OBJECTIVE: To investigate the role of α7-nicotinic acetylcholine receptor (α7-nAChR) in the regulation of neuronal activity and expression of synapse-related proteins in the thalamic reticular nucleus (TRN) of insomnia rats treated by acupuncture. METHODS: A total of 36 male Sprague-Dawley (SD) rats of clean grade were randomly divided into a control group, a model group, an acupuncture group, and an acupuncture+antagonist group, with 9 rats in each group. The model group, the acupuncture group, and the acupuncture+antagonist group were treated with intraperitoneal injection of p-chlorophenylalanine (PCPA) to establish insomnia model. After successful modeling, the acupuncture group and the acupuncture+antagonist group received acupuncture at bilateral Neiguan (PC6) and Zusanli (ST36) once daily for 5 consecutive days. Thirty min before each acupuncture session, the acupuncture+antagonist group was intraperitoneally injected with methyllycaconitine citrate (MLA), an α7-nAChR antagonist, at a dosage of 5 mg/kg while the acupuncture group received the same volume of 0.9% sodium chloride solution. The rats' daytime spontaneous activity was observed. Neuronal discharge in the TRN was detected using neuroelectrophysiological methods. Immunofluorescence staining was used to detect parvalbumin-positive (PV⁺) neurons and co-expression of PV⁺ and postsynaptic density protein-95 (PSD-95) in the TRN. RESULTS: Compared with the control group, the model group showed increased daytime spontaneous activity (P<0.01); decreased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.01), prolonged inter-discharge intervals (P<0.01) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). Compared with the model group, the acupuncture group showed decreased daytime spontaneous activity (P<0.01); increased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); increased neuronal discharge frequency (P<0.01), shortened inter-discharge intervals (P<0.01) in the TRN; increased number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.05). Compared with the acupuncture group, the acupuncture+antagonist group exhibited increased daytime spontaneous activity (P<0.01); reduced average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.05), prolonged inter-discharge intervals (P<0.05) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). CONCLUSION α7-nAChR are involved in mediating the regulatory effect of acupuncture on circadian rhythm disturbances in PCPA-induced insomnia rats. Blocking α7-nAChR attenuates the activating effect of acupuncture on TRN neurons, and reduces the expression of PSD-95 protein on GABAergic neurons.
Animals ; Male ; Acupuncture Therapy ; alpha7 Nicotinic Acetylcholine Receptor/genetics* ; Rats, Sprague-Dawley ; Rats ; Sleep Initiation and Maintenance Disorders/physiopathology* ; Neurons/metabolism* ; Humans ; Thalamic Nuclei/physiopathology* ; Acupuncture Points ; Disks Large Homolog 4 Protein

OBJECTIVES: To investigate the effects of hyperoxia on the expression of N-methyl-D-aspartate receptor 1 (NMDAR1) and its synapse-associated molecules, including cannabinoid receptor 1 (CB1R), postsynaptic density 95 (PSD95), and synapsin (SYN), in the hippocampus of neonatal rats. METHODS: One-day-old Sprague-Dawley neonatal rats were randomly divided into a hyperoxia group and a control group (n=8 per group). The hyperoxia group was exposed to 80% ± 5% oxygen continuously, while the control group was exposed to room air, for 7 days. At 1, 3, and 7 days after hyperoxia exposure, hematoxylin and eosin (HE) staining was used to observe histopathological changes in the brain. The expression levels of NMDAR1, CB1R, PSD95, and SYN proteins and mRNAs in the hippocampus were detected by immunohistochemistry, Western blotting, and quantitative real-time PCR. RESULTS: After 7 days of hyperoxia exposure, the hyperoxia group showed decreased neuronal density and disordered arrangement in brain tissue. Compared with the control group, after 1 day of hyperoxia exposure, CB1R mRNA and both NMDAR1 and CB1R protein expression in the hyperoxia group were significantly downregulated, while SYN protein expression was significantly upregulated (P<0.05). After 3 days, mRNA expression of NMDAR1, CB1R, and SYN was significantly decreased (P<0.05); NMDAR1 and CB1R protein expression was significantly downregulated (P<0.05), while PSD95 and SYN protein expression was significantly upregulated (P<0.05). After 7 days of hyperoxia, the protein expression of NMDAR1 and CB1R was significantly upregulated (P<0.05). CONCLUSIONS Continuous hyperoxia exposure induces time-dependent changes in the expression levels of NMDAR1 and its synapse-associated molecules in the hippocampus of neonatal rats.
Animals ; Receptors, N-Methyl-D-Aspartate/genetics* ; Rats, Sprague-Dawley ; Hippocampus/pathology* ; Rats ; Animals, Newborn ; Receptor, Cannabinoid, CB1/genetics* ; Hyperoxia/metabolism* ; Disks Large Homolog 4 Protein/genetics* ; Synapsins/genetics* ; Synapses ; Male ; Female ; RNA, Messenger/analysis*

OBJECTIVES: To explore the neuroprotective mechanism of electroacupuncture at the acupoints Baihui and Shenting in rats with cerebral ischemia-reperfusion (IR) injury. METHODS: Forty-eight male SD rats were equally randomized into sham operation group, cerebral IR model group, acupoint electroacupuncture group and non-acupoint acupuncture group. In the latter 3 groups, cerebral focal ischemic injury was induced using the Longa method; in the two electroacupuncture groups, electroacupuncture was performed either at the acupoints Baihui and Shenting or at non-acupoint sites for 7 days. The changes in neurological deficit scores, cerebral infarction volume, learning and memory function, pathologies in hippocampal CA1 area, neuronal and synaptic ultrastructures, and synaptic density of the rats were observed, and serum GABA level and mRNA and protein expressions of GABA_AR α1, CaMK II, SYN1 and PSD-95 in the hippocampal tissue were detected. RESULTS: Compared with those in cerebral IR model group, the rats receiving electroacupuncture at the acupoints, but not those with electroacupuncture at the non-acupoints, showed significantly decreased neurological deficit scores and cerebral infarction volume with shortened escape latency and increased platform crossings. Electroacupuncture at the acupoints significantly increased neuronal cell number, decreased the width of the synaptic gaps and increased density of synaptic bodies in the ischemic hippocampal CA1 area, resulting also in increased serum GABA levels and hippocampal expressions of GABA_ARα1, SYN1 and PSD-95 and lowered expression level of CaMK II. CONCLUSIONS Electroacupuncture at Baihui and Shenting improves learning and memory function of rats with cerebral IR injury possibly through a mechanism that promotes synaptic regeneration, upregulates hippocampal expressions of GABAAR α 1, SYN1 and PSD-95 and downregulates the expression of CaMK II.
Animals ; Electroacupuncture ; Reperfusion Injury/therapy* ; Male ; Rats ; Rats, Sprague-Dawley ; Brain Ischemia/therapy* ; Hippocampus/metabolism* ; Memory ; Learning ; Disks Large Homolog 4 Protein/metabolism* ; Synapses ; Acupuncture Points ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; Receptors, GABA-A/metabolism* ; gamma-Aminobutyric Acid/metabolism*

To investigated the mechanisms underlying the effects of modified Kaixin San(MKXS) on improving memory and synaptic damage of Alzheimer's disease(AD) mouse model with conditional presenilin 1/2 conditional double knockout(PS cDKO). Specifically, 60 PS cDKO mice(3-3.5 months old) and their age-matched wild-type(WT) littermates were randomized into three groups: WT group(n=20), PS cDKO group(n=20), and PS cDKO+MKXS group(n=20). Mice in WT and PS cDKO groups were fed with standard chow and those in PS cDKO+MKXS group were given chow containing MKXS(at 2.55 g·kg~(-1)) for 60 days. Novel object reco-gnition task was employed to detect the recognition memory of mice, and Western blot to detect the protein levels of synapse-associated proteins in the hippocampus(HPC) of mice, such as NR1, NR2 A, NR2 B, p-αCaMKⅡ, tau, and p-tau. Microglial morphology in the HPC CA1 of mice was observed based on immunohistochemistry. Quantitative real time-PCR(qRT-PCR) was employed to detect the mRNA levels of the pro-inflammatory factors and synapse-associated proteins in the HPC of mice, including COX-2, iNOS, IL-1β, IL-6, TNF-α, PSD95, NR1, NR2 A, NR2 B, and MAP2. The protein levels of IL-1β, TNF-α, and IL-6 were tested by enzyme-linked immunosorbent assay(ELISA). The interaction between PSD95 and αCaMKⅡ and between PSD95 and p-αCaMKⅡ was tested by co-immunoprecipitation(Co-IP). The results showed that PS cDKO+MKXS demonstrated significantly higher preference index and recognition index of the new objects, lower protein level of p-tau(ser 396/404) and mRNA levels of COX-2, iNOS, TNF-α, IL-1β, and IL-6 in HPC, higher protein levels of NR1, NR2 A, NR2 B, and p-αCaMKⅡ and mRNA levels of NR1, NR2 A, NR2 B, PSD95, and MAP2, and stronger interaction of αCaMKⅡ with PSD95 and interaction of p-αCaMKⅡ with PSD95 than the PS cDKO group. Immunohistoche-mical staining showed that MKXS inhibited the activation of microglia. In conclusion, MKXS improves memory and synaptic damage in mice with AD by modulating αCaMKⅡ-PSD95 protein binding through inhibition of neuroinflammation.
Animals ; Mice ; Alzheimer Disease/genetics* ; Disks Large Homolog 4 Protein/metabolism* ; Neuroinflammatory Diseases ; Tumor Necrosis Factor-alpha/metabolism* ; Cyclooxygenase 2/metabolism* ; Interleukin-6/metabolism* ; Protein Binding ; Mice, Knockout ; Hippocampus/metabolism* ; Disease Models, Animal ; RNA, Messenger/metabolism*

OBJECTIVE: To observe the expression of discs large homolog 4 (DLG4) protein in hippocampus, amygdala and frontal cortex of rats and evaluate postsynaptic density in heroin dependence. METHODS: The rat heroin dependent model was established by increasing intraperitoneal injection of heroin. DLG4 proteins in hippocampus, amygdala and frontal cortex of heroin dependent 9, 18, 36 days rats were detected with immunohistochemical staining and compared with that in the control group. RESULTS: DLG4 proteins in hippocampus, amygdala and frontal cortex were gradually reduced with extension of heroin dependent time. CONCLUSION Heroin dependence can affect postsynaptic density of hippocampus, amygdala and frontal cortex. The changes become more apparent with extension of heroin dependence time.
Amygdala/metabolism* ; Animals ; Disks Large Homolog 4 Protein ; Frontal Lobe/metabolism* ; Heroin/pharmacology* ; Heroin Dependence ; Hippocampus/metabolism* ; Injections, Intraperitoneal ; Intracellular Signaling Peptides and Proteins/metabolism* ; Membrane Proteins/metabolism* ; Rats

OBJECTIVETo investigate the effect of cerebral X-ray irradiation on learning and memory function in young rats.

METHODSFifty-four SD rats aged 35 d were randomly divided into 3 groups with 18 in each group: rats in 3-d group and 7-d group received X-ray irradiation with a dose of 28.5 mGy/d for 3 d and 7 d, respectively; rats in control group received sham X-ray irradiation. Morris water maze (MWM) was tested when animals at age of 60 d; then the animals were sacrificed and brain samples were taken. The neurodegeneration was observed by Fluro-Jade B staining; the expression of N-methyl-aspartate (NMDA) receptors subunit 2B (NR2B) and postsynaptic density protein-95 (PSD-95) in the hippocampus were analyzed by immunofluorescence and Western blot methods, respectively, and ultrastructure of CA1 region was observed with electron microscopy.

RESULTSNo significant difference in 1-4 d escape latency as shown in MWM test was noted between 3d group and control group (P>0.05); while the escape latency in 7d group was significantly longer than that in control group (P<0.01). No significant differences in lingering in the quadrant and the frequency of passing through the original platform between 3-d group and control group (P>0.05), while those in 7-d group were significantly lower than those in control group (P<0.01). Compared to control group, the number of FJB positive cells in 7-d group was increased (P<0.01); the expressions of NR2B and PSD-95 in hippocampus CA1 region were also increased (P<0.05). The ultrastructure observation in 7-d group showed that the synapse structure of some neurons was impaired.

CONCLUSIONX-ray irradiation may affect learning and memory function of young rats, which is associated with overexpression of NR2B and PSD-95 in hippocampal regions.

Animals ; Brain ; radiation effects ; CA1 Region, Hippocampal ; metabolism ; radiation effects ; Disks Large Homolog 4 Protein ; Intracellular Signaling Peptides and Proteins ; metabolism ; Learning ; radiation effects ; Membrane Proteins ; metabolism ; Memory ; radiation effects ; Neurons ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; metabolism ; Synapses ; X-Rays

OBJECTIVETo explore the effect mechanism of electroacupuncture (EA) at Changqiang (GV 1) or Baihui (GV 20) on autism based on molecular biology.

METHODSThe autism model was established by intraperitoneal injection of sodium valproate (VPA) in Wistar pregnant rats. Forty young rats with autism were selected and randomly divided into a model group, a non-acupoint group, an electroacupuncture at "Changqiang" (GV 1) (EAGV 1 for short) group and an electroacupuncture at "Baihui" (GV 20) (EAGV 20 for short) group. Another 10 normal young rats were selected as a blank group. In the EAGV 1 group, acupuncture was applied at Houhai [as Changqiang (GV 1)], then EA apparatus was connected with continuous wave, 2 Hz, 20 min, once a day for consecutive 20 days. The same EA manipulation as EAGV 1 group was used in the EAGV 20 group where "Baihui" (GV 20) was selected and non-acupoint group where non-acupoint in the right rib was selected. Blank group and model group were reared under the same conditions without any intervention. The escape latency and the ratio of swimming distance in platform quadrant to total swimming distance in each group were observed by using Morris water maze, and the PSD-95 protein expression in hippocampal CA 1 was measured by immunohistochemical techniques.

RESULTSCompared with the blank group, the escape latency in the model group and the non-acupoint group lengthened (both P < 0.05), the ratio of swimming distance in platform quadrant to total swimming distance was decreased (both P < 0.05), the PSD-95 protein expression was decreased (P < 0.05). Compared with the model group, the escape latency in the EAGV 1 group and the EAGV 20 group were decreased (both P < 0.05), the ratio of swimming distance in platform quadrant to total swimming distance was increased, the PSD-95 protein expression was increased (both P < 0.05). But the escape latency, the ratio of swimming distance in platform quadrant to total swimming distance and the PSD-95 protein expression had no significant difference between EAGV 1 group and EAGV 20 group (P > 0.05).

CONCLUSIONElectroacupuncture at Changqiang (GV 1) or Baihui (GV 20) can respectively improve learning and memory ability of rats with autism, which has no significant difference and the mechanism of action may be related to regulation of the PSD-95 protein expression.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Autistic Disorder ; genetics ; metabolism ; psychology ; therapy ; Disks Large Homolog 4 Protein ; Electroacupuncture ; Female ; Hippocampus ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Learning ; Male ; Membrane Proteins ; genetics ; metabolism ; Memory ; Rats ; Rats, Wistar

OBJECTIVETo observe the effect of curcumin on the expression of synapse-related proteins PSD-95 and Shank1 in APP/PS1 double transgenic mice.

METHODThree-month-old APP/PS1 dtg mice were randomly divided into the model group, the positive Rosiglitazone control group and curcumin high (400 mg x kg(-1) x d(-1)), medium (200 mg x kg(-1) x d(-1)) and low (100 mg x kg(-1) x d(-1)) dose groups, with non-genetically modified mice with the same background as the normal group. After the oral administration for three months, immunohistochemistry and Western blot were adopted for detection.

RESULTAccording to the behavioral detection, the treatment group and the model group showed differences in the place navigation test and the spatial probe test to varying degrees (P < 0.01 or P < 0.05). The expression of PSD-95 and Shank1-positive cells of hippocampus CA1 region significantly decreased in model mice compared with normal control group (P < 0.01); while the curcumin intervention group showed recovery to some extend. Western blot results showed that the strap of PSD-95 protein expression became significantly thinner and lighter in the model group compared with the normal control group (P < 0.01); while the curcumin intervention group showed notably thicker and darker straps of PSD-95 protein expression (P < 0.05).

CONCLUSIONCurcumin can increase the expression of synapse-related proteins PSD95 and Shank1 in APP/PS1 double transgenic mice, improve structure and plasticity of synapse in APP/PS1 double transgenic mice and enhance their learning and memory abilities.

Amyloid beta-Protein Precursor ; genetics ; Animals ; CA1 Region, Hippocampal ; drug effects ; metabolism ; Curcumin ; pharmacology ; Disks Large Homolog 4 Protein ; Gene Expression Regulation ; drug effects ; Guanylate Kinases ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Mice, Transgenic ; Nerve Tissue Proteins ; metabolism ; Presenilin-1 ; genetics ; Synapses ; drug effects ; metabolism

OBJECTIVETo observe the effects of PSD95 gene specific siRNAs on neuropathic pain relief, neuron viability, and postsynaptic calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) phosphorylation in vitro and in vivo.

METHODSGene-specific siRNAs of rat PSD95 were synthesized chemically for transfection. Adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups: naïve group (n=6), sham group (n=6), and sciatic nerve chronic constriction injury (CCI) group (n=24). The CCI group was further divided into 4 groups (n=6 in each group), which were pretreated with normal saline, transfection vehicle, negative control siRNAs, and PSD95 gene specific siRNAs respectively. All the subgroups received corresponding agents intrathecally for 3 days, started one day before the CCI of sciatic nerve. Both mechanical allodynia and thermal hyperalgesia were measured on post-operative day 3 and 7. PSD95 gene silenced NG108-15 cells were further stimulated by glutamate, with the cell viability and the expression/phosphorylation of CaMKIIα measured by MTT cell proliferation assay and Western blot, respectively.

RESULTSThe siRNAs decreased PSD95 mRNA level significantly both in vivo and in vitro. Neuropathic pain rats pretreated with PSD95 gene specific siRNAs exhibited significant elevation in the mechanical withdrawal threshold and paw withdrawal thermal latency, without affecting the baseline nociception. PSD95 gene silencing enhanced neuronal tolerance against the glutamate excitotoxicity, meanwhile the phosphorylation of CaMKIIα Thr286 was attenuated.

CONCLUSIONPre-emptive administration of PSD95 gene specific siRNAs may attenuate the central sensitization CaMKIIα-related signaling cascades, leading to the relief of neuropathic pain.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cells, Cultured ; Disks Large Homolog 4 Protein ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Membrane Proteins ; genetics ; Neuralgia ; therapy ; Neurons ; physiology ; Phosphorylation ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley

To detect the effect of PDZ1, domain of postsynaptic density 95 (PSD-95), on apoptosis of hippocampal neurons induced by oxygen-glucose deprivation (OGD), Sprague-Dawley rat hippocampal neurons were infected with PDZ1-viruses after 21 days of plating. Twenty-four hours after infection, cells were treated with OGD for 1.5 h, then were incubated with DAPI and apoptosis-like cells were characterized, or were collected for co-immunoprecipitation and Western blot analyses. The results showed that: (1) PDZ1 overexpression was observed in hippocampal neurons; (2) Apoptosis induced by OGD was obviously decreased in neurons overexpressing PDZ1 (P<0.05); (3) Overexpression of PDZ1 prevented the binding of GluR6 to PSD-95; (4) Overexpression of PDZ1 inhibited MLK3 and JNK1/2 activation induced by OGD. These results indicate that overexpression of PDZ1 may prevent hippocampal neurons from apoptosis induced by OGD.
Animals ; Apoptosis ; Cells, Cultured ; Culture Media ; chemistry ; Disks Large Homolog 4 Protein ; Glucose ; chemistry ; Hippocampus ; cytology ; Intracellular Signaling Peptides and Proteins ; metabolism ; Membrane Proteins ; metabolism ; Neurons ; cytology ; pathology ; Oxygen ; chemistry ; PDZ Domains ; Rats ; Rats, Sprague-Dawley