pCREB Expression Research Articles

Melatonin improves d‐galactose‐induced aging effects on behavior, neurogenesis, and lipid peroxidation in the mouse dentate gyrus via increasing pCREB expression

Melatonin (N-acetyl-5-methoxytryptamine) has multiple functions. In this study, we investigated the effects of melatonin on memory, cell proliferation, and neuroblast differentiation in the dentate gyrus of a mouse model of D-galactose-induced aging. D-galactose was subcutaneously administered to 7-wk-old mice for 10 wk, and age-matched mice were used as controls. Seven weeks after D-galactose administration, vehicle (water) or melatonin (6 mg/L in water) was administered ad libitum to the mice for 3 wk. The administration of D-galactose significantly increased the escape latency compared with that in the control mice on days 1-3. In addition, cells in the subgranular zone and in the granule cell layer of the dentate gyrus showed severe damage (cytoplasmic condensation) in the D-galactose-treated mice. However, melatonin supplementation to these mice for 3 wk significantly ameliorated the D-galactose-induced increase in escape latency and neuronal damage compared with the vehicle-treated group. The administration of melatonin also significantly restored the D-galactose-induced reduction of proliferating cells (Ki67-positive cells) and differentiating neuroblasts (doublecortin-positive neuroblasts) in the dentate gyrus. Furthermore, the administration of melatonin significantly increased Ser133-phosphorylated cyclic AMP response element binding protein in the dentate gyrus. The administration of melatonin significantly reduced D-galactose-induced lipid peroxidation in the dentate gyrus. These results suggest that melatonin may be helpful in reducing age-related phenomena in the brain.

Triptans attenuate capsaicin-induced CREB phosphorylation within the trigeminal nucleus caudalis: a mechanism to prevent central sensitization?

The c-AMP-responsive element binding protein (CREB) and its phosphorylated product (P-CREB) are nuclear proteins expressed after stimulation of pain-producing areas of the spinal cord. There is evidence indicating that central sensitization within dorsal horn neurons is dependent on P-CREB transcriptional regulation. The objectives of the study were to investigate the expression of P-CREB in cells in rat trigeminal nucleus caudalis after noxious stimulation and to determine whether pre-treatment with specific anti-migraine agents modulate this expression. CREB and P-CREB labelling was investigated within the trigeminal caudalis by immunohistochemistry after capsaicin stimulation. Subsequently, the effect of i.v. pre-treatment with either sumatriptan (n = 5), or naratriptan (n = 7) on P-CREB expression was studied. Five animals pre-treated with i.v. normal saline were served as controls. CREB and P-CREB labelling was robust in all animal groups within Sp5C. Both naratriptan and sumatriptan decreased P-CREB expression (p = 0.0003 and 0.0013) within the Sp5C. Triptans attenuate activation of CREB within the central parts of the trigeminal system, thereby leading to potential inhibition of central sensitization. P-CREB may serve as a new marker for post-synaptic neuronal activation within Sp5C in animal models relevant to migraine.

Effects of tianeptine and lithium on expression of pCREB in hippocampus of chronic stress depression rats

Objective To research the effects of tianeptine and lithium on expression of pCREB in hippocampus of chronic stress depression rats. Methods All the experimental rats were divided by random into : Group of depression,Group of tianeptine,Group of lithium and Group of control. The rats of Group of depression, Group of tianeptine and Group of lithium were applied stress for 21 days,and meanwhile Group of control had no stress. The rats of Group of tianeptine were fed with tianeptine (50 mg/kg) , Group of lithium were fed with lithium (60 mg/kg) , while another groups were fed with normal sodium of the same volume. The ethology examination was performed by using method of open-field and experiment of fluid consumption. The expression of pCREB was detected by Western-blotting method. Results After the chronic stress,the horizontal crossing numbers,the erection times,the modification times and the percentage of sacchar-consumption of the rats of Group of depression were 23.2±23.0;8. 1 ±7.2; 3.6 ±3.5 and (55.4 ±11.7)% respectively, which were less than Group of control (46.0±18.9;20.3±11.3;8.4±2.7 and (68.5 ±8.2)% ; P＜0.01). The horizontal crossing numbers(28. 1 ±23.0) ,the erection times(12. 1 ± 9.4) and the modification times(5.5 ±3.2) of Group of tianeptine are less than those of Group of control (P ＜ 0. 05), but no significant difference compared with Group of depression; the percentage of sacchar-consumption(62.7 ± 10.6) % ,Group of tianeptine was more than Group of depression (P＜ 0.05 ) , but no obvious difference with Group of control. The horizontal crossing numbers, the erection times, the modification times and the percentage of sacchar-consumption of Group of lithium were less than those of Group of control (P ＜ 0.05), more than those of Group of depression but no significant difference (P ＞ 0.05). In Westernblotting method,the level of pCREB in the hippocampus of Group of depression was less than that of Group of control (P＜ 0.01); that of Group of tianeptine was more than that of Group of depression (P ＜ 0.01) but no obvious difference with Group of control; that of Group of lithium was less than that of Group of control (P＜0. 01) and more than Group of depression (P＜0.01). Conclusion Tianeptine could reverse the reduction of expression of pCREB in hippocampus of chronic stress depression rats and lithium partly did it. Key words: pCREB ; Chronic stress ; Hippocampus ; Tianeptine ; Lithium ;

New insights into “GPR40‐CREB interaction inadult neurogenesis” specific for primates

Polyunsaturated fatty acids (PUFA), such as docosahexaenoic (DHA) and arachidonic acids (ARA) are known to be closely related to the brain development and also have beneficial effects on adult neurogenesis, learning, and mental disorders. Although PUFA were demonstrated as ligands for G protein-coupled receptor 40 (GPR40), their signaling mechanism in the brain, especially in the neurogenic niche, remains unknown. Using a monkey model of ischemia-enhanced hippocampal neurogenesis, we studied the spatial correlation between GPR40 and the phosphorylated cAMP response element-binding protein (pCREB), a transcription factor involved in adult neurogenesis, learning and memory. Furthermore, the brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB), both being downstream gene transcripts of pCREB, were studied. Similar to the dynamic change of GPR40 as the authors reported previously, pCREB was up-regulated significantly after transient global brain ischemia on Western blots, and this was associated with an enhanced hippocampal neurogenesis. Immunofluorescence microscopic analysis showed that GPR40 and pCREB expression patterns were completely identical, and they were coexpressed in both mature and newborn neurons as well as in the astrocytes residing in the subgranular zone (SGZ). GPR40/pCREB double-positive cells significantly increased in the SGZ on day 15 after ischemia. The mature form of BDNF (mBDNF) and TrkB receptor showed no remarkable changes on Western blots, although proBDNF (precursor of mBDNF) was maximal on day 9. Immunofluorescence microscopy showed that the newborn neurons expressed BDNF, but not TrkB. These results altogether suggest that PUFA, GPR40, pCREB, and BDNF may be engaged in the same signaling pathway to promote neurogenesis in the adult primate hippocampus.

Effect of intrathecal dexmedetomidine on expression of cAMP response element-Wnding protein phosphorylation in spinal dorsal horn in a rat model of bone cancer pain

Objective To investigate the effect of intrathecal (IT) dexmedetomidine on the expression of cAMP response element-binding protein phosphorylation (p-CREB) in spinal dorsal horn in a rat model of bone cancer pain. Methods Sixty-four adult female Wistar rats weighing 200-240 g were randomly divided into 4 groups (n = 16 each): sham operation group (group S); bone cancer pain group (group BP); normal saline group ( group NS) ; dexmedetomidine group (group D) . Bone cancer pain was induced by injecting Walker 2S6 mammary gland carcinoma cell suspension (2 ×106 cells/ml) 10μl into the medullary cavity of the tibia in BP, NS and D groups. Groups S and BP received no IT injection. Croups NS and D received IT injection of NS 10 μl and dexme detomidine 5 μg/kg respectively 7 days after successful establishment of the model. Ten animals were selected from each group at 1 day before IT administration (T0), immediately before IT administration (T1 ) and at 1, 6, 12 and 24 h after IT administration (T2-5 ) and paw withdrawal threshold (PWT) to mechanical stimuli was measured with von Frey filaments. The other 6 rats in each group were sacrificed at T4 and the spinal cord was removed for determination of p-CREB expression in the spinal dorsal horn.Results PWT was significantly decreased at T1-5 and pCREB expression up-regulated at T4 in BP, NS and D groups compared with group S ( P ＜ 0.05) . Compared with group BP, PWT was significantly decreased at T2-5 and p-CREB expression down-regulated at T4 in group D ( P ＜0.03), while no significant change in PWT and p-CREB expression was found in group NS (P ＞ 0.05) .Conclusion IT dexmedetomidine can reduce the bone cancer pain through inhibiting the phosphorylation of CREB in rat spinal dorsal horn. Key words: Dexmedetomidine; Bone neoplasms; Cyclic AMP response element-binding protein; Spinal cord

Abstract 1605: Social stress stimulates the progression of pancreatic cancer xenografts in nude mice

Abstract Pancreatic ductal adenocarcinoma (PDAC) cancer is a highly aggressive cancer with poor prognosis due to limited chemosensitivity to currently available therapeutic regimens. Smoking, diabetes and pancreatitis are risk factors for PDAC. Our laboratory has shown that agonist for β-ARs promoted the progression of pancreatic cancer in hamsters while the beta-blocker propranolol had strong cancer preventive effects. The stress neurotransmitters noradrenaline and adrenaline are the physiological agonists for β-ARs. They are released from nerve endings and the adrenal medulla in response to nicotinic acetylcholine receptor (nAChR) stimulation by acetylcholine. The neurotransmitter γ-aminobutyric acid (GABA) acts as the physiological inhibitor of β-adrenergic signaling by blocking Gα1-mediated activation of adenylyl cyclase. The present study has tested the hypothesis that social stress stimulates the growth of human PDAC xenografts via nAChR-mediated release of stress neurotransmitters and the associated activation of β-adrenergic signaling. Groups of nude mice were exposed to social stress 4 weeks prior to subcutaneous injection with PDAC cell lines Panc-1 (with activating point mutation in K-ras) and BxPc-3 (without K-ras mutation) and stress continued for another 4 weeks. Mice not exposed to stress served as controls. The developing xenografts were measured weekly. At the end of the experiment, the systemic levels of noradrenaline, adrenaline, cortisol GABA in serum and xenograft tissues and cAMP in the cellular fraction of blood and in xenograft tissues were assessed by immunoassays. In addition, the protein expression of nAChR subunits α7, α4, α3, α5, the expression of β-adrenergic signaling proteins p-CREB and p-ERK and of the GABA synthesizing enzymes GAD67 and GAD65 were assessed by Western blotting in xenograft tissues. Our results show a significant stimulation of xenograft growth by social stress accompanied by elevated levels of stress neurotransmitters, cortisol and cAMP. An enhanced expression of nAChR subunits α3, α4, α5 and α7 as well as of β-adrenergic pathway proteins were found in xenografts of stressed animals. By contrast the inhibitory neurotransmitter GABA and both GAD enzymes were downregulated in animals exposed to stress. These findings suggest that social stress stimulates the progression of PDAC via nAChR-dependent upregulation of cancer stimulating noradrenaline/adrenaline and their associated β-adrenergic signaling while simultaneously reducing inhibitory GABA thereby counteracting the efficacy of cancer therapeutics. Psychological stress reduction strategies as well as pharmacological inhibition of this PDAC stimulating pathway by beta-blockers or GABA-ergic agents should be considered to improve the responsiveness to cancer therapeutics in PDAC patients. Supported by Challenge grant 1RC1CA144640 with the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1605. doi:10.1158/1538-7445.AM2011-1605

Time-course of changes in phosphorylated CREB in neuroblasts and BDNF in the mouse dentate gyrus at early postnatal stages.

Cyclic AMP (cAMP) response element-binding protein (CREB) is involved in memory, learning, and synaptic transmission. In this study, we observed changes of phosphorylated CREB (pCREB) immunoreactivity and its protein levels as well as brain-derived neurotrophic factor (BDNF) levels in the hippocampal dentate gyrus at postnatal (P) 1, 7, 14, and 21 in mice. In addition, we also investigated pCREB expression in doublecortin (DCX, a marker for neuronal progenitors) immunoreactive neuroblasts at P21. pCREB immunoreaction at P1 was detected in most of cells in the dentate gyrus, thereafter pCREB immunoreactivity was decreased in all the layers of the dentate gyrus with time, however, strong pCREB immunoreactivity was shown in cells confined to the subgranular zone of the dentate gyrus at P21. In this group, many pCREB immunoreactive cells were co-localized with DCX immunoreactive neuroblasts. In addition, pCREB protein levels were decreased with age, showing that their levels were very low at P21, while BDNF protein levels were increased with age. These results suggest that pCREB may play important roles in functional maturity of granule cells in mice.

Novel Control by the CA3 Region of the Hippocampus on Neurogenesis in the Dentate Gyrus of the Adult Rat

The dentate gyrus is a site of continued neurogenesis in the adult brain. The CA3 region of the hippocampus is the major projection area from the dentate gyrus. CA3 sends reciprocal projections back to the dentate gyrus. Does this imply that CA3 exerts some control over neurogenesis? We studied the effects of lesions of CA3 on neurogenesis in the dentate gyrus, and on the ability of fluoxetine to stimulate mitotic activity in the progenitor cells. Unilateral ibotenic-acid generated lesions were made in CA3. Four days later there was no change on the number of either BrdU or Ki67-positive progenitor cells in the dentate gyrus. However, after 15 or 28 days, there was a marked reduction in surviving BrdU-labelled cells on the lesioned side (but no change in Ki-67+ cells). pCREB or Wnt3a did not co-localise with Ki-67 but with NeuN, a marker of mature neurons. Lesions had no effect on the basal expression of either pCREB or Wnt3a. Subcutaneous fluoxetine (10 mg/kg/day) for 14 days increased the number of Ki67+ cells as expected on the control (non-lesioned) side but not on that with a CA3 lesion. Nevertheless, the expected increase in BDNF, pCREB and Wnt3a still occurred on the lesioned side following fluoxetine treatment. Fluoxetine has been reported to decrease the number of “mature” calbindin-positive cells in the dentate gyrus; we found this still occurred on the side of a CA3 lesion. We then showed that the expression GAP-43 was reduced in the dentate gyrus on the lesioned side, confirming the existence of a synaptic connection between CA3 and the dentate gyrus. These results show that CA3 has a hitherto unsuspected role in regulating neurogenesis in the dentate gyrus of the adult rat.

Long lasting effects of predator stress on pCREB expression in brain regions involved in fearful and anxious behavior

Predator stress is one animal model of posttraumatic stress disorder (PTSD). Neural plasticity in amygdala afferent and efferent pathways underlies anxiogenic effects of predator stress. Predator stress increases pCREB expression in these pathways 20 min after stress, implicating pCREB in stress-induced neural plasticity. Here we examined impact of predator stress on pCREB expression 6–24 h and 7 days after stress in amygdala pathways and in the supramammillary nucleus (SuM). Patterns of change in pCREB expression were complex, time dependent, column dependent in the periaqueductal gray (PAG), and AP plane dependent in the amygdala. In contrast to past work at 20 min after stress, there were no stress-induced increases in pCREB in the amygdala in the anterior AP plane or in the lateral PAG at 6 h onward after stress. However, dorsal PAG pCREB was increased bilaterally at 24 h and 7 days after stress. In the mid AP plane of all amygdala nuclei there were bilateral stress-induced increases in pCREB at 6 h followed by decreases at 24 h post stress. A similar pattern was observed in the posterior AP plane. In addition, we found a persistent increase (6 h to 7 days after stress) in pCREB expression in the SuM. Further study of this nucleus as a contributor to fear sensitization following predator stress is warranted. Overall, these data highlight persistent neuroplastic changes in key brain areas following traumatic stress. Identification of these changes may aid in understanding the neural mechanisms underlying acquired anxiety disorders such as PTSD.

Cilostazol enhances integrin‐dependent homing of progenitor cells by activation of camp‐dependent protein kinase in synergy with Epac1

Recruitment and adhesion of exogenous endothelial progenitor cells (EPCs) or endogenously mobilized bone marrow mononuclear cells (BM MNCs) to the sites of ischemia is an important focus of cell therapy. This study sought to determine whether cilostazol enhances integrin-dependent homing of progenitor cells both in vitro and in vivo. In the in vitro experiments with human umbilical cord blood (HUCB)-derived EPCs, cilostazol (10 μM) stimulated up-regulation of integrins β1, α1, and αv as well as 8-pCPT-2'-O-Me-cAMP (100 μM; 8-pCPT, Epac activator). Cilostazol and 8-pCPT significantly enhanced migration and adhesion of HUCB EPCs to a fibronectin-coated plate and endothelial cells, which were inhibited by KT5720 (PKA inhibitor, 1 μM) and GGTI-298 (Rap1 inhibitor, 20 μM). Cilostazol stimulated Epac1 expression and up-regulated the active Rap1, as did 8-pCPT, and they were suppressed by KT5720 (P < 0.001) and GGTI-298 (P < 0.001). 8-pCPT increased p-CREB expression and stimulated PKA activity, which was inhibited by KT5720, Rp-cAMPS, and GGTI-298. In addition, N(6)-benzoyl-cAMP (100 μM) increased Rap1 GTP expression, as did 8-pCPT; they were suppressed by Rp-cAMPS and GGTI-298. The in vivo experiments showed that cilostazol (30 mg/kg/day, orally for 7 days) significantly enhanced the integrin β1 expression in the molecular layer and up-regulated homing of BM MNCs to the injured molecular layer with increased capillary density in mouse brain subjected to transient forebrain ischemia (n = 6, P < 0.001). In conclusion, cilostazol stimulated integrin expression and enhanced migration and adhesion of progenitor cells through cooperative activation of PKA and Epac signals; such activity may improve the efficacy of cell therapy for ischemic disease.

Dose-related effects of venlafaxine on pCREB and brain-derived neurotrophic factor (BDNF) in the hippocampus of the rat by chronic unpredictable stress.

Background: The molecular pathogenesis of depression and psychopharmacology of antidepressants remain elusive. Recent hypotheses suggest that changes in neurogenesis and plasticity may underlie the aetiology of depression. The hippocampus is affected by depression and shows neuronal remodelling during adulthood.Objective: The present study on the adult rat hippocampus, was to evaluate the dose-related effects of chronic venlafaxine on the expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cyclic-AMP response element binding protein (pCREB).Methods: Sprague-Dawley rats were exposed to a variety of chronic unpredictable stressors (CUSs) to establish a depression model. Rats were treated for either 14 or 28 days with venlafaxine (5 and 10 mg/kg, respectively). The hippocampal expression of pCREB and BDNF mRNA and protein was assessed by using immunohistochemistry, western blotting and reverse transcription polymerase chain reaction (RT-PCR).Results: Rats subjected to CUS procedure consumed less sucrose solution compared with non-stressed rats. The CUS influenced exploratory activity resulting in a reduction of the motility counts. Chronic low dose (5 mg/kg, 14 and 28 days), but not high dose (10 mg/kg, 14 and 28 days) of venlafaxine treatment increased the expression of pCREB and BDNF mRNA and protein in the CUS rat hippocampus.Conclusion: Neuronal plasticity-associated proteins such as pCREB and BDNF play an important role both in stress-related depression and in antidepressant effect.

Effects of dexmedetomidine on the release of glial cell line-derived neurotrophic factor from rat astrocyte cells

Dexmedetomidine (DEX) has been found to improve neuronal survival after transient global or focal cerebral ischemia in rats. Astrocyte cells may possess beneficial properties that promote neuronal recovery by secreting neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF). The purpose of this study was to investigate the effects of DEX on GDNF release from astrocytes and the possible mechanisms involved. Astrocyte cells were treated with DEX, and GDNF level in the conditioned media was determined by ELISA assay. The expression of CREB, p-CREB and PKCα was analyzed by Western blotting to explore the mechanisms involved in GDNF release. Our results showed that DEX stimulated GDNF release in a time- and dose-dependent manner; and this stimulation was blocked by the α2-adrenoreceptor antagonist yohimbine, but not by α1-adrenoreceptor antagonist prasozin, demonstrating that DEX induced GDNF release likely acts via activating the α2A adrenoreceptor. In addition, DEX-stimulated GDNF release was also blocked by the universal PKC inhibitor Ro-318220 and PKCα/β inhibitor Gö 6976, but not by PKCδ inhibitor rottlerin and PKCβ inhibitor LY333531. Interestingly, DEX also activated CREB phosphorylation, which was inhibited by Ro-318220, Gö 697 and ERK kinase inhibitor PD98059. Silencing CREB by siRNA decreased the DEX-stimulated GDNF release. In addition, the membrane translocation of PKCα was enhanced following DEX treatment. Furthermore, we found that DEX stimulated GDNF release rescued neurons against OGD-induced neurotoxicity; this effect was partly abolished by GDNF antibody. Thus, through α2A adrenergic receptors, DEX may activate astrocytes, and promote GDNF release to protect neurons after stroke, and this signaling is possibly dependent on PKCα and CREB activation.

Brain tissue energy dependence of CaM kinase IV cascade activation during hypoxia in the cerebral cortex of newborn piglets

The present study aims to investigate the dependence of CaM kinase IV cascade activation during hypoxia and tests the hypothesis that hypoxia-induced tyrosine phosphorylation of CaM and CaM kinase IV, activation of CaM kinase IV and phosphorylation of CREB protein during hypoxia increases as a function of increase in cerebral tissue hypoxia as measured by decrease in tissue ATP and phosphocreatine (PCr). 3–5 days old newborn piglets were divided into normoxic (Nx, FiO 2 of 0.21 for 1 h) and hypoxic (Hx, FiO 2 of 0.07 for 1 h) groups. Cerebral tissue hypoxia was documented by determining the levels of high energy phosphates ATP and phosphocreatine (PCr). Cerebral cortical neuronal nuclei were isolated and purified, and tyrosine phosphorylation of calmodulin (Tyr 99), the activator of CaM kinase IV, and CaM kinase IV determined by Western blot using anti-phospho-(pTyr 99)-calmodulin, anti-pTyrosine and anti-CaM kinase IV antibodies. The activity of CaM kinase IV and its consequence the phosphorylation of CREB protein at Ser 133 were determined. The levels of ATP (μmole/g brain) ranged from 3.48 to 5.28 in Nx, and 0.41 to 2.26 in Hx. The levels of PCr (μmole/g brain) ranged from 2.46 to 3.91 in Nx and 0.72 to 1.20 in Hx. The pTyr 99 calmodulin (OD × mm 2) ranged from 20.35 to 54.47.60 in Nx, and 84.52 to 181.42 in Hx ( r 2 = 0.5309 vs ATP and r 2 = 0.6899 vs PCr). Expression of tyrosine phosphorylated CaM kinase IV ranged from 32.86 to 82.46 in Nx and 96.70 to 131.62 in Hx ( r 2 = 0.5132 vs ATP and r 2 = 0.4335 vs PCr). The activity of CaM kinase IV (pmole/mg protein/min) ranged from 1263 to 3448 in Nx and 3767 to 6633 in Hx ( r 2 = 0.7113 vs ATP and r 2 = 0.6182 vs PCr). The expression of p-CREB at Ser 133 ranged from 44.26 to 70.28 in Nx and 82.70 to 182.86 in Hx ( r 2 = 0.6621 vs ATP and r 2 = 0.5485 vs PCr). The data show that hypoxia results in increased tyrosine phosphorylation of calmodulin (Tyr 99), increased tyrosine phosphorylation of CaM kinase IV, increased activity of CaM kinase IV and increased phosphorylation of CREB at Ser 133 as an inverse function of cerebral concentration of high energy phosphates, ATP and PCr. We conclude that the hypoxia-induced increased activation of CaM kinase IV cascade increases with the increase in the degree of cerebral tissue hypoxia as measured by cerebral tissue high energy phosphates in a curvilinear manner. The tyrosine kinases (Src kinase and EGFR kinase) mediated activation of CaM kinase IV cascade potentially results in increased CREB phosphorylation that triggers transcription of proapoptotic proteins during hypoxia.

Anti-hyperalgesic effect of CaMKII inhibitor is associated with downregulation of phosphorylated CREB in rat spinal cord

Calcium/calmodulin-dependent protein kinase (CaMK) II and its downstream effector cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB) may be involved in the development of neuropathic pain. The aim of this study was to examine the effect of the CaMKII inhibitor AIP on the association of CaMKII and CREB in a partial sciatic nerve ligation neuropathic pain model in rats. Male Sprague-Dawley rats were randomly divided into six groups: Sham operation; partial sciatic nerve ligation (SNL); AIP administration prior to or after SNL; and normal saline (NS) administration prior to or after SNL. NS or 10μmol/L AIP (10μl) were injected intrathecally in the NS group and AIP group, respectively, 20min before or 4days after nerve injury. The mechanical withdrawal threshold of the hindpaw was measured before and after SNL. In another cohort treated as above, the lumbar spinal cord was removed on post-surgery days 1, 3, and 7 to detect the expression of phosphorylated CaMKII (pCaMKII) and phosphorylated CREB (pCREB), by in situ immunostaining and western blot, respectively. AIP significantly suppressed tactile allodynia in the SNL rats, and its effects lasted for 3days when given prior to nerve injury. In contrast, it had a transitory effect when given after nerve injury. AIP decreased the expression of pCaMKII and pCREB and its effect was sustained for up to 3days after the experiments. Intrathecal injection of a CaMKII inhibitor attenuated neuropathic pain. This benefit may have been due to the downmodulation of its downstream effector pCREB.

A critical time window for the survival of neural progenitor cells by HDAC inhibitors in the hippocampus.

Histone deacetylase inhibitors (HDACIs) that modulate gene expression by inhibiting HDAC enzymes may contribute to the survival of immature hippocampal neurons. However, it remains unknown how and when HDACIs regulate the survival of newly generated immature hippocampal neurons. In the present study, if the treatment of valproic acid (VPA) and sodium butyrate (SBt) in the specific time window during the development of newly generated n eurons r esulted in the i ncreased survival of bromodeoxyuridine (BrdU)(+) neurons in the dentate gyrus (DG) of hippocampus in mice was investigated. It was found that the number of BrdU(+) cells, the expressions of anti-apoptotic Bcl-2 family members and pCREB [D1] were increased by HDACIs when HDACIs were treated no later than 2-3 weeks after BrdU labeling. This suggests that epigenetic modification within a specific time window is critical for the survival of newborn hippocampal neurons by inhibiting the apoptotic pathway.

SiRNA‐mediated GABAB receptor at early fetal rat brain upon acute and chronic ethanol exposure: Down regulation of PKA and p‐CREB expression

To observe the modulatory role of GABA(B1)R upon ethanol's effect during early brain development, we studied the effects of chronic maternal (10% ethanol during pregnancy) and acute (in vitro) ethanol exposure on the neuronal protein kinase A (PKA-α) and phosphorylation of cAMP-response element binding protein (p-CREB), using a system where GABA(B1)R were specifically knocked down in the primary cells cultured at gestational day (GD) 12.5. The results showed that upon acute and chronic ethanol treatment the GABA(B1)R expression was decreased and further decreased when GABA(B1)R was transfection with siRNA, while increased upon exposure of baclofen, and baclofen plus phaclofen treatment. PKA expression was also decreased with acute and chronic ethanol treatment, whereas it showed increase upon exposure of baclofen and baclofen with phaclofen. Furthermore, intracellular Ca(2+) concentration was increased upon ethanol, baclofen, phaclofen exposure but showed decrease in GABA(B1)R siRNA group. Finally, these effects could lead to changes of p-CREB expression, which showed same expression pattern as PKA. We speculate that GABA(B)R activity upon ethanol exposure could modulate intracellular calcium homeostasis and the expressional changes of PKA and p-CREB, which cause various negative effects on fetal brain development and modulation of GABA(B)R upon ethanol exposure may underlying cause of ethanol's effects.

Effect of ketamine administration on memory consolidation, p-CREB and c-fos expression in the hippocampal slices of minor rats

To investigate the effect of ketamine administration on memory consolidation, p-CREB and c-fos expression in hippocampus of infant rats and the possible mechanism. Ninety-six SD rats of 21 days old were divided into seven groups, Y-maze was used to test the ability of learning and memory for minor rats. p-CREB and c-fos protein expression was tested by immunhistochemistry. The results showed that the memory consolidation rate in normal saline group mice was higher than that in k1a and k7a group (P<0.05). However, normal saline group between k1b and k7b group was not significant difference. The p-CREB and c-fos protein expressing cells of normal saline group were higher than those in passive control group, pseudotraining group, k1a and k7a group (P<0.05). However, there was not significant difference between normal saline group and k1b or k7b group. The c-fos protein expressing cells of pseudotraining group was higher than those in passive control group, however, the p-CREB protein expressing cells was not significant difference. Therefore, administration of ketamine may temporally affect the ability of memory consolidation rate of minor rats through suppressing the expression of p-CREB and c-fos protein in hippocampus.

The Roles of BDNF, pCREB and Wnt3a in the Latent Period Preceding Activation of Progenitor Cell Mitosis in The Adult Dentate Gyrus by Fluoxetine

The formation of new neurons continues into adult life in the dentate gyrus of the rat hippocampus, as in many other species. Neurogenesis itself turns out to be highly labile, and is regulated by a number of factors. One of these is the serotoninergic system: treatment with drugs (such as the SSRI fluoxetine) markedly stimulates mitosis in the progenitor cells of the dentate gyrus. But this process has one remarkable feature: it takes at least 14 days of continuous treatment to be effective. This is despite the fact that the pharmacological action of fluoxetine occurs within an hour or so of first administration. This paper explores the role of BDNF in this process, using the effect of a Trk antagonist (K252a) on the labelling of progenitor cells with the mitosis marker Ki67 and the associated expression of pCREB and Wnt3a. These experiments show that (i) Fluoxetine increased Ki67 counts, as well as pCREB and Wnt3a expression in the dentate gyrus. The action of fluoxetine on the progenitor cells and on pCREB (but not Wnt3a) depends upon Trk receptor activation, since it was prevented by icv infusion of K252a. (ii) These receptors are required for both the first 7 days of fluoxetine action, during which no apparent change in progenitor mitosis occurs, as well as the second 7 days. Increased pCREB was always associated with progenitor cell mitosis, but Wnt3a expression may be necessary but not sufficient for increased progenitor cell proliferation. These results shed new light on the action of fluoxetine on neurogenesis in the adult dentate gyrus, and have both clinical and experimental interest.

Psychophysical stress increases the expression of phospho-CREB, Fos protein and neurokinin-1 receptors in superficial laminae of trigeminal subnucleus caudalis in female rats

Psychological stress and estrogen status are risk factors to develop painful temporomandibular joint disorders (TMJD); however, the neural basis for this relationship is not known. This study tested the hypothesis that repeated forced swim stress and estradiol treatment alter the phosphorylation of cAMP responsive element-binding protein (pCREB) in trigeminal subnucleus caudalis (Vc), the initial site of sensory input from the TMJ. Ovariectomized female rats were given low or high dose estradiol and subjected to repeated forced swim stress for 3 days and on day 4 an intra-TMJ injection of mustard oil or vehicle was given. Forced swim alone increased the number of pCREB-positive neurons, independent of estradiol treatment or TMJ stimulation, in superficial and deep laminae of Vc. Forced swim also increased the number of Fos-positive neurons in superficial laminae and neurokinin-1 receptor mRNA in whole dorsal Vc, independent of estradiol treatment. These results indicated that persistent psychophysical stress alone was sufficient to increase the expression of pCREB and downstream regulated genes associated with enhanced excitability in the caudal medullary dorsal horn, a brainstem region thought to be critical for TMJD pain.

C/EBPβ is AMP kinase sensitive and up-regulates PEPCK in response to ER stress in hepatoma cells

Diabetes and obesity are associated with activation of endoplasmic reticulum (ER) stress; however a direct link between ER stress and increased hepatic gluconeogenesis remains unclear. Here we show that ER stress triggers a significant increase in expression of CCAAT/enhancer-binding protein (C/EBPβ) and phosphorylated CREB together with reduced phospho-AMP-activated protein kinase (pAMPK) in hepatoma cells. ER stress contributed to transcriptional activation of the gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK) promoter in Huh7 and HepG2 cells via cAMP binding motif (CRE site). Chromatin immunoprecipitation assays demonstrate that C/EBPβ is recruited to the PEPCK promoter during ER stress and is reversed by pre-treatment with a JNK inhibitor that relieves ER stress. C/EBPβ but not pCREB was suppressed by the AMPK-activator AICAR or constitutively active AMPK, while dominant negative AMPK increased C/EBPβ expression. These data suggest that ER stress triggers suppression of AMPK while increasing C/EBPβ and pCREB expression which activates PEPCK gene transcription. Understanding how ER stress suppresses AMPK activation and increases C/EBPβ expression could lead to a potentially novel pathway for treatment of diabetes.