Age-dependent decline in hippocampal neurogenesis is not altered by chronic treatment with fluoxetine.

There has been ongoing controversy as to whether selective serotonin reuptake inhibitors (SSRIs) exhibit the same antidepressant efficacy and risk profile within different age groups. Although the etiology of such potential differences is currently not clear, age-dependent differences in the rate of hippocampal neurogenesis offer one possibility. In the current studies we have therefore examined whether fluoxetine, the prototypical selective serotonin reuptake inhibitor, differentially modulates neurogenesis in adolescent, young adult, and aged rats. Proliferation in the dentate gyrus was measured by assaying expression of the endogenous proliferative marker, Ki67. Survival of proliferating cells was assayed by staining with BrdU. We confirmed previous reports that the rate of neurogenesis, as well as the survival of proliferating cells, decreases significantly with age. Moderate decreases were found in young adult rats relative to adolescent rats, and profound decreases were found in aged rats. We additionally found that age did not alter the response to 25 days of treatment with fluoxetine. In fact, we did not observe enhancement of hippocampal neurogenesis, nor enhancement of proliferating cell survival, in any of the three age groups despite using doses of fluoxetine which have been reported to be effective. In addition to finding no age-dependent effects, our data question the general reproducibility of previously reported fluoxetine effects in animals.

Interaction of FGF-2 with IGF-1 and BDNF in stimulating Akt, ERK, and neuronal survival in hippocampal cultures.

The significance of multiple growth factors acting on individual neurons in the central nervous system is presently unclear. Cultured hippocampal neurons were used in the present study to compare the neurotrophic actions of fibroblast growth factor-2 (FGF-2) with the better characterized growth factors, insulin-like growth factor (IGF)-1 and brain-derived neurotrophic factor (BDNF). Additionally, cultures were utilized to identify possible interactions between FGF-2 and the other growth factors. Activation of the ERK and Akt pro-survival pathways, as well as neuronal survival itself, were studied. The maximal magnitude of Akt activation stimulated by FGF-2 was found to be similar to that stimulated by IGF-1 and BDNF. In contrast, IGF-1 was less effective at inducing ERK activation than were BDNF and FGF-2. All three agents were found to promote survival of neurons cultured under serum-free, low-insulin conditions, with FGF-2 surprisingly being significantly more effective than the other two peptides. Co-treatment with maximal concentrations of either IGF-1 or BDNF enhanced FGF-2-stimulated Akt and ERK activation. However, no enhancement of survival beyond that stimulated by FGF-2 was observed with co-treatment. These findings suggest that FGF-2 may play an important role in promoting the survival of hippocampal neurons. Additionally, an interesting dissociation was identified between the positive interaction of FGF-2 with both IGF-1 and BDNF in activating Akt and ERK, and the lack of enhancement of FGF-2-induced neuroprotection.

Serotonin and neuronal growth factors - a convergence of signaling pathways.

Monoamines, including serotonin (5-HT), have traditionally been associated with short-term signaling pathways in neurons, such as the modulation of cAMP and Ca(2+) levels. In contrast, neuronal growth factors, such as neurotrophins, have been traditionally associated with signaling pathways, such as those for activation of extracellular-regulated kinase (ERK) and Akt (protein kinase B), which are known to induce long-term protective changes. It has therefore been unclear how antidepressants that increase serotonin (5-HT), induce such changes as hippocampal neuroprotection and neurogenesis. It has been hypothesized, that the actions of 5-HT may be mediated indirectly through increased synthesis of peptide growth factors. However, there is increasing evidence that some subtypes of 5-HT receptors can directly couple to activation of the ERK and Akt pathways. Such coupling suggests a more direct potential role for 5-HT in mediating the long-term actions induced by antidepressants.

Staffing and case scheduling for anesthesia in geographically dispersed locations outside of operating rooms.

PURPOSE OF REVIEW: Scheduling and staffing for anesthetics outside of the operating room that are geographically dispersed is different than for operating room cases. Whereas methods to predict how long such cases take were published recently, this article reviews staffing and case scheduling. RECENT FINDINGS: Methods have been developed based on the assumption that physicians doing procedures requiring anesthesia are provided open access to anesthesia time within a reasonable number of days (e.g., 2 weeks) or on any future workday. The latter is commonly used in operating rooms. Outside of operating rooms, the former is more practical economically. Statistical forecasting of anesthesia staffing months ahead is conducted by using billing data with the objective of maximizing the efficiency of use of anesthesia time. Calculations assume that anesthesia time that would otherwise be underutilized is released for use by services that would otherwise work in overutilized anesthesia time. Forecasting is different for services with many patients hospitalized preoperatively (e.g., electroconvulsive therapy). Implementation encourages longer-term changes benefiting the anesthesia group (e.g., services choose to work longer hours for fewer days of the week). SUMMARY: Plan staffing based on providing open access to anesthesia time within a reasonable number of days (e.g., 2 weeks). Schedule cases and release allocated time based on reducing overutilized anesthesia time.

Cumulative activation of akt and consequent inhibition of glycogen synthase kinase-3 by brain-derived neurotrophic factor and insulin-like growth factor-1 in cultured hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) seem to play key roles in mediating neuronal plasticity in the hippocampus. In the current studies, we have used cultured hippocampal neurons to study possible interactions between the two growth factors in modulating neuronal signaling pathways. BDNF and IGF-1 were found to each effectively activate the neuroprotective Akt pathway, with the magnitude of activation being at least additive when cultures were simultaneously treated with supramaximal concentrations of peptides. Likewise, a cumulative inhibitory Akt-dependent phosphorylation of proapoptotic glycogen synthase kinase-3 was observed. Immunofluorescent studies demonstrated that a single population of neurons responded to BDNF and IGF-1. In contrast, the magnitude of BDNF-stimulated extracellular signal-regulated kinase (ERK) activation was found to be much greater than that of IGF-1-stimulated ERK, such that the difference in magnitude stimulated by BDNF in the presence and absence of IGF-1 did not reach statistical significance. Consistent with the observed agonist-stimulated activation of Akt, BDNF and IGF-1 were both found to act as neurotrophins, enhancing neuronal survival under low-insulin culture conditions. Maximal survival was achieved when both growth factors were present. These findings provide insight into the significance of multiple growth factors stimulating activation of ERK and Akt in the central nervous system. In some cases, the magnitude of activation required to elicit biological responses may be achieved only with a combination of compounds.

Mesangial cell apoptosis induced by stimulation of the adenosine A3 receptor: signaling and apoptotic events.

Mesangial cell apoptosis has been proposed as a means of resolution of glomerular hypercellularity in proliferative forms of glomerular disease. We previously demonstrated that adenosine causes mesangial cell apoptosis by stimulating the A3-type adenosine receptor. This is a G protein-coupled receptor shown to activate kinases involved in apoptotic signaling. In this work, we assessed changes in phosphorylation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2 and in levels of specific pro- and antiapoptotic proteins following exposure of mesangial cells to the A3 adenosine receptor agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA). Cultured mesangial cells were incubated with IB-MECA for 30 minutes and 6, 24, and 48 hours. IB-MECA was used at a concentration (30 microM) that induces a reproducible degree of mesangial cell apoptosis. Changes in ERK1/2 phosphorylation and in protein levels of Bcl-2, Bax, and caspase 3 were assessed by Western blot analysis. IB-MECA markedly increased phosphorylation of ERK1/2. This effect peaked at 5 minutes, dissipated by 20 minutes, and was abolished by the inhibitor of ERK phosphorylation, compound U0126, in a dose-dependent manner. This inhibitor had no effect on the extent of IB-MECA-induced apoptosis. Bcl-2 levels progressively declined, whereas those of Bax and activated caspase 3 increased. These observations indicate that stimulation of the A3-type adenosine receptor causes mesangial cell apoptosis via mechanisms independent of ERK activation. The observations also point to an imbalance in the expression of antiapoptotic (Bcl-2) and proapoptotic (Bax, caspase 3) proteins as a potential mechanism underlying adenosine-induced mesangial cell apoptosis.

5-HT receptors couple to activation of Akt, but not extracellular-regulated kinase (ERK), in cultured hippocampal neurons.

5-HT(1A) receptors have been hypothesized to mediate some of the neuronal plasticity and behavioral responses stimulated by serotonin selective reuptake inhibitors. Although the cellular signaling pathways required for inducing these actions have not yet been determined, roles for the neuroprotective extracellular-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and Akt pathways have been suggested. In the current studies we have utilized primary cultures to directly determine whether hippocampal 5-HT(1A) receptors couple to activation of Akt and ERK. We found that E18 hippocampal neurons exhibit a twofold activation of Akt when exposed to nanomolar concentrations of 5-HT. The 5-HT(1/7) receptor-selective agonist 5-carboxamidotryptamine maleate (5-CT) and the 5-HT(1A/7) receptor-selective agonist 8-hydroxy-N,N-dipropyl-aminotetralin (8-OH-DPAT) maleate were found to activate Akt with equal efficacy, and similar potency, to 5-HT. p-MPPI and WAY-100635, antagonists selective for 5-HT(1A) receptors, completely inhibited 5-CT- stimulated Akt activation. Activation of Akt was also inhibited by pretreatment with pertussis toxin as well as the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002. In contrast, the 5-HT selective antagonist, SB269970, caused no inhibition. Although the density of 5-HT(1A) receptors expressed by cultured neurons was sufficient to activate Akt, no activation of ERK was observed. These findings suggest that Akt, and not ERK, may be relevant to previous reports of hippocampal 5-HT(1A) receptors mediating neurotrophic responses.

Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells.

The most commonly prescribed antidepressants, the serotonin (5-HT) selective reuptake inhibitors, increase 5-HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by Gs-coupled 5-HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular-regulated kinase (ERK) and Akt pathways by Gs-coupled 5-HT7A receptors and Gq-coupled 5-HT2A receptors, which are co-expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5-HT2A receptor-mediated activation of the two pathways was found to be Ca2+-dependent. In contrast, 5-HT7A receptor-mediated activation of Akt required increases in both [cAMP] and intracellular [Ca2+], while activation of ERK was inhibited by Ca2+. The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5-HT2A and 5-HT7A receptor agonists was found to be at least additive. Cell-permeable cAMP analogs mimicked 5-HT7A receptor agonists in enhancing 5-HT2A receptor-mediated activation of ERK and Akt. A role was identified for the cAMP-guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5-HT2A and 5-HT7A receptors may also be relevant to the interaction of other neuronally expressed Gq- and Gs-coupled receptors.

Coupling of neuronal 5-HT7 receptors to activation of extracellular-regulated kinase through a protein kinase A-independent pathway that can utilize Epac.

The roles of 3',5'-cyclic adenosine monophosphate (cAMP) and protein kinase A in 5-hydroxytryptamine (5-HT)7 receptor-mediated activation of extracellular-regulated kinase (ERK) were studied in cultured hippocampal neurons and transfected PC12 cells. Activation of ERK by neuronal Gs-coupled receptors has been thought to proceed through a protein kinase A-dependent pathway. In fact we identified coupling of 5-HT7 receptors to activation of adenylyl cyclase and protein kinase A. However, no inhibition of agonist-stimulated ERK activation was found when cells were treated with H-89 and KT5720 at concentrations sufficient to completely inhibit activation of protein kinase A. However, activation of ERK was found to be sensitive to the adenylyl cyclase inhibitor 9-(tetrahydrofuryl)-adenine, suggesting a possible role for a cAMP-guanine nucleotide exchange factor (cAMP-GEF). Co-treatment of cells with 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate, a direct activator of the cAMP-GEFs Epac1 and 2, reversed the inhibition of agonist-stimulated ERK activation induced by adenylyl cyclase inhibition. Additionally, over-expression of Epac1 enhanced 5-HT7 receptor-mediated activation of ERK. These results demonstrate that the activation of ERK mediated by neuronal Gs-coupled receptors can proceed through cAMP-dependent pathways that utilize cAMP-GEFs rather than protein kinase A.

SptP, a Salmonella typhimurium type III-secreted protein, inhibits the mitogen-activated protein kinase pathway by inhibiting Raf activation.

Salmonella has developed ways to modulate host cellular response in order to survive. Although the steps required for such modulation have been incompletely characterized, there is increasing evidence for a role for SptP, a type III secretion protein. In part, the actions of SptP are thought to be mediated through its reported inhibition of the extracellular-regulated kinase (ERK) MAP kinase pathway. In the present studies, a series of transfections were performed in which various constitutively activated components of the MAP kinase pathway were co-transfected with SptP in order to determine the mechanism by which SptP inhibits this MAP kinase activation. SptP was found to inhibit the activation of ERK stimulated by both a constitutively active form of Ras and a partially activated form of Raf-1 containing a phospho-mimetic mutation (Raf Y340D). In contrast, the activation of ERK by constitutively active forms of MAP kinase kinase (MEK) was not inhibited, suggesting that the actions of SptP were mediated by Raf-1. In order to determine how SptP might interfere with activation of Raf, we utilized a membrane-localized form of Raf. Constitutive membrane-localization of Raf (RafCAAX), resulting in partial activation, did not prevent inhibition by SptP. However, introduction of an additional, partially activating (Y340D) phospho-mimetic mutation, to RafCAAX, dramatically reduced the ability of SptP to inhibit Raf action. Comparison of SptP mutants, lacking either GTPase-activating protein (GAP) activity or tyrosine phosphatase activity, further suggested that SptP inhibits both the membrane localization and subsequent phosphorylation required for activation of Raf. Both tyrosine phosphatase activity and GAP activity were responsible for SptP inhibition of Raf(Y340D)-induced ERK activation, but only GAP activity was responsible for inhibition of the membrane localized forms of Raf-1. To assess the biological significance of SptP, we examined tumour necrosis factor (TNF)-alpha induction following Salmonella infection. SptP gene deletion enhanced the capacity of Salmonella to induce TNF-alpha secretion following infection of J774A.1 macrophage cells.

Activation of extracellular-regulated kinase by 5-hydroxytryptamine(2A) receptors in PC12 cells is protein kinase C-independent and requires calmodulin and tyrosine kinases.

5-Hydroxytryptamine (5-HT)(2A) receptors have been implicated to play a role in both the treatment and pathophysiology of a number of psychiatric disorders. Therefore, the coupling of this receptor to signals, such as extracellular signal-regulated kinase (ERK), that elicit long-term neuronal changes may be relevant. In the present study we examined the coupling of the G(q)-coupled receptor to ERK in PC12 cells, a cell line commonly used as a neuronal model system. Activation of ERK occurred through a pathway different than the protein kinase C-dependent pathways described previously in studies of non-neuronal cells. Activation of ERK, in PC12 cells, was inhibited by both chelation of extracellular Ca(2+) and by depletion of intracellular Ca(2+) stores. Surprisingly, activation was not inhibited, but actually potentiated, by a variety of protein kinase C inhibitors covering all known protein kinase C isoforms. In contrast, the coupling of receptor to activation of ERK was found to be sensitive to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (W13), inhibitors of calmodulin, but not to 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN62) and 2-[N-(2-hydroxyethyl)]-N-4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN93), inhibitors of calmodulin-dependent protein kinase. Additionally, the general tyrosine kinase inhibitor genistein, as well as the Src inhibitor PP1 and the epidermal growth factor receptor kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG 1478), inhibited receptor-mediated activation of ERK, suggesting a role for tyrosine kinases. In fact, 5-HT was found to stimulate tyrosine phosphorylation of a number of proteins, and this phosphorylation was inhibited by W7. 5-HT(2A) receptor-activation of ERK through a protein kinase C-independent pathway requiring Ca(2+)/calmodulin/tyrosine kinases represents a pathway distinct from those described in studies of non-neuronal cells.

Differential coupling of 5-HT(1) receptors to G proteins of the G(i) family.

1: Since all 5-HT(1) receptors couple to G(i)-type G proteins and inhibit adenylyl cyclase, the functional significance of five distinct subtypes of 5-HT(1) receptors has been unclear. 2: In previous studies we have used transfected cells to demonstrate that 5-HT(1B) receptors can couple more efficiently than 5-HT(1A) receptors to activation of extracellular signal-regulated kinase (ERK) and to inhibition of adenylyl cyclase. These findings suggested the possibility that individual 5-HT(1) receptors differentially couple to isoforms of G(ialpha). 3: In the present study we utilized a model system in which pertussis toxin resistant forms of human G(ialpha1), G(ialpha2), and G(ialpha3) were used to directly compare the coupling of human 5-HT(1A), 5-HT(1B), and 5-HT(1D) receptors to each G(ialpha) in transfected human HeLa cells. 4: 5-HT(1A) receptors displayed a preference for G(ialpha1) and G(ialpha2), relative to G(ialpha3). Pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 73%, 76%, and 44%, respectively, of the ERK activation stimulated by 5-HT in the absence of pertussis toxin. 5: In contrast, pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 32%, 118%, and 35% of 5-HT(1B) receptor-stimulated activity, respectively, indicating that 5-HT(1B) receptors coupled primarily through G(ialpha2). A similar preference for G(ialpha2) was found in studies of the 5-HT(1D) receptor, where toxin resistant G(ialpha1), G(ialpha2), and G(ialpha3) rescued 30%, 70%, and 40% of activity, respectively. 6: In conclusion, the observed differential coupling of 5-HT(1) receptors to isoforms of G(ialpha), provides additional evidence for our previous findings that the subtypes of 5-HT(1) receptors exhibit similar, but distinct, functions.