Incubation Of Slices Research Articles

Counteracting vasopressin-mediated water reabsorption by ATP, dopamine, and phorbol esters: mechanisms of action

Water homeostasis is regulated by a wide variety of hormones. When in need for water conservation, vasopressin, released from the brain, binds renal principal cells and initiates a signaling cascade resulting in the insertion of aquaporin-2 (AQP2) water channels in the apical membrane and water reabsorption. Conversely, hormones, including extracellular purines and dopamine, antagonize AVP-induced water permeability, but their mechanism of action is largely unknown, which was investigated here. Addition of these hormones to mpkCCD cells decreased total and plasma membrane abundance of AVP-induced AQP2, partly by increasing its internalization to vesicles and lysosomal degradation. This internalization was ubiquitin dependent, because the hormones increased AQP2 ubiquitination, and the plasma membrane localization of AQP2-K270R, which cannot be monoubiquitinated, was unaffected by these hormones. Both hormones also increased AQP2 phosphorylation at S261, which followed ubiquitination, but was not essential for hormone-induced AQP2 degradation. A similar process occurs in vivo, as incubation of dDAVP-treated kidney slices with both hormones also resulted in the internalization and S261 phosphorylation of AQP2. Both hormones also reduced cAMP and AQP2 mRNA levels, suggesting an additional effect on AQP2 gene transcription. Interestingly, phorbol esters only reduced AQP2 through the first pathway. Together, our results indicate that ATP and dopamine counteract AVP-induced water permeability by increasing AQP2 degradation in lysosomes, preceded by ubiquitin-dependent internalization, and by decreasing AQP2 gene transcription by reducing the AVP-induced cAMP levels.

Temperature elevation increases GABAA-mediated cortical inhibition in a mouse model of genetic epilepsy

A missense mutation (R43Q) in the γ2 subunit of the γ-aminobutyric acid (GABA)(A) receptor is associated with generalized (genetic) epilepsy with febrile seizures plus (GEFS+). Heterozygous GABA(A) γ2(R43Q) mice displayed a lower temperature threshold for thermal seizures as compared to wild-type littermates. Temperature-dependent internalization of GABA(A) γ2(R43Q)-containing receptors has been proposed as a mechanism underlying febrile seizure genesis in patients with this mutation. We tested this idea using the GABA(A) γ2(R43Q) knockin mouse model and analyzed GABAergic miniature postsynaptic inhibitory currents (mIPSCs) in acute brain slices after exposure to varying temperatures. Incubation of slices at an elevated temperature increased mIPSC amplitude in neurons from heterozygous mice, with no change seen in wild-type controls. [³H]Flumazenil binding measured in whole-brain homogenates from mutant and control mice following elevation of body temperature showed no temperature-dependent differences in γ2-containing receptor density. Therefore, in vivo mouse data do not support earlier in vitro observations that proposed temperature-dependent internalization of γ2 R43Q containing GABA(A) receptors as the cellular mechanism underlying febrile seizure genesis in patients with the GABA(A) γ2(R43Q) mutation.

Exposure of nicotine to ventral tegmental area slices induces glutamatergic synaptic plasticity on dopamine neurons

Nicotine promotes glutamatergic synaptic plasticity in dopaminergic (DA) neurons in the ventral tegmental area (VTA), which is thought to be an important mechanism underlying nicotine reward. However, it is unclear whether exposure of nicotine alone to VTA slice is sufficient to increase glutamatergic synaptic strength on DA neurons and which nicotinic acetylcholine receptor (nAChR) subtype mediates this effect. Here, we report that the incubation of rat VTA slices with 500 nM nicotine induces glutamatergic synaptic plasticity in DA neurons. We measure the ratio of AMPA and NMDA receptor-mediated currents (AMPA/NMDA) and compare these ratios between nicotine-treated and -untreated slices. Our results demonstrate that the incubation of VTA slices with 500 nM nicotine for 1 h (but not for 10 min) significantly increases the AMPA/NMDA ratio when compared with controls. Preincubation with 10 nM of the α7-nAChR antagonist, methyllycaconitine (MLA) but not 1 μM α4-containing nAChR antagonist, dihydro-β-erythroidine (DHβE) prevents nicotinic effect, suggesting that α7-nAChRs are mainly mediated this nicotinic effect. This finding is further supported by the disappearance of this nicotinic effect in nAChR α7 knockout (KO) mice. Furthermore, nicotine reduced paired-pulse ratio (PPR) of evoked excitatory postsynaptic potential (eEPSP) in the VTA slices prepared from wild-type (WT) mice but not α7 KO mice. Collectively, these findings suggest that exposure of smoking-relevant concentrations of nicotine to VTA slices is sufficient to increase glutamatergic synaptic strength on DA neurons and that α7-nAChRs likely mediate this nicotinic effect through increasing presynaptic release of glutamate.

Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies

Precision-cut tissue slices (PCTS) are viable ex vivo explants of tissue with a reproducible, well defined thickness. They represent a mini-model of the organ under study and contain all cells of the tissue in their natural environment, leaving intercellular and cell-matrix interactions intact, and are therefore highly appropriate for studying multicellular processes. PCTS are mainly used to study the metabolism and toxicity of xenobiotics, but they are suitable for many other purposes. Here we describe the protocols to prepare and incubate rat and human liver and intestinal slices. Slices are prepared from fresh liver by making a cylindrical core using a drill with a hollow bit, from which slices are cut with a specially designed tissue slicer. Intestinal tissue is embedded in cylinders of agarose before slicing. Slices remain viable for 24 h (intestine) and up to 96 h (liver) when incubated in 6- or 12-well plates under 95% O(2)/5% CO(2) atmosphere.

β‐Amyloid regulates leptin expression and tau phosphorylation through the mTORC1 signaling pathway

High levels of the adipocytokine leptin are associated with reduced risk of Alzheimer's disease. Leptin treatment also reduces β-amyloid (Aβ) levels in in vivo and in vitro models of Alzheimer's disease. Aβ and leptin interact with the Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Akt/mTORC1 activation reduces tau phosphorylation through the inhibition of the downstream enzyme GSK-3β. mTORC1 also regulates translation of many proteins including leptin. While Aβ has been shown to inactivate Akt, inhibit mTORC1, and facilitate the phosphorylation of tau, leptin activates both Akt and mTORC1 and reduces tau phosphorylation. However, the extent to which Aβ may modulate leptin expression and increase tau phosphorylation involving Akt/mTORC1 has not been determined. In this study, we show that incubation of organotypic slices from rabbit hippocampus with Aβ down-regulates leptin expression, inhibits Akt, activates GSK-3β, increases tau phosphorylation, and inactivates mTORC1. Leptin treatment reverses Aβ effects by alleviating Akt inhibition, preventing GSK-3β activation, reducing tau phosphorylation, and activating mTORC1. On the other hand, Rapamycin, an allosteric inhibitor of mTORC1, down-regulates leptin expression, increases tau phosphorylation, and does not affect Akt and GSK-3β. Our results demonstrate for the first time that Aβ regulates leptin expression and tau phosphorylation through mTORC1.

In vitro and in vivo analysis of fatty acid effects on metabolism of 17β-estradiol and progesterone in dairy cows

Some studies have reported improved reproductive performance with dietary fat supplementation. This study examined effects of fatty acids with different lengths, or desaturation, or both, on metabolism of estradiol (E2) and progesterone (P4) in bovine liver slice incubations (experiments 1 and 2) and in vivo (experiment 3). In experiment 1, effects of fatty acids C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:1 (oleic acid), and C18:3 (linolenic acid) were evaluated at 30, 100, and 300 μM on P4 and E2 metabolism in vitro. In experiment 2, stearic acid (C18:0) and C18:3 were evaluated in the same incubation conditions. In experiment 1, all of the fatty acids had some significant inhibitory effect on metabolism of P4, E2, or both (300 μM C16:0 on E2; 100 μM C16:1 on E2; 300 μM C16:1 on both P4 and E2; 300 μM C18:1 on P4; and 100 and 300 μM C18:3 on both P4 and E2). In experiment 2, C18:3 (100 and 300 μM) but not C18:0 decreased P4 and E2 metabolism. Overall, the most profound increase (∼60%) in half-life of P4 and E2 was observed with incubations of 300 μM C18:3 in both in vitro experiments. Based on these in vitro results, in experiment 3 linseed oil (rich in C18:3) was supplemented into the abomasum and acute effects on metabolism of E2 and P4 were evaluated. Cows (n=4) had endogenous E2 and P4 minimized (corpus luteum regressed, follicles aspirated) before receiving continuous intravenous infusion of E2 and P4 to analyze metabolic clearance rate for these hormones during abomasal infusion of saline (control) or 70mL of linseed oil every 4h for 28h. Linseed oil infusion increased C18:3 in plasma by 46%; however, metabolic clearance rate for E2 and P4 were similar for control cows compared with linseed-treated cows. Thus, in vitro experiments indicated that E2 and P4 metabolism can be inhibited by high concentrations of C18:3. Nevertheless, in vivo, linseed oil did not acutely inhibit E2 and P4 metabolism, perhaps because insufficient C18:3 concentrations (increased to ∼8 μM) were achieved. Further research is needed to determine the mechanism(s) of fatty acid inhibition of P4 and E2 metabolism and to discover practical methods to mimic this effect in vivo.

Ascorbate regulation of 2-Cys peroxiredoxin-A promoter activity is light-dependent

The 2-Cys peroxiredoxin-A (2CPA) promoter is a model promoter to study redox and ABA-dependent stress signaling. Here, an Arabidopsis reporter gene line expressing luciferase under control of the 2CPA promoter was used to study the impact of ascorbate on reporter gene transcription in a series of protoplast and leaf slice incubation experiments. It was shown that ascorbate has a dual function on gene expression regulation. First, a comparison of responses to ascorbate, dehydroascorbate and reduced and oxidized glutathione demonstrated that ascorbate feeding supports gene expression regulation by increasing the catalytic capacity in redox signaling, as defined by the concentration of low molecular weight antioxidants and their oxidized counterparts. Second, ascorbate had a specific and light-dependent effect on 2CPA transcription, which cannot be substituted by reduced glutathione. Based on the differences between ascorbate and glutathione in the subcellular redox-cycling capacities, it is concluded that ascorbate feeding modulates chloroplast-specific regulation of 2CPA expression.

Mechanism of S100b Release from Rat Cortical Slices Determined Under Basal and Stimulated Conditions

Incubation of rat cortical slices in a medium that was not containing oxygen and glucose (oxygen-glucose deprivation, OGD) caused a 200% increase in the release of S100B. However, when slices were transferred to a medium containing oxygen and glucose (reoxygenation conditions, or REO), S100B release reached 500% of its control value. Neither inhibition of nitric oxide (NO) synthase by L-NAME nor addition of the NO donors sodium nitroprussid (SNP) or hydroxylamine (HA) to the medium altered basal S100B release. Similarly, the presence of SNP, HA or NO precursor L: -arginine in the medium, or inhibition of NO synthase by L-NAME also failed to alter OGD- and REO-induced S100B outputs. Moreover, individual inhibition of PKC, PLA(2) or PLC all failed to attenuate the S100B release determined under control condition or enhanced by either OGD or REO. Blockade of calcium channels with verapamil, chelating the Ca(+2) ions with BAPTA or blockade of sodium channels with tetrodotoxin (TTX) did not alter OGD- and REO-induced S100B release. In contrast to the pharmacologic manipulations mentioned above, glutamate and alpha-ketoglutarate added at high concentrations to the medium prevented both OGD- and REO-induced S100B outputs. These results indicate that neither NO nor the activation of PKC, PLA(2) or PLC seem to be involved in basal or OGD- and REO-induced S100B outputs. Additionally, calcium and sodium currents that are sensitive to verapamil and TTX, respectively, are unlikely to contribute to the enhanced S100B release observed under these conditions.

Microfluidic biochip for the perifusion of precision‐cut rat liver slices for metabolism and toxicology studies

Early detection of kinetic, metabolic, and toxicity (ADME-Tox) profiles for new drug candidates is of crucial importance during drug development. This article describes a novel in vitro system for the incubation of precision-cut liver slices (PCLS) under flow conditions, based on a poly(dimethylsiloxane) (PDMS) device containing 25-microL microchambers for integration of the slices. The microdevice is coupled to a perifusion system, which enables a constant delivery of nutrients and oxygen and a continuous removal of waste products. Both a highly controlled incubation environment and high metabolite detection sensitivity could be achieved using microfluidics. Liver slices were viable for at least 24 h in the microdevice. The compound, 7-ethoxycoumarin (7-EC), was chosen to test metabolism, since its metabolism includes both phase I and phase II metabolism and when tested in the conventional well plate system, correlates well with the in vivo situation (De Kanter et al. 2004. Xenobiotica 34(3): 229-241.). The metabolic rate of 7-EC was found to be 214 +/- 5 pmol/min/mg protein in the microdevice, comparable to well plates, and was constant over time for at least 3 h. This perifusion system better mimics the in vivo situation, and has the potential to significantly contribute to drug metabolism and toxicology studies of novel chemical entities.

Calcium imaging in single neurons from brain slices using bioluminescent reporters

Responses of three bioluminescent Ca(2+) sensors were studied in vitro and in neurons from brain slices. These sensors consisted of tandem fusions of green fluorescent protein (GFP) with the photoproteins aequorin, obelin, or a mutant aequorin with high Ca(2+) sensitivity. Kinetics of GFP-obelin responses to a saturating Ca(2+) concentration were faster than those of GFP-aequorin at all Mg(2+) concentrations tested, whereas GFP-mutant aequorin responses were the slowest. GFP-photoproteins were efficiently expressed in pyramidal neurons following overnight incubation of acute neocortical slices with recombinant Sindbis viruses. Expression of GFP-photoproteins did not result in conspicuous modification of morphological or electrophysiological properties of layer V pyramidal cells. The three sensors allowed the detection of Ca(2+) transients associated with action potential discharge in single layer V pyramidal neurons. In these neurons, depolarizing steps of increasing amplitude elicited action potential discharge of increasing frequency. Bioluminescent responses of the three sensors were similar in several respects: detection thresholds, an exponential increase with stimulus intensity, photoprotein consumptions, and kinetic properties. These responses, which were markedly slower than kinetics measured in vitro, increased linearly during the action potential discharge and decayed exponentially at the end of the discharge. Onset slopes increased with stimulus intensity, whereas decay kinetics remained constant. Dendritic light emission contributed to whole-field responses, but the spatial resolution of bioluminescence imaging was limited to the soma and proximal apical dendrite. Nonetheless, the high signal-to-background ratio of GFP-photoproteins allowed the detection of Ca(2+) transients associated with 5 action potentials in single neurons upon whole-field bioluminescence recordings.

Purinergic modulation in the development of the rat uncrossed retinotectal pathway

Adenosine is a neuromodulator implicated in nervous system development and plasticity and its effects are mediated by inhibitory (A1, A3) and excitatory (A2a, A2b) receptors. The role of adenosine in the synaptic activity depends mainly on a balanced activation of A1 and A2a receptors which are activated by various ranges of adenosine concentrations. Herein, we investigated the expression of A1 and A2a receptors and also the accumulation of cAMP in the superior colliculus at different stages of development. Furthermore, we examined the effects of an acute in vivo blockade of adenosine deaminase during the critical period when the elimination of misplaced axons/terminals takes place with a simultaneous fine tuning of terminal arbors into appropriate terminal zones. Lister Hooded rats ranging from postnatal days (PND) 0–70 were used for ontogeny studies. Our results indicate that A1 expression in the visual layers of the superior colliculus is higher until PND 28, while A2a expression increases after PND 28 in a complementary developmental pattern. Accordingly, the incubation of collicular slices with 5′-N-ethylcarboxamido-adenosine, a non-specific adenosine receptor agonist, showed a significant reduction in cAMP accumulation at PND 14 and an increase in adults. For the anatomical studies, the uncrossed retinotectal projections were traced after the intraocular injection of horseradish peroxidase. One group received daily injections of an adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl adenine), 10 mg/kg i.p.) between PND 10 and 13, while control groups were treated with vehicle injections (NaCl 0.9%, i.p.). We found that a short-term blockade of adenosine deaminase during the second postnatal week induced an expansion of retinotectal terminal fields in the rostrocaudal axis of the tectum. Taken together, the results suggest that a balance of purinergic A1 and A2a receptors through cAMP signaling plays a pivotal role during the development of topographic order in the retinotectal pathway.

Rapid and fine adjustment of corticosteroidogenesis operating in the adrenal gland of the Mongolian gerbil (Meriones unguiculatus) superfused in-vitro.

Corticosteroid release from adrenals of male adult Mongolian gerbils (Meriones unguiculatus) has been studied during continuous and discontinuous in-vitro superfusion. Corticosteroid output from glands of untreated animals with plasma corticosteroid levels below 300 ng/ml was 3.3 +/- 0.3 ng/adrenal pair/min and decreased only slightly with the length of superfusion (60 min: 3.1 +2- 0.3 ng/adrenal pair/min). In-vitro secretion was significantly higher from adrenals of animals which had corticosteroid plasma levels of over 300 ng/ml, underwent clonic-tonic seizures, or were injected with 6 IU/animal (1-24) ACTH. On the other hand, injection of 2 X 50 micrograms dexamethasone markedly decreased corticosteroid plasma levels but had no significant effect on in-vitro secretion of corticosteroids. In contrast to the slow and small, but long-lasting stimulation of corticosteroid secretion elicited by (1-24) ACTH, secretion could be changed within much shorter time periods, either by the addition of plasma proteins to superfusion medium or by stops of superfusion flow. While a significant stimulation of corticosteroid output occurred after the addition of 1% or 10% BSA or rat plasma, stops of superfusion flow for 1.5 or 10 min resulted in a strong inhibition of steroidogenesis, as is evident from corticosteroid amounts found in the first 1-min samples after re-start of superfusion. Within 4-5 min after re-start of superfusion, secretion returned to basal values. Corticosteroid amounts secreted from adrenals superfused in-vitro were significantly higher than those secreted from adrenals incubated in-vitro. In addition, prolonged incubation suppressed corticosteroidogenesis (30 min: 100%, 60 min: 64%, 90 min: 56%, 120 min: 59%). The results demonstrate that superfusion of sliced adrenal tissue gives insights into aspects of adrenal function, including the rapid changes in synthesis and secretion after flow stops which cannot be investigated by incubation of either tissue slices or isolated cells. The possibility that the observed decline in corticosteroidogenesis during flow stops may be due to a feedback inhibition resulting from corticosteroids accumulating within slices is discussed.

Precision-cut slice cultures of tumors from MMTV-neu mice for the study of the ex vivo response to cytokines and cytotoxic drugs

Ex vivo analysis of signaling pathways operating in tumor tissue is complicated by the three-dimensional structure, in particular by stroma-epithelial interactions. Studies performed with pure populations of tumor cells usually do not take into account this issue. One possibility to preserve the tissue architecture is the use of tumor slices. However, diffusion of oxygen and nutrients may become limiting factors, resulting in decreased cell viability and change of tissue morphology, especially after long-term incubation of slices. By using precision cut slices of defined thickness, we were able to establish culture conditions for tumor material obtained from MMTV-neu transgenic mice, which allow the study of the action of cytokines and cytotoxic drugs for up to 24 h. A slice thickness of 160 mum was found to be optimal for viability and handling of material. These slices were highly responsive to the action of the cytokine IFN-gamma, as evident form the increase of pY701 STAT1, detected by both immunohistochemistry and western blotting, and by the increase of mRNA levels of the IFN-gamma response genes IRF-1, SOCS-1, and STAT1, analyzed by reverse transcriptase-polymerase chain reaction. Furthermore, induction of apoptosis and increase of DNA damage could be monitored after treatment with IFN-gamma or doxorubicin. The slices were also a convenient source for the establishment of explant cultures of tumor epithelial cells. It is concluded that cultivation of precision-cut tumor slices provides a convenient way for the ex vivo molecular analysis of MMTV-neu tumor tissue under conditions which closely simulate the situation in vivo and can provide an alternative to in vivo experiments.

Changes in Neuropil Ultrastructure in Hippocampal Field CA1 in Rat Pups after Application of Hyaluronidase

Electron microscopy and electrophysiological studies were performed on cross-sectional slices of the hippocampus of four-week-old male rat pups (n = 35) to detect ultrastructural changes in hippocampal field CA1 and the characteristics of the formation of excitatory postsynaptic potentials in this area of the brain after incubation of slices in hyaluronidase solution (10 U/ml), whose specific substrate is the extracellular matrix glycosaminoglycan hyaluronic acid. At 1.5 min after enzyme application, there were reductions in synaptic cleft widths in axodendritic contacts of the striatum radiatum of hippocampal field CA1 by 15-25%, which were consistent with increases seen in the amplitudes of excitatory postsynaptic potentials. At 4.5 min of incubation, the lumens of synaptic clefts decreased by 45-55%: during this time there was blockade of signal transmission via Schäffer collaterals to hippocampal field CA1. Thus, the structural-functional state of glycosaminoglycans is among the factors determining the efficiency of synaptic transmission in the brain.

Andrenogonadal interactions in nicotine-induced alteration of steroidogenesis in male rat.

Chronic nicotine administration in larger doses inhibits steroidogenesis in testis. This inhibition is possibly secondary to increased adrenomedullary norepinephrine activity, since the anti-steroidogenic effect of nicotine can be prevented by alpha-adrenergic blocker like phentolamine. In vitro incubation of testicular slices with nicotine failed to elicit any appreciable effect on delta 5-3 beta-hydroxysteroid dehydrogenase activity indicating lack of direct action of nicotine on testicular steroidogenesis.

In vitro metabolism of (3H)-androstenedione in the rat epididymis and vas deferens.

The in vitro metabolism of (3H)-androstenedione in the epididymis and vas deferens of intact and castrated rats was investigated and the metabolites formed were identified by radio gas chromatography. Incubation of slices of caput epididymidis for 2 hr at 34 degrees C metabolised 90% androstenedione. Similar incubations of tissue samples from cauda epididymidis and vas deferens metabolized 60 and 25% of androstenedione respectively. The major metabolites formed in the epididymis were androstanedione (caput: 48%; cauda: 33%) and androsterone (caput: 35%; cauda: 13%). These metabolites appeared in much less concentration in the incubations with vas deferens (about 8% each). In general, conversion to testosterone and dihydrotesterone was low in all the three organs examined. Castration did not significantly alter the metabolic pattern in the caput epididymidis and vas deferens but promoted the formation of androsterone (38%) in the cauda epididymidis. The conversion of androstenedione, a weak androgen to testosterone, dihydrotestosterone and 3 alpha/3 beta-diols in the epididymidis and vas deferens of castrated rats may be of physiological significance. In addition, androsterone appears to be an important androgenic metabolite in the epididymis.

Prostaglandin E2 modulates Na+,K+‐ATPase activity in rat hippocampus: implications for neurological diseases

Prostaglandin E(2) (PGE(2)) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na(+),K(+)-ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE(2) decreases Na(+),K(+)-ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE(2). Na(+),K(+)-ATPase activity was determined by assessing ouabain-sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE(2) (0.1-10 microM) for 30 min decreased Na(+),K(+)-ATPase activity in a concentration-dependent manner. However, PGE(2) did not alter Na(+),K(+)-ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE(2) on Na(+),K(+)-ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic alpha subunit of Na(+),K(+)-ATPase. We found that the inhibitory effect of PGE(2) (1 microM) on Na(+),K(+)-ATPase activity was receptor-mediated, as incubation with selective antagonists for EP1 (SC-19220, 10 microM), EP3 (L-826266, 1 microM) or EP4 (L-161982, 1 microM) receptors prevented the PGE(2)-induced decrease of Na(+),K(+)-ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1-10 microM) increased enzyme activity per se in a concentration-dependent manner, but did not prevent the inhibitory effect of PGE(2). Incubation with a protein kinase A (PKA) inhibitor (H-89, 1 microM) and a protein kinase C (PKC) inhibitor (GF-109203X, 300 nM) also prevented PGE(2)-induced decrease of Na(+),K(+)-ATPase activity. Accordingly, PGE(2) increased phosphorylation of Ser943 at the alpha subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE(2) decreases Na(+),K(+)-ATPase activity in vivo. The results presented here imply Na(+),K(+)-ATPase as a target for PGE(2)-mediated signaling, which may underlie PGE(2)-induced increase of brain excitability.

Dopamine D4 Receptors Regulate GABAA Receptor Trafficking via an Actin/Cofilin/Myosin-dependent Mechanism

The GABA(A) receptor-mediated inhibitory transmission in prefrontal cortex (PFC) is implicated in cognitive processes such as working memory. Our previous study has found that GABA(A)R current is subject to the regulation of dopamine D(4) receptors, a PFC-enriched neuromodulator critically involved in various mental disorders associated with PFC dysfunction. In this study, we have investigated the cellular mechanism underlying D(4) modulation of GABA(A)Rs. We found that the density of surface clusters of GABA(A)R beta2/3 subunits was reduced by D(4), suggesting that the D(4) reduction of GABA(A)R current is associated with a decrease in functional GABA(A)Rs at the plasma membrane. Moreover, the D(4) reduction of GABA(A)R current was blocked by the actin stabilizer phalloidin and was occluded by the actin destabilizer latrunculin, suggesting that D(4) regulates GABA(A)R trafficking via an actin-dependent mechanism. Cofilin, a major actin depolymerizing factor whose activity is strongly increased by dephosphorylation at Ser(3), provides the possible link between D(4) signaling and the actin dynamics. Because myosin motor proteins are important for the transport of vesicles along actin filaments, we also tested the potential involvement of myosin in D(4) regulation of GABA(A)R trafficking. We found that dialysis with a myosin peptide, which competes with endogenous myosin proteins for actin-binding sites, prevented the D(4) reduction of GABA(A)R current. These results suggest that D(4) receptor activation increases cofilin activity presumably via its dephosphorylation, resulting in actin depolymerization, thus causing a decrease in the myosin-based transport of GABA(A)R clusters to the surface.

Distribution and functional characterization of pituitary adenylate cyclase–activating polypeptide receptors in the brain of non-human primates

The distribution and density of pituitary adenylate cyclase–activating polypeptide (PACAP) binding sites have been investigated in the brain of the primates Jacchus callithrix (marmoset) and Macaca fascicularis (macaque) using [ 125I]-PACAP27 as a radioligand. PACAP binding sites were widely expressed in the brain of these two species with particularly high densities in the septum, hypothalamus and habenula. A moderate density of recognition sites was seen in all subdivisions of the cerebral cortex with a heterogenous distribution, the highest concentrations occurring in layers I and VI while the underlying white matter was almost devoid of binding sites. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis revealed intense expression of the mRNAs encoding the short and hop-1 variants of pituitary adenylate cyclase–activating polypeptide–specific receptor (PAC1-R) in the cortex of both marmoset and macaque, whereas vasoactive intestinal polypeptide/pituitary adenylate cyclase–activating polypeptide mutual receptor, subtype 1 (VPAC1-R) and vasoactive intestinal polypeptide/pituitary adenylate cyclase–activating polypeptide mutual receptor, subtype 2 (VPAC2-R) mRNAs were expressed at a much lower level. In situ hybridization histochemistry showed intense expression of PAC1-R and weak expression of VPAC1-R mRNAs in layer IV of the cerebral cortex. Incubation of cortical tissue slices with PACAP induced a dose-dependent stimulation of cyclic AMP formation, indicating that PACAP binding sites correspond to functional receptors. Moreover, treatment of primate cortical slices with 100 nM PACAP significantly reduced the activity of caspase-3, a key enzyme of the apoptotic cascade. The present results indicate that PACAP should exert the same neuroprotective effect in the brain of primates as in rodents and suggest that PAC1-R agonists may have a therapeutic value to prevent neuronal cell death after stroke or in specific neurodegenerative diseases.

Normobaric hyperoxia attenuates early blood–brain barrier disruption by inhibiting MMP‐9‐mediated occludin degradation in focal cerebral ischemia

Early blood-brain barrier (BBB) disruption resulting from excessive neurovascular proteolysis by matrix metalloproteinases (MMPs) is closely associated with hemorrhagic transformation events in ischemic stroke. We have shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 increase in the ischemic brain. The aim of this study was to determine whether NBO could attenuate MMP-9-mediated early BBB disruption following ischemic stroke. Rats were exposed to NBO (95% O(2)) or normoxia (30% O(2)) during 90-min middle cerebral artery occlusion, followed by 3-hour reperfusion. NBO-treated rats showed a significant reduction in Evan's blue extravasation in the ischemic hemisphere compared with normoxic rats. Topographically, Evan's blue leakage was mainly seen in the subcortical regions including the striatum, which was accompanied by increased gelatinolytic activity and reduced immunostaining for tight-junction protein, occludin. Increased gelatinolytic activities and occludin protein loss were also observed in isolated ischemic microvessels. Gel gelatin zymography identified that MMP-9 was the main enzymatic source in the cerebral microvessels. Incubation of brain slices or isolated microvessels with purified MMP-9 revealed specific degradation of occludin. Inhibition of MMP-9 by NBO or MMP-inhibitor, BB1101, significantly reduced occludin protein loss in ischemic microvessels. These results suggest that NBO attenuates early BBB disruption, and inhibition of MMP-9-mediated occludin degradation is an important mechanism for this protection.