Immediate-early genes (IEGs) have long been used to visualize neural activations induced by sensory and behavioral stimuli. Recent advances in imaging techniques have made it possible to use endogenous IEG signals to visualize and discriminate neural ensembles activated by multiple stimuli, and to map whole-brain-scale neural activation at single-neuron resolution. In addition, a collection of IEG-dependent molecular tools has been developed that can be used to complement the labeling of endogenous IEG genes and, especially, to manipulate activated neural ensembles in order to reveal the circuits and mechanisms underlying different behaviors. Here, we review these techniques and tools in terms of their utility in studying functional neural circuits. In addition, we provide an experimental strategy to measure the signal-to-noise ratio of IEG-dependent molecular tools, for evaluating their suitability for investigating relevant circuits and behaviors.
Animals ; Brain ; metabolism ; Gene Expression Profiling ; methods ; Genes, Immediate-Early ; Humans ; Molecular Imaging ; methods ; Neural Pathways ; metabolism ; Neurons ; metabolism ; Signal-To-Noise Ratio

Memantine, a noncompetitive antagonist of the N-methyl-d-aspartate (NMDA) receptor, suppresses the release of excessive levels of glutamate that may induce neuronal excitation. Here we investigated the effects of memantine on salicylate-induced tinnitus model. The expressions of the activity-regulated cytoskeleton-associated protein (ARC) and tumor necrosis factor-alpha (TNF α)genes; as well as the NMDA receptor subunit 2B (NR2B) gene and protein, were examined in the SH-SY5Y cells and the animal model. We also used gap-prepulse inhibition of the acoustic startle reflex (GPIAS) and noise burst prepulse inhibition of acoustic startle, and the auditory brainstem level (electrophysiological recordings of auditory brainstem responses, ABR) and NR2B expression level in the auditory cortex to evaluate whether memantine could reduce salicylate-mediated behavioral disturbances. NR2B was significantly upregulated in salicylate-treated cells, but downregulated after memantine treatment. Similarly, expression of the inflammatory cytokine genes TNFα and immediate-early gene ARC was significantly increased in the salicylate-treated cells, and decreased when the cells were treated with memantine. These results were confirmed by NR2B immunocytochemistry. GPIAS was attenuated to a significantly lesser extent in rats treated with a combination of salicylate and memantine than in those treated with salicylate only. The mean ABR threshold in both groups was not significant different before and 1 day after the end of treatment. Additionally, NR2B protein expression in the auditory cortex was markedly increased in the salicylate-treated group, whereas it was reduced in the memantine-treated group. These results indicate that memantine is useful for the treatment of salicylate-induced tinnitus.
Acoustics ; Animals ; Auditory Cortex ; Brain Stem ; Evoked Potentials, Auditory, Brain Stem ; Genes, Immediate-Early ; Glutamic Acid ; Immunohistochemistry ; Integrin alpha2 ; Memantine ; Models, Animal ; N-Methylaspartate ; Neurons ; Noise ; Prepulse Inhibition ; Rats ; Reflex, Startle ; Tinnitus ; Tumor Necrosis Factor-alpha

OBJECTIVETo screen the learning and memory mutant from N-ethyl-N-nitrosourea (ENU) mutagenic zebrafish F1, and to get the new model animal to study the mechanism of learning and memory.

METHODSZebrafish mutant was screened by inhibitory avoidance behavioral test and identified by the expression of gene c-fos with qRT-PCR.

RESULTSWe isolated a zebrafish mutant related to learning and memory, fgt. In this fgt zebrafish mutant long-term memory was much lower than that in wild-type when tested at 24 h after training. The 24 h long-term memory in about half of fgt mutant F2 (13/30) were significantly lower than those in wild-type, and the others relatively normal. Compared with the expression in wild-type fishes, the expression of immediate-early genes (IEGs) c-fos in half of fgt mutant F2 (13/30) after exploring in a novel environment increased distinctly from the basal control levels statistically, and the others relatively normal, which were in accordance with the behavioral results.

CONCLUSIONThe zebrafish mutant fgt is a dominant mutant with defect in long-term memory.

Animals ; Disease Models, Animal ; Female ; Genes, Immediate-Early ; Genetic Testing ; Male ; Memory, Long-Term ; Zebrafish ; genetics

OBJECTIVEThe effect of allitridin on the transcription levels of immediate-early (ie), early(e) and late (1) genes of human cytomegalovirus (HCMV) was investigated in order to explore the mechanism of allitridin against HCMV.

METHODEstablished the models of HCMV AD169 strain infected cells and AD169 strain infected cells treated with allitridin (9.6 mg x L(-1)), and they were compared with the appropriate dose(2.3 mg x L(-1)) of ganciclovir (GCV). All groups of cells were infected at 2.5 multiplicity of infection (MOI), using SYBR Green real-time PCR method to detect the dynamic change of ul122, ul123, ul54 and ul83 mRNA expression at 0.5, 2, 4, 6, 12, 24 h post-infection.

RESULTThe mRNA levels of ul122 and ul123 in AD169 infected cells treated with allitridin at all time points were markedly lower than those of AD169 infected controls (P<0.05), but there were no significant difference of ul122 gene in AD169 infected cells treated with GCV and AD169 infected cells at 0.5-6 h post-infection. The inhibitory rates of allitridin to AD169 ul122 and ul123 mRNA reached 75.2% and 70.4% at 24 h post-infection, respectively. The expression of ul54 mRNA in two drug-treatment groups at all time points were lower than those of AD169 infected cells group (P<0.05). The inhibitory rates of alltridin and GCV to AD169 ul54 mRNA were 45.4% and 27.2% at 24 h post-infection,respectively. The expression of HCMV ul83 mRNA in all groups rapidly increased after 6 h of infection,which is most obvious in AD169 infected cells group. The inhibitory rates of alltridin and GCV to AD169 ul83 mRNA were 45.9% and 26.2% at 24 h post-infection, respectively.

CONCLUSIONAllitridin could effectively suppress the transcription of ie genes (ul122 and ul123) of HCMV AD169 strain, led the expression of mRNA significantly lowerd. It was able to supress the transcription of egene (ul54) and l gene (ul83) too, indicating that HCMV ie genes may be the key target of allitridin against HCMV.

Allyl Compounds ; pharmacology ; Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; genetics ; Genes, Immediate-Early ; genetics ; Genes, Viral ; genetics ; Humans ; Sulfides ; pharmacology ; Transcription, Genetic ; drug effects

OBJECTIVETo study influence of lanthanum chloride (LaCl(3)) on the expression of immediate early genes (IEGs) including c-jun, early growth response gene 1 (Egr1) and activity-regulated cytoskeletal gene (Arc) in the hippocampus of rats, and discuss the mechanism of LaCl(3) undermining learning and memory capability.

METHODSForty female Wistar adult rats were divided into control group, low LaCl(3)-contaminated group (0.25%), medium LaCl(3)-contaminated group (0.50%), and high LaCl(3)-contaminated group (1.00%) by randomized design. Each group had ten female rats along with five male rats and mated by the ratio of 2:1. The amounts of pups in the above four groups were 80, 83, 78 and 75 separately. The pups in respective group were La-dyed by lactation, and then the pups in LaCl(3)-contaminated groups drank 0.25%, 0.50% and 1.00% LaCl(3) separately for one month. Learning and memory capability of pups were measured in jumping stairs experiment. Hippocampal lanthanum content was determined by inductively coupled plasma mass spectrometry (ICP-MS). Hippocampal c-jun, Egr1 and Arc mRNA expression was detected by RT-PCR, and corresponding protein expression was measured by Western blotting method.

RESULTSIn the jumping stairs experiment, pups in 0.25%, 0.50% and 1.00% LaCl(3)-contaminated groups respectively made (1.75 ± 0.71), (2.38 ± 0.92) and (3.00 ± 0.76) mistakes; significantly higher than control group (1.25 ± 0.46) (q values were 4.386, 6.793, P < 0.05). However, the incubation period of 0.25%, 0.50% and 1.00% LaCl(3)-contaminated groups were (174.13 ± 33.72), (139.25 ± 45.83) and (75.50 ± 18.56) respectively, which were all significantly lower than that of control group (206.75 ± 20.47) (q values were 2.958, 6.121, 11.902, P < 0.05). Hippocampal c-jun mRNA expression were (0.89 ± 0.08), (0.77 ± 0.12), (0.58 ± 0.14) and (0.29 ± 0.10); while the c-jun protein expression were (0.72 ± 0.13), (0.64 ± 0.11), (0.43 ± 0.11) and (0.31 ± 0.14), and the Egr1 mRNA expression were (0.78 ± 0.09), (0.61 ± 0.13), (0.53 ± 0.10) and (0.22 ± 0.08), Egr1 protein expression were (0.65 ± 0.18), (0.40 ± 0.15), (0.32 ± 0.13) and (0.14 ± 0.09) in 0.25%, 0.50% and 1.00% LaCl(3)-contaminated groups; and all of which presented a dose-effect relationship that the correlation coefficients of these parameters with dose were -0.900 (t = 11.309, P = 0.000), -0.969 (t = 7.058, P = 0.000), -0.898 (t = 11.179, P = 0.000) and -0.962 (t = 6.739, P = 0.000).

CONCLUSIONLaCl(3) undermines the learning and memory capability of rats, which is possibly related to lower expression of c-jun and Egr1 gene and protein induced by lanthanum in hippocampus.

Animals ; Early Growth Response Protein 1 ; metabolism ; Female ; Gene Expression ; Genes, Immediate-Early ; drug effects ; genetics ; Hippocampus ; drug effects ; metabolism ; Lanthanum ; pharmacology ; Learning ; drug effects ; Male ; Memory ; drug effects ; Proto-Oncogene Proteins c-jun ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of RNA interference targeting human cytomegalovirus immediate early gene 1 (HCMV- IE1) on the gene expression in vitro.

METHODSAccording to the sequence of HCMV-IE1 gene, the small interfering RNA (siRNA) sequences were designed and introduced into the eukaryotic expression vector containing the U6 promoter. After verification by sequence analysis, the recombinant eukaryotic plasmid (pHCMV-IE1i) was transfected into HEL HCMVAD169 cells. The effectiveness of HCMV-IE1 gene silencing was investigated by fluorescence microscopy, flow cytometry and RT-PCR.

RESULTSSequence analysis confirmed successful construction of the recombinant eukaryotic expression plasmid pHCMV-IE1i. The expression of HCMV-IE1 was effectively suppressed by pHCMV-IE1i transfection in HEL cells as shown by fluorescence microscopy, flow cytometry (P < 0.05) and RT-PCR (P < 0.05).

CONCLUSIONThe expression of HCMV-IE1 can be effectively suppressed by RNA interference technique in vitro, which provides experimental data for prevention and treatment of HCMV infection.

Antigens, Viral ; biosynthesis ; genetics ; Cell Line ; Genes, Immediate-Early ; Genetic Vectors ; genetics ; Humans ; Immediate-Early Proteins ; biosynthesis ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

BACKGROUND: General anesthetics were known to induce expression of immediate early genes (IEGs), including c-fos and c-jun. However, mechanisms of IEG induction by general anesthetics were not fully understood. METHODS: IEG induction by propofol, a kind of intravenous anesthetics, and signal transduction pathways for propofol-induced IEG expression were investigated in human neuroblastoma cell line IMR32 and CHP134 with Northern and Western blot analysis. RESULTS: Cell viability was significantly decreased in IMR32 and CHP134 treated with increasing concentrations of propofol. IMR32 was more sensitive to propofol-induced cytotoxicity than CHP134. Propofol did not affect the cell cycle profile of IMR32. Expression of cyclin A, cyclin B1, CDK4 and CDK6 was increased in IMR32 by propofol treatment in a time-dependent manner. However, expression of cyclin A and CDK4 was decreased in CHP134. Proliferating cell nuclear antigen (PCNA) was increased in both IMR32 and CHP134 treated with propofol from 6 h to 24 h. c-fos and c-jun were induced by propofol treatment in both cells. Propofol also induced extracellular signal-regulated kinase (ERK) phosphorylation in both cells. Pretreatment of PD98059, an MEK inhibitor, blocked propofol-induced c-fos and c-jun expression.Propofol treatment was decreased nuclear transcription factor-kappa B (NF-kappa B) expression in IMR32, but not in CHP134. CONCLUSIONS: Propofol-induced c-fos expression might be mediated through ERK phosphorylation in both IMR32 and CHP134. Propofol-induced cytotoxicity, changes in expressions of cell cycle regulatory proteins, expression of IEGs, ERK phosphorylation, and NF-kappa B expression were different between IMR32 and CHP134.
Anesthetics, General ; Anesthetics, Intravenous ; Blotting, Western ; Cell Cycle ; Cell Cycle Proteins ; Cell Line ; Cell Survival ; Cyclin A ; Cyclin B1 ; Cyclins ; Flavonoids ; Gene Expression ; Genes, Immediate-Early ; Humans ; Neuroblastoma ; NF-kappa B ; Phosphorylation ; Phosphotransferases ; Proliferating Cell Nuclear Antigen ; Propofol ; Signal Transduction

It has been demonstrated that some of immediate early genes (IEGs) such as c-Jun or fos are induced immediately following neuronal injury and they play an important role in determining the fate of the injured neurons. Of IEGs, the activating transcription factor 3 (ATF3) is focused by many investigators, because they are expressed in various types of neural insults and have been known to serve a diverse function in both neuronal survival and death. However, little is known about the functional role of ATF3 in ischemic brain injury. So in this study, the authors examined the expression pattern of the activating transcription factor 3 (ATF3) following middle cerebral artery (MCA) occlusion-reperfusion injury. According to the findings obtained by triphenyltetrazolium chloride (TTC) stains, the authors have classified the infarcted area into two regions, the ischemic core region and the ischemic penumbra region. In both regions, many neurons underwent neuronal degeneration, characterized by the shrunken nuclei with eosinophilic perikaryon. The H & E stain also demonstrated the increased number of probable activated astrocytes and microglia in the ischemic brain regions and this was confirmed by GFAP- and OX42-immunohistochemistry. Immunohistochemical study for ATF3 also demonstrated the specific upregulation of ATF3 in the nuclei of neurons under ischemic injury, but not in those of the contralateral regions. Interestingly, the number of the ATF3 positive neurons in the ischemic penumbra regions outnumbered that of the ischemic core regions. Based on many reports that the neuronal death in ischemic penumbra region is caused by programed cell death rather than by necrosis which is main cause of neuronal death in ischemic core region, our results could suggest that the ATF3 is an important IEGs which determine the fate of the ischemic neurons.
Activating Transcription Factor 3 ; Astrocytes ; Brain ; Brain Injuries ; Brain Ischemia ; Cell Death ; Coloring Agents ; Eosinophils ; Genes, Immediate-Early ; Humans ; Microglia ; Middle Cerebral Artery ; Necrosis ; Neurons ; Research Personnel ; Tetrazolium Salts ; Up-Regulation

OBJECTIVE: To characterize the antisense c-fos oligonucleotides that control the expression of immediate-early gene c-fos in retina in order to better understand the mechanism by which antisense c-fos oligonucleotides induced myopia. In this study the signal transduction in the pathway linking visual experience and the regulation of the eye's growth was investigated. METHODS: Thirty-one 3-week guinea pigs were assigned into 3 groups: antisense and sense c-fos oligonucleotides were intravitreally injected every 3 days to the eyes of the experimental guinea pigs at different concentrations; and saline vehicle to control guinea pigs in the same way. The refraction and axial length of the eyes were measured before and after the treatment, and the immediate-early gene c-fos expression in the retina was quantified by immunohistochemistry and RT-PCR. RESULTS: The moderate myopia was induced in high (1 nmol) and low (0.1 nmol) level of antisense c-fos oligonucleotide intravitreous injection (-5.425 D and -5.575 D, respectively) compared with the control ateral eyes. The refraction and axial length of the treated eyes increased, and the expression of immediate-early gene c-fos decreased significantly in the antisense c-fos oligonucleotides intravitreously injected eyes compared with the sense c-fos oligonucleotide intravitreously and saline vehicle injected eyes (P<0.01). The refraction and axial length were of no statistically significant differences among the sense c-fos oligonucleotides-treated eyes and saline-treated eyes and non-treated eyes (P>0.05). CONCLUSION The obvious myopia can be induced by antisense c-fos oligonucleotides in guinea pigs; antisense c-fos oligonucleotides inhibit c-fos expression in the retina. Immediate-early gene c-fos may be a potential factor in the prevention of myopia and plays an important role in the signal transduction of the retina.
Animals ; Genes, Immediate-Early ; genetics ; Guinea Pigs ; Immunohistochemistry ; Microinjections ; Myopia ; chemically induced ; genetics ; physiopathology ; Oligonucleotides, Antisense ; administration & dosage ; genetics ; toxicity ; Proto-Oncogene Proteins c-fos ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Retina ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; physiology

In order to investigate the mechanism of mechanical stress-mediated arterial remodeling, we studied the pressure-induced expression of immediate-early response gene product c-Jun in common carotid arteries in rats. The common carotid arteries were perfused with both high pressure (160 mmHg) and normal pressure (80 mmHg) for 0.5, 1, 3 and 6 hours. Expression of immediate-early response gene product c-Jun in the arteries was examined by immunohistochemistry and computer image processing. c-Jun was weakly expressed at 1 h, then increased at 3 h and 6 h after exposure of the arteries to normal pressure. Positive immunohistochemical product of c-Jun appeared in the arteries at 0.5 h after the onset of high pressure, then it increased markedly till 6 h. There was significant difference between the two groups. These results indicate that expression of c-Jun of the arteries can be induced by pressure, which may play an important role in mechanical stress-mediated arterial remodeling.
Animals ; Biomechanical Phenomena ; Carotid Artery, Common ; cytology ; metabolism ; physiology ; Genes, Immediate-Early ; Male ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Pressure ; Proto-Oncogene Proteins c-jun ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical