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.     

CREB activity maintains the survival of cingulate cortical pyramidal neurons in the adult mouse brain

Cyclic AMP-responsive element binding protein (CREB) activity is known to contribute to important neuronal functions, such as synaptic plasticity, learning and memory. Using a microelectroporation technique to overexpress dominant negative mutant CREB (mCREB) in the adult mouse brain, we found that overexpression of mCREB in the forebrain cortex induced neuronal degeneration. Our findings suggest that constitutively active CREB phosphorylation is important for the survival of mammalian cells in the brain.     

Enhancement of Behavioral Sensitization,Anxiety-Like Behavior,and Hippocampal and Frontal Cortical CREB Levels Following Cocaine Abstinence in Mice Exposed to Cocaine during Adolescence

Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p.) for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC) and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous system.     

Aggression frequency and intensity, independent of testosterone levels, relate to neural activation within the dorsolateral subdivision of the ventromedial hypothalamus in the tree lizard Urosaurus ornatus

The mechanisms by which testosterone regulates aggression are unclear and may involve changes that alter the activity levels of one or more brain nuclei. We estimate neural activity by counting immunopositive cells against phosphorylated cyclic AMP response element binding protein (pCREB). We demonstrate increased pCREB immunoreactivity within the dorsolateral subdivision of the ventromedial hypothalamus (VMHdl) following an aggressive encounter in male tree lizards Urosaurus ornatus. This immunoreactivity is induced both by exposure to and performance of aggressive behaviors. This dual activation of the VMHdl suggests its possible role as an integration center for assessment and expression of aggressive behavior. Furthermore, pCREB induction was greater in encounters involving higher frequency and intensity of aggressive display, demonstrating a direct relationship between neural activation and behavior. The VMHdl is also rich in steroid receptors. In a second experiment involving hormone manipulations, testosterone treatment increased aggression levels, though it did not increase the number of pCREB positive cells within the VMHdl. This lack of an effect of testosterone on pCREB induction within the VMHdl may be due to induction arising from the behaviors of conspecifics (especially in low-testosterone, low-aggression individuals), variation in aggression mediated by other variables, or regulation of aggression by circuits outside of the VMHdl. Together, these findings support a notion of the VMHdl as a nucleus involved in integrating afferent and efferent information within the neural aggression-control circuit.     

Correlation of glutamate binding activity with glutamate-binding protein immunoreactivity in the brain of control and alcohol-treated rats

Specific antibodies raised against a glutamate binding protein purified from bovine brain were used to trace the immunoreactivity of this protein in rat brain subcellular fractions. In the subcellular fractions obtained from whole brain homogenates, the synaptic membranes had the highest immunochemical reactivity towards the anti-glutamate-binding protein antibodies. The combination of measurements of glutamate binding activity and glutamate-binding protein immunoreactivity indicated that in brain synaptic membranes from control animals the highest activity in these two measures was associated with a synaptic plasma membrane subfraction that was enriched with synaptic junctions. In animals treated with ethanol for 14 days, there was a significant increase in the density of synaptic membrane glutamate binding sites. This increase in glutamate binding capacity was correlated with a greater than two-fold increase in the glutamate binding activity and binding protein immunoreactivity of the light synaptic membrane subfraction, a subfraction which does not contain many recognizable synaptic junctions. Acute administration of ethanol to rats produced a moderate but non-significant decrease in glutamate binding capacity of synaptic membranes. The increase in the number of glutamate binding protein subunits in brain plasma membranes may be an adaptive response of central nervous system neurons to the acute effects of ethanol on glutamate synaptic transmission.     

In vivo CREB phosphorylation mediated by dopamine and NMDA receptor activation in mouse hippocampus and caudate nucleus

The pattern of CREB phosphorylation was investigated in the caudate nucleus and hippocampus 10 min or 3 h after i.p. injection of dopamine or NMDA receptor agonists alone, or in combination with antagonists. Ten minutes after C57BL/6 J mice were injected with either the dopamine D1 receptor agonist SKF-38393 hydrobromide or NMDA, immunoreactivity of phosphorylated CREB (pCREB) was significantly increased in all parts of the caudate nucleus but not in hippocampal regions. However, 3 h after the injection of SKF-38393, pCREB levels in the caudate nucleus did not differ significantly from the pCREB levels in control animals, whereas pCREB levels were still elevated 3 h after NMDA injection. Except for the D1 receptor antagonist SCH-23390, which induced CREB phosphorylation in the caudate nucleus, dopamine and NMDA receptor antagonists had little effect on pCREB levels by themselves. However, the NMDA receptor antagonist CGS-19755 injected i.p. blocked both the NMDA- and SKF-38393-induced rise of pCREB levels in the caudate nucleus. Similarly, the D1 receptor antagonist SCH-23390 inhibited the effects produced by SKF-38393 or NMDA. Interestingly, the D2 receptor antagonist sulpiride also blocked the SKF-38393-triggered rise of pCREB. The results demonstrated that NMDA and dopamine receptors modulate pCREB levels in the caudate nucleus and suggest mutual permissive roles for both receptors.     

Exercise Prevents Memory Impairment Induced by Arsenic Exposure in Mice: Implication of Hippocampal BDNF and CREB

High concentrations of arsenic, which can be occasionally found in drinking water, have been recognized as a global health problem. Exposure to arsenic can disrupt spatial memory; however, the underlying mechanism remains unclear. In the present study, we tested whether exercise could interfere with the effect of arsenic exposure on the long-term memory (LTM) of object recognition in mice. Arsenic (0, 1, 3, and 10 mg/ kg, i.g.) was administered daily for 12 weeks. We found that arsenic at dosages of 1, 3, and 10 mg/kg decreased body weight and increased the arsenic content in the brain. The object recognition LTM (tested 24 h after training) was disrupted by 3 mg/ kg and 10 mg/ kg, but not 1 mg/ kg arsenic exposure. Swimming exercise also prevented LTM impairment induced by 3 mg/ kg, but not with 10 mg/ kg, of arsenic exposure. The expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP-response element binding protein (pCREB) in the CA1 and dentate gyrus areas (DG) of the dorsal hippocampus were decreased by 3 mg/ kg and 10 mg/ kg, but not by 1 mg/ kg, of arsenic exposure. The decrease in BDNF and pCREB in the CA1 and DG induced by 3 mg/ kg, but not 10 mg/ kg, of arsenic exposure were prevented by swimming exercise. Arsenic exposure did not affect the total CREB expression in the CA1 or DG. Taken together, these results indicated that swimming exercise prevented the impairment of object recognition LTM induced by arsenic exposure, which may be mediated by BDNF and CREB in the dorsal hippocampus.