ABSTRACT
Neuronal cell death contributes significantly to the pathology of traumatic brain injury (TBI) irrespective of the mode or severity of the injury. Activation of a pro-survival protein, Akt, is known to be regulated by an E3 ligase TRAF6 through a process of ubiquitination-coupled phosphorylation at its T308 residue. Here we show that upregulation of a pro-apototic protein, GADD34, attenuates TRAF6-mediated Akt activation in a controlled cortical impact model of TBI in mice. TBI induces the expression of GADD34 by stimulating binding of a stress inducible transcription factor, ATF4, to the GADD34 promoter. GADD34 then binds with TRAF6 and prevents its interaction with Akt. This event leads to retention of Akt in the cytosol and prevents phosphorylation at the T308 position. Finally, in vivo depletion of GADD34 using a lentiviral knockdown approach leads to a rescue of Akt activation and markedly attenuates TBI-induced cell death.
Subject(s)
Apoptosis , Brain Injuries/enzymology , Protein Phosphatase 1/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Activating Transcription Factor 4/metabolism , Animals , Cells, Cultured , Cerebral Cortex/enzymology , Cerebral Cortex/pathology , Endoplasmic Reticulum Stress , Excitatory Amino Acid Agonists/pharmacology , Homeostasis , Male , Mice , Mice, Inbred C57BL , N-Methylaspartate/pharmacology , Neurons/physiology , Phosphorylation , Primary Cell Culture , Promoter Regions, Genetic , Protein Binding , Protein Phosphatase 1/genetics , Reactive Oxygen Species/metabolism , TNF Receptor-Associated Factor 6/metabolism , Transcriptional Activation , UbiquitinationABSTRACT
This study investigated the behavioral mechanisms underlying the anxiogenic, or anxiolytic mediated effects of CCK(2) receptor mediated agonist (CCK-4) and antagonist drugs (LY225910, LY288513, CR2945) in PVG hooded and Sprague-Dawley (SD) rats using the elevated plus maze test apparatus. In addition, the effects of a CCK(1) antagonist (CR1409) were investigated for its possible mediation in anxiety behavior between PVG hooded and SD rats. PVG hooded rats treated with CCK-4, decreased the time spent in the open arm and increased the time spent in the closed arm and correspondingly showed increase in the number of entries in the open arms while the number of entries in closed arm was insignificant, whereas SD rats decreased the time spent in the closed arm, while other parameters remained insignificant. PVG hooded rats administered with various CCK(2) antagonists (LY225910, LY288513, and CR2945) significantly increased the time spent in the open arm and correspondingly decreased the time spent in the closed arm, while the number of entries in the open or closed arm was insignificant, in contrast, SD rats failed to show any reliable significance. PVG hooded rats administered with the CCK(1) antagonist (CR1409), failed to show any reliable significance, in contrast, SD rats significantly increased the time spent in the open arm. The strain differences observed in this study suggests that CCK plays mainly as a neuromodulator, in which the various CCK(2) antagonists may not affect baseline anxiety state, but instead they modulate heightened states of anxiety through differential effects of CCK(1)/CCK(2) receptors.
Subject(s)
Anti-Anxiety Agents/pharmacology , Anxiety/metabolism , Brain/metabolism , Cholecystokinin/metabolism , Proglumide/analogs & derivatives , Receptors, Cholecystokinin/metabolism , Species Specificity , Animals , Anxiety/chemically induced , Anxiety/drug therapy , Behavior, Animal/drug effects , Behavior, Animal/physiology , Benzodiazepines , Brain/drug effects , Chemokines/agonists , Chemokines/antagonists & inhibitors , Chemokines/metabolism , Chemokines, CC , Cholecystokinin/agonists , Cholecystokinin/antagonists & inhibitors , Male , Maze Learning/drug effects , Maze Learning/physiology , Proglumide/pharmacology , Pyrazoles/pharmacology , Quinazolines/pharmacology , Quinazolinones , Rats , Rats, Sprague-Dawley , Receptor, Cholecystokinin B/agonists , Receptor, Cholecystokinin B/antagonists & inhibitors , Receptor, Cholecystokinin B/metabolism , Receptors, Cholecystokinin/agonists , Receptors, Cholecystokinin/antagonists & inhibitors , Tetragastrin/pharmacologyABSTRACT
Our previous studies (NeuroReport 12 (2001) 2717) showed that PVG hooded and not Sprague-Dawley (SD) rats exhibit remarkable freezing behavior on exposure to a cat in the cat freezing test apparatus. In the present study, we further examined the differences between these two strains of rats in response to repeated daily exposure to a cat in the cat freezing test apparatus. Freezing behavior habituation was observed in both PVG hooded (days 5-7) and SD rats (days 3-7). A selective CCK(2) agonist (BC264, 0.3 microg/kg, i.p.) on day 8 reversed habituated freezing behavior and locomotor activity in PVG hooded rats, but not in SD rats. These results suggest that CCK2 receptors mediate habituation to an anxiety-inducing stimulus in PVG hooded rats and further suggest that differential expression of these CCK2 receptors underlies this strain difference.
Subject(s)
Cholecystokinin/analogs & derivatives , Cholecystokinin/pharmacology , Habituation, Psychophysiologic/drug effects , Immobilization/physiology , Motor Activity/drug effects , Peptide Fragments/pharmacology , Receptor, Cholecystokinin B/agonists , Animals , Cats , Habituation, Psychophysiologic/physiology , Male , Motor Activity/physiology , Rats , Rats, Sprague-Dawley , Receptor, Cholecystokinin B/physiology , Species SpecificityABSTRACT
The periaqueductal gray (PAG) is considered to be an exit relay for defensive responses. Studies have shown that the ventrolateral periaqueductal gray (vlPAG) plays a role in the expression of freezing behavior whereas dorsolateral periaqueductal gray (dlPAG) is involved on both freezing and active forms of defensive behaviors. To further elucidate this theory, lesioned vlPAG and dlPAG rats were exposed to a cat in the cat-freezing test apparatus. Subsequently, a 7-day repeated exposure to a cat was done on the vlPAG and dlPAG lesioned rats. Results showed that the vlPAG lesioned rats demonstrated significant decrease in freezing behavior and corresponding increase in locomotor activity, while the dlPAG lesioned rats failed to show any significance. Subsequent repeated exposure of the vlPAG lesioned rats to a cat for 7 days showed a gradual decrease in freezing behavior with significance shown at days 5, 6 and 7 while the dlPAG lesioned rats failed to show any changes. These results suggest that vlPAG regulates freezing behavior in hooded PVG rats.
Subject(s)
Behavior, Animal/physiology , Fear/physiology , Neural Pathways/physiology , Periaqueductal Gray/physiology , Animals , Axotomy , Cats , Habituation, Psychophysiologic/physiology , Male , Motor Activity/physiology , Neural Pathways/cytology , Periaqueductal Gray/cytology , RatsABSTRACT
To identify genes involved in the development of anxiety or fear, we analyzed the gene expression profiles of the cortex of anxious hooded PVG and Sprague-Dawley (SD) rats after exposure to the cat-freezing test apparatus. These two rat strains showed a marked difference in the extent of anxious behavior on the cat-freezing test; the hooded PVG rats showed highly anxious behavior while a low anxiety state was observed in SD rats. A cDNA microarray consisting of 5,931 genes was employed to investigate the global mRNA expression profiles of anxiety-related genes. According to the assumption that an abundance ratio of > or =1.5 is indicative of a change in gene expression, we detected 16 upregulated and 38 downregulated genes in PVG hooded and SD rats. Some of these genes have not yet been associated with anxiety (e.g. FGF), while other genes were recently found to be expressed in an anxious state (e.g., rat nerve growth factor-induced gene, NGFI-A). Our study also focused on the expression of some neurotransmitter receptors that have already been proven to be relevant to anxiety or fear, e.g., gamma-aminobutyric acid (GABA), cholecystokinin (CCK) and 5-HT(3) receptors. To further confirm the microarray data, the mRNA expressions of three genes: rat activity-regulated cytoskeleton-associated gene (Arc), rat NGFI-A gene and rat 5-HT(3) receptor (5-HT(3)R) mRNA, were studied by reverse transcription-polymerase chain reaction (RT-PCR). The results of RT-PCR were basically consistent with those from cDNA microarray. Our study therefore demonstrated that the microarray technique is an efficient tool for analyzing global expression profiles of anxiety-related genes, which may also provide further insight into the molecular mechanisms underlying the states of anxiety and fear.
Subject(s)
Anxiety/genetics , Anxiety/metabolism , Cerebral Cortex/metabolism , Gene Expression Regulation , Oligonucleotide Array Sequence Analysis , Animals , Down-Regulation , Fear , Male , Rats , Rats, Inbred Strains , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Up-RegulationABSTRACT
This study examined differential freezing behavior, mediated by cholecystokinin-2 (CCK2) receptors (J. M. Farook et al., 2001), in PVG hooded and Sprague-Dawley (SD) rats exposed to a predator. The authors confirmed by reverse transcription polymerase chain reaction that CCK2 receptor expression in the PVG rats was increased in the hippocampus and cerebral cortex compared with that of SD rats. In addition, 4 variations in the coding region of the CCK2 receptor gene were detected between the PVG hooded and SD rats: 1 in Exon 4, 1 in Intron 2, and 2 in Intron 3. Acute treatment with a CCK2 agonist (CCK-4) or antagonist (LY225910) did not alter the level of CCK2 receptor expression, indicating no difference between the 2 strains in sensitivity of the CCK2 receptor to drugs.
Subject(s)
Gene Expression Regulation , Genetic Variation , Receptors, Cholecystokinin/genetics , Animals , Behavior, Animal/physiology , Brain/anatomy & histology , Brain/drug effects , Cats , Exons , Fear/physiology , Introns , Male , Quinazolines/pharmacology , Quinazolinones , RNA, Messenger/biosynthesis , Rats , Rats, Inbred Strains , Rats, Sprague-Dawley , Receptor, Cholecystokinin B , Receptors, Cholecystokinin/antagonists & inhibitors , Receptors, Cholecystokinin/drug effects , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Analysis, DNA , Species Specificity , Tetragastrin/pharmacologyABSTRACT
PVG hooded, but not Sprague-Dawley (SD), rats showed remarkable freezing behavior on cat exposure. Experiments using cDNA microarray and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that cholecystokinin (CCK)2 receptors were expressed to a greater extent in the PVG hooded rats. A CCK2 antagonist, LY225910, inhibited freezing behavior in the PVG hooded rats while a CCK2 agonist, CCK-4, increased freezing behavior in the SD rats. These results strongly suggest that CCK2 receptors mediate the freezing behavior and the differential expression of these receptors underlie the strain difference in such behavior.
