Neurogenomic signatures of spatiotemporal memories in time-trained forager honey bees.

Honey bees can form distinct spatiotemporal memories that allow them to return repeatedly to different food sources at different times of day. Although it is becoming increasingly clear that different behavioral states are associated with different profiles of brain gene expression, it is not known whether this relationship extends to states that are as dynamic and specific as those associated with foraging-related spatiotemporal memories. We tested this hypothesis by training different groups of foragers from the same colony to collect sucrose solution from one of two artificial feeders; each feeder was in a different location and had sucrose available at a different time, either in the morning or afternoon. Bees from both training groups were collected at both the morning and afternoon training times to result in one set of bees that was undergoing stereotypical food anticipatory behavior and another that was inactive for each time of day. Between the two groups with the different spatiotemporal memories, microarray analysis revealed that 1329 genes were differentially expressed in the brains of honey bees. Many of these genes also varied with time of day, time of training or state of food anticipation. Some of these genes are known to be involved in a variety of biological processes, including metabolism and behavior. These results indicate that distinct spatiotemporal foraging memories in honey bees are associated with distinct neurogenomic signatures, and the decomposition of these signatures into sets of genes that are also influenced by time or activity state hints at the modular composition of this complex neurogenomic phenotype.

Absence of consistent diel rhythmicity in mated honey bee queen behavior.

Relatively little is known about the temporal control of behavior of honey bee queens under natural conditions. To determine if mated honey bee queens possess diel rhythmicity in behavior, we observed them in glass-sided observation hives, employing two focal studies involving continuous observations of individual queens as well as a scan-sampling study of multiple queens. In all cases, all behaviors were observed at all times of the day and night. In four of the five queens examined in focal studies, there were no consistent occurrences of diel periodicity for any of the individual behaviors. A more encompassing measure for periodicity, in which the behaviors were characterized as active (walking, inspecting, egg-laying, begging for food, feeding, and grooming self) or inactive (standing), also failed to reveal consistent diel rhythmicity. Furthermore, there were no consistent diel differences in the number of workers in the queen's retinue. Behavioral arrhythmicity persisted across seasons and despite daily changes in both light and temperature levels. Both day and night levels of behavioral activity were correlated with daytime, but not with nighttime, ambient temperatures. The behavior of the one exceptional queen was not consistent: diurnal activity patterns were present during two 24-h observation sessions but arrhythmicity during another. Based on the behavior observed by all but one of the queens examined in this work, the arrhythmic behavior by the mated honey bee queen inside the colony appears to be similar to that exhibited by worker bees before they approach the age of onset of foraging behavior.

Impairment of myocardial angiogenic response in the absence of osteopontin.

OBJECTIVE: Osteopontin (OPN), increased in the heart following myocardial infarction (MI), plays an important role in post-MI remodeling. Angiogenesis, an important feature of tissue repair, begins in the infarcted myocardium within 3 days post-MI. Here, the authors studied the role of OPN in myocardial angiogenesis using wild-type (WT) and OPN knockout (KO) mice. RESULTS: Measurement of angiogenic response using Griffonia simplicifolia lectin-1 (GSL-1) staining indicated reduced capillary density in the infarcted region of the OPN KO hearts as compared to WT hearts 7 and 14 days post-MI. Arteriolar density was lower in OPN KO hearts 14 days post-MI. The number of CD31 positive cells was also lower in the infarcted region of the OPN KO hearts as compared to WT hearts 14 days post-MI. In contrast, capillary and arteriolar densities in the noninfarcted regions of OPN KO and WT hearts were not significantly different. In vivo myocardial angiogenesis measured using Matrigel implantation in the left ventricular myocardium indicated significant decrease in the percentage of vessel-like areas in the OPN KO vs. WT hearts. Furthermore, in vitro Matrigel tube formation assay demonstrated a significant decrease in total tube length in cardiac microvascular endothelial cells (CMECs) isolated from OPN KO hearts as compared to CMECs from WT hearts. Treatment of OPN KO CMECs with purified OPN protein significantly increased total tube length, while bovine serum albumin had no effect. CONCLUSION: Lack of OPN impairs myocardial angiogenic response, leading to adverse remodeling post-MI.

Glycogen synthase kinase-3beta plays a pro-apoptotic role in beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes: Role of beta1 integrins.

Beta-adrenergic receptor (beta-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVM). beta1 integrin signaling plays a protective role in beta-AR-stimulated apoptosis. Glycogen synthase kinase-3beta (GSK-3beta), a multifunctional serine/threonine kinase, negatively regulates cardiac hypertrophy. Here we show that beta-AR stimulation (isoproterenol; 15 min) increases tyr(216) phosphorylation and GSK-3beta activity. Inclusion of LiCl, inhibitor of GSK-3beta, in the reaction mix or expression of catalytically inactive GSK-3beta (KM-GSK) inhibited beta-AR-stimulated GSK-3beta activity. Inhibition of tyrosine kinase using genistein or chelation of intracellular Ca(2+) using BAPTA-AM inhibited beta-AR-stimulated increases in tyr(216) phosphorylation and GSK-3beta activity. Inhibition of GSK-3beta using pharmacological inhibitors or infection with KM-GSK decreased beta-AR-stimulated cytosolic cytochrome C release and apoptosis. Expression of beta1 integrins increased ser(9) phosphorylation and inhibited beta-AR-stimulated increase in GSK-3beta activity. Wortmannin, inhibitor of PI3-kinase, reversed the effects of beta1 integrins on GSK-3beta activity and apoptosis. Purified active matrix metalloproteinase-2 (MMP-2), shown to interfere with beta1 integrin signaling, increased GSK-3beta activity, while inhibition of MMP-2 inhibited beta-AR-stimulated increases in GSK-3beta activity. beta-AR stimulation induced nuclear accumulation of GSK-3beta. beta-AR stimulation (3 h) increased the expression of transcription factor Gadd153 (growth arrest- and DNA damage-inducible gene 153). These data suggest that beta-AR stimulation increases GSK-3beta activity. Activation of GSK-3beta plays a pro-apoptotic role in beta-AR-stimulated apoptosis via the involvement of mitochondrial death pathway. beta1 integrins inactivate GSK-3beta and play an anti-apoptotic role via the involvement of PI3-kinase pathway. The apoptotic effects of GSK-3beta may be mediated, at least in part, via its nuclear localization and induction of pro-apoptotic genes, such as Gadd153.

Expression of the cytoplasmic domain of beta1 integrin induces apoptosis in adult rat ventricular myocytes (ARVM) via the involvement of caspase-8 and mitochondrial death pathway.

Stimulation of beta-adrenergic receptor (beta-AR) induces cardiac myocyte apoptosis. Integrins, a family of cell-surface receptors, play an important role in the regulation of cardiac myocyte apoptosis and ventricular remodeling. Cleavage of extracellular domain of beta1 integrin, also called integrin shedding, is observed during cardiac hypertrophy and progression to early heart failure. Here we show that stimulation of beta-AR induces beta1 integrin fragmentation in mouse heart. To examine the role of intracellular domain of beta1 integrin in cardiac myocyte apoptosis, a chimeric receptor consisting of the cytoplasmic tail domain of beta(1A) integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (TAC-beta1) was expressed in adult rat ventricular myocytes (ARVM) using adenoviruses. TAC-beta1 increased the percentage of apoptotic ARVM as measured by TUNEL-staining assay. TAC-beta1-induced apoptosis was found to be associated with increased cytosolic cytochrome c and decreased mitochondrial membrane potential. TAC-beta1 increased caspase-8 activity. Z-IETD-FMK, a specific caspase-8 inhibitor, significantly inhibited TAC-beta1-induced apoptosis. TAC-beta1 expression also increased cleavage of Bid, a pro-apoptotic Bcl-2 family protein. These data suggest that shedding of beta1 integrin may be a mechanism of induction of apoptosis during beta-AR-stimulated cardiac remodeling.

beta-Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of beta1 integrin signaling and mitochondrial pathway.

Stimulation of beta-adrenergic receptors (beta-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits beta-AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with beta1 integrins. Herein we tested the hypothesis that MMP-2 impairs beta1 integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited beta-AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated beta-AR-stimulated decreases in mitochondrial membrane potential. Overexpression of beta1 integrins using adenoviruses expressing the human beta1A-integrin decreased beta-AR-stimulated cytochrome c release and apoptosis. Overexpression of beta1 integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the beta1 integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis.