combined with confocal microscopy. EtOH decreased ERK activation in vivo and decreased nuclear translocation of BDNF-stimulated ERK in situ. These data suggest EtOH-induced inhibition of growth factor signaling may contribute to the development of fetal alcohol syndrome and alcohol-related birth defects.

Organotypic brain slice cultures for functional analysis of alcohol-related disorders: novel versus conventional preparations.

Assessment of long-term alterations in neural function and phenotype has usually involved culture techniques that utilize dissociated preparations. Recently, we have approached such topics in alcohol research by using brain slice cultures, also known as explant or organotypic preparations. In this symposium presentation, two preparations will be discussed, and examples of the particular advantages of these preparations will be presented in relation to alcohol research. First, we use the hippocampal explant preparation for assessment of long-term alterations in N-methyl-D-aspartate receptor (NMDAR) function due to chronic ethanol exposure and subsequent withdrawal. This preparation displays many synaptic, structural, and enzymatic phenotypes indicative of normal neural preparations. Patch clamp recordings reveal NMDAR-mediated excitatory postsynaptic current (EPSC) elicited upon stimulation of Schaffer collateral fibers and recorded from CA1 pyramidal cells. Long-term ethanol exposure followed by subsequent withdrawal resulted in a specific enhancement of NMDAR-mediated synaptic responses which preceded the expression of epileptiform events that occurred after prolonged withdrawal periods. Second, we describe a novel explant preparation, derived from horizontal slices of the entire forebrain and midbrain of the rat. These long-term explants displayed multiple normal phenotypes including Nissl, AChE, TH, and GFAP staining. Electrophysiologically, these explants displayed a functional corticostriatal pathway recorded with field and patch clamp techniques and elicited by synaptic stimulation. Taken together, these explant preparations display utility for long-term study of ethanol effects on neural systems, especially relating to withdrawal hyperexcitability as well as systems involved in drug-seeking behavior.