Fluoxetine depresses glutamate exocytosis in the rat cerebrocortical nerve terminals (synaptosomes) via inhibition of P/Q-type Ca2+ channels

Fluoxetine, an antidepressant that is used clinically in the treatment of mood disorders, is a selective serotonin reuptake inhibitor. In the present study we investigated the effects of fluoxetine on 4-aminopyridine (4AP)-evoked glutamate release in cerebrocortical nerve terminals (synaptosomes). Fluoxetine suppressed the release of glutamate evoked by 4AP in a concentration-dependent manner. This effect was associated with a reduction in the depolarization-evoked increase in cytosolic free calcium levels in the absence of significant effect on the synaptosomal membrane potential. In addition, both ionomycin- and sucrose-evoked glutamate releases were not affected by fluoxetine, indicating that fluoxetine-mediated inhibition of glutamate release is not a direct effect on the exocytotic machinery. Furthermore, the inhibitory action of fluoxetine was completely abolished in synaptosomes pretreated with P/Q type Ca(2+) channel blocker omega-agatoxin IVA (omega-AgTX IVA) or protein kinase C (PKC) stimulator 4beta-phorbol 12, 13-dibutyrate (PDBu). These results suggest that, in cerebrocortical nerve terminals, fluoxetine inhibits glutamate release through the suppression of P/Q type Ca(2+) channel activity. The presynaptic action of fluoxetine is mediated by a PKC-sensitive signaling pathway. Synapse 48:170-177, 2003.     

Presynaptic quantal plasticity: Katz's original hypothesis revisited

Changes in the amplitudes of signals conveyed at synaptic contacts between neurons underlie many brain functions and pathologies. Here we review the possible determinants of the amplitude and plasticity of the elementary postsynaptic signal, the miniature. In the absence of a definite understanding of the molecular mechanism releasing transmitters, we investigated a possible alternative interpretation. Classically, both the quantal theory and the vesicle theory predict that the amount of transmitter producing a miniature is determined presynaptically prior to release and that rapid changes in miniature amplitude reflect essentially postsynaptic alterations. However, recent data indicates that short-term and long-lasting changes in miniature amplitude are in large part due to changes in the amount of transmitter in individual released packets that show no evidence of preformation. Current representations of transmitter release derive from basic properties of neuromuscular transmission and endocrine secretion. Reexamination of overlooked properties of these two systems indicate that the amplitude of miniatures may depend as much, if not more, on the Ca(2+) signals in the presynaptic terminal than on the number of postsynaptic receptors available or on vesicle's contents. Rapid recycling of transmitter and its possible adsorption at plasma and vesicle lumenal membrane surfaces suggest that exocytosis may reflect membrane traffic rather than actual transmitter release. This led us to reconsider the disregarded hypothesis introduced by Fatt and Katz (1952; J Physiol 117:109-128) that the excitability of the release site may account for the "quantal effect" in fast synaptic transmission. In this case, changes in excitability of release sites would contribute to the presynaptic quantal plasticity that is often recorded.     

Bovine mastitis and intramammary drug delivery: review and perspectives

Intramammary infections (IMIs) represent a major feature in bovine pathology. The treatment of IMIs concern antimicrobial substances. Therapeutic strategies involve administration of immediate release formulations during lactation with or without long-acting formulations during the dry period. Current treatments are not very successful and cure rates are poor, especially towards Staphylococcus aureus which is responsible for chronic infections and huge economic losses. New strategies have recently been investigated. These include particular immunomodulators like lysostaphin or cytokines, and novel formulations (e.g. liposomes, microparticles or nanoparticles) that allow uptake of the active component by phagocytes and thus prolong an enhanced antibacterial activity.     

The power of single channel recording and analysis: its application to ryanodine receptors in lipid bilayers

1. Since the inception of the patch-clamp technique, single-channel recording has made an enormous impact on our understanding of ion channel function and its role in membrane transport and cell physiology. 2. However, the impact of single-channel recording methods on our understanding of intracellular Ca2+ regulation by internal stores is not as broadly recognized. There are several possible reasons for this. 3. First, ion channels in the membranes of intracellular organelles are not directly accessible to patch pipettes, requiring other methods that are not as widely known as the patch-clamp techniques. 4. Second, bulk assays for channel activity have proved successful in advancing our knowledge of Ca2+ handling by intracellular stores. These assays include Ca2+ imaging, ryanodine binding assays and measurements of muscle tension and Ca2+ release and uptake by vesicles that have been isolated from internal stores. 5. The present review describes methods used for single- channel recording and analysis, as applied to the calcium release channels in striated muscle, and details some of the unique contributions that single-channel recording and analysis have made to our current understanding of the release of Ca2+ from the internal stores of muscle. 6. With this in mind, the review focuses on three aspects of channel function and shows how single-channel investigations have led to an improved understanding of physiological processes in muscle. 7. Finally, the review describes some of the latest improvements in membrane technology that will underpin future advances in single-channel recording.     

Effects of Food on the Pharmacokinetics of Methylphenidate

Purpose. To test the hypothesis that the pharmacokinetics of d-meth- ylphenidate (d-MPH) would be altered by food ingested before administration of an immediate release formulation (dl-MPH- IR) but not when food is ingested before a slow release formulation (dl-MPH-SR). Methods. A randomized, four-phase, open label, crossover design was conducted in 24 healthy men who each received, on separate occasions, dl-MPH-IR and dl-MPH-SR taken after an overnight fast and 15 min after a standardized breakfast (20% protein, 21% fat, 59% carbohydrate). Plasma MPH levels were monitored by a validated, stereoselective, GLC-ECD method. Results. For plasma d-MPH, there were significant differences (ANOVA) between dl-MPH-IR and dl-MPH-SR in tmax, Cmax (peak exposure), and Cmax/AUC (sensitive to rate of absorption). Dl-MPH-SR on average delayed tmax from 2.3 to 3.7 h and lowered Cmax 34%. There was no significant difference between the formulations in AUC (extent of absorption). For dl-MPH-IR, food significantly increased Cmax (23%) and AUC (15%) and for dl-MPH-SR the corresponding increases were Cmax (17%) and AUC (14%). After dl-MPH-IR, food delayed average tmax from 2.0 to 2.5 but had no effect on tmax after dl-MPH-SR. There was no effect of food on Cmax/AUC (rate of absorption). Conclusions. Food caused a significant increase in extent of absorption but had no effect on rate of absorption of d-MPH after either dl-MPHIR or dl-MPH-SR.     

Study of the Breakup Under Shear of a New Thermally Reversible Water-in-Oil-in-Water (W/O/W) Multiple Emulsion

Purpose. Thickening of the external aqueous phase of W/O/W multiple emulsions is essential to increase the release under shear. However, it leads to globules bursting during fabrication. To reduce this problem, we have tested a novel thermally reversible hydrogel, EMP hydrogel. This way, the corresponding multiple emulsion (EMPME) would gel only at skin temperature, which may increase the active ingredient delivery when topically applied. Methods. Samples were sheared at different shear rates and temperatures (20, 30, and 35°C) with a controlled rheometer. A granulometric analysis was then performed with a laser diffraction granulometer, to assess the break up as a function of the shear rate at the three temperatures. Conductometric measurements (CDM 230 conductometer) provided the corresponding release curves. Results. As we expected, EMPME exhibited a thermally reversible behavior. Compared to a reference emulsion thickened by carbopol, this new thermo–sensitive multiple emulsion displayed higher break up and fraction released at 35°C. Conclusion. The first thermally reversible multiple emulsion has been developed in the present work. This one presents interesting advantages: (1) an easy fabrication process with a higher entrapment yield and (2) a higher fraction released at 35°C compared with the reference emulsion.     

Biological characterization and optical imaging of brain-derived neurotrophic factor-green fluorescent protein suggest an activity-dependent local release of brain-derived neurotrophic factor in neurites of cultured hippocampal neurons

To visualize the release dynamics of the brain-derived neurotrophic factor (BDNF) involved in neural plasticity, we constructed a plasmid encoding green fluorescent protein (GFP) fused with BDNF. First, several biological studies confirmed that this fusion protein (BDNF-GFP) mimics the biological functions and the release kinetics of unfused (native) BDNF. Second, when BDNF-GFP was expressed in cultured hippocampal neurons, we observed that this protein formed striking clusters in the neurites of mature neurons and colocalized with the PSD-95 immunoreactivity. Such a clustered BDNF-GFP rapidly disappeared in response to depolarization with KCl, as revealed by confocal microscopic studies. These data suggest that BDNF is locally and rapidly released at synaptic sites in an activity-dependent manner. Optical studies using BDNF-GFP may provide important evidence regarding the participation of BDNF in synaptic plasticity.     

Complexities in the neurotoxic actions of 6-hydroxydopamine in relation to the cytoprotective properties of taurine

The neurotoxin 6-hydroxydopamine was shown to cause an imbalance between the direct and indirect pathways of the striato-nigral system as evidenced by a decreased release of gamma-aminobutyric acid and taurine in the substantia nigra but not in the globus pallidus following neostriatal stimulation with kainate (100 microM). The neurotoxicity of 6-hydroxydopamine is generally believed to result from reactive-oxygen radical formation, although it is also known to inhibit mitochondrial NADH dehydrogenase. The release of Fe(II) from the unactivated form [3Fe(III)-4S] of cytoplasmic aconitase (EC(50) < 8 microM) was shown to be followed by the slower oxidation of thiol groups in the protein. Complete loss of -SH groups, and enzyme activity, was seen after incubation of glyceraldenyde-3-phosphate dehydrogenase with 200 microM 6-hydroxydopamine for 75 min at 37 degrees C (IC(50) = 70.8 +/- 0.3 microM). Thus the cellular effects of 6-hydroxydopamine are complex, involving impairment of mitochondrial function, iron- release, sulphydryl-group oxidation, and enzyme inhibition in addition to direct generation of reactive oxygen radicals. Taurine, which is known to be neuroprotective in some other systems, only affords protection against some of these effects, thereby explaining its reported ineffectiveness against 6-hydroxydopamine toxicity.     

Secretory characteristics and viability of human term placental tissue after overnight cold preservation

Collection of human term placentae for research purposes is generally limited during working hours. Preserving placental tissue overnight might help to postpone experiments and, by extent, to increase material availability. In this study, fragments from normal placentae were incubated at 37 degrees C either immediately after delivery or after preservation at 4 degrees C in a HEPES-buffered solution or in a Roswell Park Memorial Institute (RPMI) 1640 culture medium. Protein, human chorionic gonadotrophin (HCG), human placental lactogen (HPL) and lactate dehydrogenase (LDH) contents within preserved explants were similar to those within freshly delivered ones. In contrast, HCG and HPL amounts released during incubation of preserved tissue were lower than with freshly delivered tissue. Differences were significant only during the first 3 h of incubation. Hormone releases were similarly Ca(2+)-stimulated, and Co(2+)- and low temperature-inhibited in preserved and freshly delivered tissues. After preservation, LDH leakage was also reduced. Furthermore, before and after 37 degrees C incubation during 6 h, preserved tissue was morphologically indistinguishable from freshly delivered tissue and showed neither higher incidence of DNA fragmentation, nor elevated caspase-3 activity, both of which are markers of apoptosis. This study validates an original, useful and rapid method to preserve placental tissue. Consequently, this preservation model may facilitate the study of physiological processes regulating placental hormone secretion in normal and pathological conditions.