GluR1 Expression Research Articles

Autophagy-regulated AMPAR subunit upregulation in in vitro oxygen glucose deprivation/reoxygenation-induced hippocampal injury

Autophagy has been implicated to mediate experimental cerebral ischemia/reperfusion-induced neuronal death; the underlying molecular mechanisms, though, are poorly understood. In this study, we investigated the role of autophagy in regulating the expression of AMPAR subunits (GluR1, GluR2, and GluR3) in oxygen glucose deprivation/reperfusion (OGD/R)-mediated injury of hippocampal neurons. Our results showed that, OGD/R-induced hippocampal neuron injury was accompanied by accumulation of autophagosomes and autolysosomes in cytoplasm alongside a dramatic increase in expression of autophagy-related genes, LC3 and Beclin 1 and increased intracellular Ca2+ levels. Pre-treatment with autophagy inhibitor 3-methyladenine (3-MA) significantly reduced this effect. Moreover, the OGD/R-induced upregulation of mRNA and protein expressions of GluR1, GluR2, and GluR3 were also effectively reversed in cells pretreated with 3-MA. Our findings indicate that OGD/R induced the expression of GluRs by activating autophagy in in vitro cultured hippocampal neurons, which could be effectively reversed by the administration of 3-MA.

Behavioral alterations are associated with vitamin B12 deficiency in the transcobalamin receptor/CD320 KO mouse.

Vitamin B12 (cobalamin) deficiency is prevalent worldwide and causes megaloblastic anemia and neurologic deficits. While the anemia can be treated, the neurologic deficits can become refractive to treatment as the disease progresses. Therefore, timely intervention is critical for a favorable outcome. Moreover, the metabolic basis for the neuro-pathologic changes and the role of cobalamin deficiency in the pathology still remains unexplained. Using a transcobalamin receptor / CD320 knockout mouse that lacks the receptor for cellular uptake of transcobalamin bound cobalamin, we aimed to determine whether cobalamin deficiency in the central nervous system produced functional neurologic deficits in the mouse that would parallel those observed in humans. Our behavioral analyses indicate elevated anxiety and deficits in learning, memory and set-shifting of a spatial memory task in the KO mouse. Consistent with the behavioral deficits, the knockout mouse shows impaired expression of the early phase of hippocampal long-term potentiation along with reduced expression of GluR1, decreased brain mass and a significant reduction in the size of nuclei of the hippocampal pyramidal neurons. Our study suggests that the CD320 knockout mouse develops behavioral deficits associated with cobalamin deficiency and therefore could provide a model to understand the metabolic and genetic basis of neuro-pathologic changes due to cobalamin deficiency.

Effect of isoflurane preconditioning on expression of hippocampal GluR1 subunits-containing AMPA receptors in a rat model of focal cerebral ischemia-reperfusion

Objective To evaluate the effect of isoflurane preconditioning on the expression of hippocampal GluR1 subunits-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate(AMPA)receptors in a rat model of focal cerebral ischemia-reperfusion(I/R). Methods Forty-eight pathogen-free healthy male Sprague-Dawley rats, weighing 250-280 g, aged 7-8 weeks, were divided into 3 groups(n=16 each)using a random number table: sham operation group(group S), focal cerebral I/R group(group I/R)and isoflurane preconditioning group(group IPC). Focal cerebral I/R was induced by occlusion of the middle cerebral artery for 2 h followed by reperfusion to induce cognitive decline in I/R and IPC groups. Rats were exposed to 1.5% isoflurane for 1 h every day for 5 consecutive days, and the model was established at 24 h after the last exposure in group IPC. Eight rats in each group were selected to perform Morris water maze test for 6 consecutive days starting from 9 or 23 days after operation. The rats were sacrificed at 14 and 28 days after operation, and the hippocampal tissues were obtained for determination of the expression of GluR1 mRNA(by using real-time polymerase chain reaction)and GluR1 protein(by Western blot). Results Compared with group S, the escape latency was significantly prolonged at each time point after operation, the frequency of crossing the original platform quadrant and percentage of swimming distance at the original platform quadrant were decreased at 14 and 28 days after operation, and the expression of GluR1 protein and mRNA was down-regulated at 14 days after operation in I/R and IPC groups(P<0.05). Compared with group I/R, the escape latency was significantly shortened at 10-13 days after operation, the percentage of swimming distance at the original platform quadrant and frequency of crossing the original platform quadrant were increased at 14 days after the operation, and the expression of GluR1 protein and mRNA was up-regulated at 14 days after operation in group IPC(P<0.05). Conclusion The mechanism by which isoflurane preconditioninig improves the cognitive function is related to up-regulation of the expression of hippocampal GluR1 subunits-containing AMPA receptors in a rat model of focal cerebral I/R. Key words: Isoflurane; Ischemic preconditioning; Brain; Reperfusion injury; Hippocampus; Receptors, AMPA

Hippocampal AMPARs involve the central sensitization of rats with irritable bowel syndrome.

ObjectiveThe roles of hippocampal AMPARs were investigated in irritable bowel syndrome (IBS)‐like rats to clarify the central sensitization mechanisms.Methods IBS model was induced by neonatal maternal separation. The effects of AMPARs on visceral hypersensitivity were examined by the responses of abdominal muscle to colorectal distension after the bilateral intrahippocampal injections of CNQX (an AMPAR inhibitor). The expressions of hippocampal AMPARs (GluR1 and GluR2) were determined by Western blot.ResultsThe IBS‐like rats showed visceral hypersensitivity when compared with controls. Bilateral intrahippocampal injections of CNQX alleviated the visceral pain in IBS‐like rats. The maximal effect appeared at the time point of 30 min, and the duration lasted for 90 min after CNQX application, under 40 and 60 mmHg CRD. The expressions of hippocampal GluR2 significantly increased in IBS‐like rats when compared with controls (p < .05). However, the levels of hippocampal GluR1 had no significant differences in rats. Hippocampal LTP induced by HFS was significantly enhanced when compared with controls (p < .05). The expressions of GluR2 significantly increased in the control and IBS‐like rats after 60 min LTP of recordings (p < .05), but not GluR1.ConclusionNeonatal maternal separation enhances the expression of GluR2 and facilitates the LTP in the hippocampus, which could lead to the formation of visceral hypersensitivity when grown up.

Melatonin Alleviates the Epilepsy-Associated Impairments in Hippocampal LTP and Spatial Learning Through Rescue of Surface GluR2 Expression at Hippocampal CA1 Synapses.

Epilepsy-associated cognitive impairment is common, and negatively impacts patients' quality of life. However, most antiepileptic drugs focus on the suppression of seizures, and fewer emphasize treatment of cognitive dysfunction. Melatonin, an indolamine synthesized primarily in the pineal grand, is reported to be neuroprotective against several central nervous system disorders. In this study, we investigated whether melatonin could reverse cognitive dysfunction in lithium-pilocarpine treated rats. Chronic treatment with melatonin (8mg/kg daily for 15 days) after induction of status epilepticus significantly alleviated seizure severity, reduced neuronal death in the CA1 region of the hippocampus, improved spatial learning (as measured by the Morris water maze test), and reversed LTP impairments, compared to vehicle treatment. Furthermore, we found that melatonin rescued the decreased surface levels of GluR2 in the CA1 region observed in epilepsy, which might be the underlying mechanism of the neuroprotective and synapse-modulating function of melatonin. Our study provides experimental evidence for the possible clinical utility of melatonin as an adjunctive therapy to prevent epilepsy-associated cognitive impairments.

Maternal fluoride exposure during gestation and lactation decreased learning and memory ability, and glutamate receptor mRNA expressions of mouse pups.

Previous investigations demonstrated that high fluoride (F) exposure may adversely affect the neurodevelopment and learning and memory ability. However, whether maternal F exposure during gestation and lactation can influence the learning, memory ability, and glutamate receptor expressions of offspring has not yet been elucidated. Hence, in the present study, maternal mice were exposed to F (25, 50, or 100 mg/L sodium fluoride (NaF) in drinking water) during gestation and lactation. Results showed that exposure to 100 mg/L NaF significantly enhanced the number of total arm entries and working memory errors of offspring in the radial arm maze test compared to the control group. However, no difference was observed in open-field behaviors. For the subtypes of glutamate receptors in hippocampus, expression of GluR2 mRNA was significantly reduced by 25, 50, and 100 mg/L NaF. Besides, F exposure also suppressed the expression of NR2A, NR2B, and mGluR2 mRNA levels in a dose-dependent manner, where NR2A was significantly suppressed by 50 mg/L NaF and NR2B and mGluR2 by 100 mg/L NaF. However, no significant changes were observed in GluR1 and mGluR5 mRNA expression levels. Collectively, these findings suggested that F can pass through the cord blood and breast milk and may have deleterious impact on learning and memory of the mouse pups, which was mediated by reduced mRNA expression of glutamate receptor subunits.

Genistein inhibits hypoxia, ischemic-induced death, and apoptosis in PC12 cells.

A hypoxia/ischemia neuronal model was established in PC12 cells using oxygen-glucose deprivation (OGD). OGD-induced neuronal death, apoptosis, glutamate receptor subunit GluR2 expression, and potassium channel currents were evaluated in the present study to determine the effects of genistein in mediating the neuronal death and apoptosis induced by hypoxia and ischemia, as well as its underlying mechanism. OGD exposure reduced the cell viability, increased apoptosis, decreased the GluR2 expression, and decreased the voltage-activated potassium currents. Genistein partially reversed the effects induced by OGD. Therefore, genistein may prevent hypoxia/ischemic-induced neuronal apoptosis that is mediated by alterations in GluR2 expression and voltage-activated potassium currents.

Early-Life Social Isolation-Induced Depressive-Like Behavior in Rats Results in Microglial Activation and Neuronal Histone Methylation that Are Mitigated by Minocycline.

Early-life stress is a potent risk factor for development of psychiatric conditions such as depression. The underlying mechanisms remain poorly understood. Here, we used the early-life social isolation (ESI) model of early-life stress in rats to characterize development of depressive-like behavior, the role of microglia, levels of histone methylation, as well as expression of glutamate receptor subunits in the hippocampus. We found that depressive-like behavior was induced after ESI as determined by sucrose preference and forced swimming tests. Increased expression of microglial activation marker, Iba1, was observed in the hippocampus of the ESI group, while expression of the microglial CD200 receptor, which promotes microglial quiescence, significantly decreased. In addition, increased levels of proinflammatory cytokines, interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were observed in the hippocampus of the ESI group. Moreover, ESI increased levels of neuronal H3K9me2 (a repressive marker of transcription) and its associated "writer" enzymes, G9a and G9a-like protein, in the hippocampus. ESI also decreased expression of hippocampal NMDA receptor subunits, NR1, and AMPA receptor subunits, GluR1 and GluR2, which are involved in synaptic plasticity, but it did not affect expression of PSD95 and NR2B. Interestingly, treatment with minocycline to block microglial activation induced by ESI inhibited increases in hippocampal microglia and prevented ESI-induced depressive-like behavior as well as increases in IL-1β, IL-6, and TNF-α. Notably, minocycline also triggered downregulation of H3K9me2 expression and restored expression of NR1, GluR1, and GluR2. These results suggest that ESI induces depressive-like behavior, which may be mediated by microglial signaling.

Prenatal Exposure to Tributyltin Decreases GluR2 Expression in the Mouse Brain.

Tributyltin (TBT), a common environmental contaminant, is widely used as an antifouling agent in paint. We previously reported that exposure of primary cortical neurons to TBT in vitro decreased the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit glutamate receptor 2 (GluR2) expression and subsequently increased neuronal vulnerability to glutamate. Therefore, to identify whether GluR2 expression also decreases after TBT exposure in vivo, we evaluated the changes in GluR2 expression in the mouse brain after prenatal or postnatal exposure to 10 and 25 ppm TBT through pellet diets. Although the mean feed intake and body weight did not decrease in TBT-exposed mice compared with that in control mice, GluR2 expression in the cerebral cortex and hippocampus decreased after TBT exposure during the prenatal period. These results indicate that a decrease in neuronal GluR2 may be involved in TBT-induced neurotoxicity, especially during the fetal period.

Neuroprotective effects of autophagy inhibition on hippocampal glutamate receptor subunits after hypoxia-ischemia-induced brain damage in newborn rats.

Autophagy has been suggested to participate in the pathology of hypoxic-ischemic brain damage (HIBD). However, its regulatory role in HIBD remains unclear and was thus examined here using a rat model. To induce HIBD, the left common carotid artery was ligated in neonatal rats, and the rats were subjected to hypoxia for 2 hours. Some of these rats were intraperitoneally pretreated with the autophagy inhibitor 3-methyladenine (10 mM in 10 μL) or the autophagy stimulator rapamycin (1 g/kg) 1 hour before artery ligation. Our findings demonstrated that hypoxia-ischemia-induced hippocampal injury in neonatal rats was accompanied by increased expression levels of the autophagy-related proteins light chain 3 and Beclin-1 as well as of the AMPA receptor subunit GluR1, but by reduced expression of GluR2. Pretreatment with the autophagy inhibitor 3-methyladenine blocked hypoxia-ischemia-induced hippocampal injury, whereas pretreatment with the autophagy stimulator rapamycin significantly augmented hippocampal injury. Additionally, 3-methyladenine pretreatment blocked the hypoxia-ischemia-induced upregulation of GluR1 and downregulation of GluR2 in the hippocampus. By contrast, rapamycin further elevated hippocampal GluR1 levels and exacerbated decreased GluR2 expression levels in neonates with HIBD. Our results indicate that autophagy inhibition favors the prevention of HIBD in neonatal rats, at least in part, through normalizing GluR1 and GluR2 expression.

Lead-Induced ERK Activation Is Mediated by GluR2 Non-containing AMPA Receptor in Cortical Neurons.

Lead is a persistent environmental pollutant and exposure to high environmental levels causes various deleterious toxicities, especially to the central nervous system (CNS). The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor that is devoid of the glutamate receptor 2 (GluR2) subunit is Ca2+-permeable, which increases the neuronal vulnerability to excitotoxicity. We have previously reported that long-term exposure of rat cortical neurons to lead acetate induces decrease of GluR2 expression. However, it is not clarified whether lead-induced GluR2 decrease is involved in neurotoxicity. Therefore, we investigated the contribution of GluR2 non-containing AMPA receptor to lead-induced neurotoxic events. Although the expression of four AMPA receptor subunits (GluR1, GluR2, GluR3, and GluR4) was decreased by lead exposure, the decrease in GluR2 expression was remarkable among four subunits. Lead-induced neuronal cell death was rescued by three glutamate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a non-selective AMPA receptor blocker), MK-801 (N-methyl-D-aspartate (NMDA) receptor blocker), and 1-naphthyl acetyl spermine (NAS, a specific Ca2+-permeable AMPA receptor blocker). Lead exposure activated extracellular signal-regulated protein kinase (ERK) 1/2, which was significantly ameliorated by CNQX. In addition, lead exposure activated p38 mitogen-activated protein kinase (MAPK p38), and protein kinase C (PKC), which was partially ameliorated by CNQX. Our findings indicate that Ca2+-permeable AMPA receptors resulting from GluR2 decrease may be involved in lead-induced neurotoxicity.

Persistent gating deficit and increased sensitivity to NMDA receptor antagonism after puberty in a new mouse model of the human 22q11.2 microdeletion syndrome: a study in male mice.

The hemizygous 22q11.2 microdeletion is a common copy number variant in humans. The deletion confers high risk for neurodevelopmental disorders, including autism and schizophrenia. Up to 41% of deletion carriers experience psychotic symptoms. We present a new mouse model (Df(h22q11)/+) of the deletion syndrome (22q11.2DS) and report on, to our knowledge, the most comprehensive study undertaken to date in 22q11.2DS models. The study was conducted in male mice. We found elevated postpubertal N-methyl-D-aspartate (NMDA) receptor antagonist-induced hyperlocomotion, age-independent prepulse inhibition (PPI) deficits and increased acoustic startle response (ASR). The PPI deficit and increased ASR were resistant to antipsychotic treatment. The PPI deficit was not a consequence of impaired hearing measured by auditory brain stem responses. The Df(h22q11)/+ mice also displayed increased amplitude of loudness-dependent auditory evoked potentials. Prefrontal cortex and dorsal striatal elevations of the dopamine metabolite DOPAC and increased dorsal striatal expression of the AMPA receptor subunit GluR1 was found. The Df(h22q11)/+ mice did not deviate from wild-type mice in a wide range of other behavioural and biochemical assays. The 22q11.2 microdeletion has incomplete penetrance in humans, and the severity of disease depends on the complete genetic makeup in concert with environmental factors. In order to obtain more marked phenotypes reflecting the severe conditions related to 22q11.2DS it is suggested to expose the Df(h22q11)/+ mice to environmental stressors that may unmask latent psychopathology. The Df(h22q11)/+ model will be a valuable tool for increasing our understanding of the etiology of schizophrenia and other psychiatric disorders associated with the 22q11DS.

The Effect of Electroacupuncture on PKMzeta in the ACC in Regulating Anxiety-Like Behaviors in Rats Experiencing Chronic Inflammatory Pain.

Chronic inflammatory pain can induce emotional diseases. Electroacupuncture (EA) has effects on chronic pain and pain-related anxiety. Protein kinase Mzeta (PKMzeta) has been proposed to be essential for the maintenance of pain and may interact with GluR1 to maintain CNS plasticity in the anterior cingulate cortex (ACC). We hypothesized that the PKMzeta-GluR1 pathway in the ACC may be involved in anxiety-like behaviors of chronic inflammatory pain and that the mechanism of EA regulation of pain emotion may involve the PKMzeta pathway in the ACC. Our results showed that chronic inflammatory pain model decreased the paw withdrawal threshold (PWT) and increased anxiety-like behaviors. The protein expression of PKCzeta, p-PKCzeta (T560), PKMzeta, p-PKMzeta (T560), and GluR1 in the ACC of the model group were remarkably enhanced. EA increased PWT and alleviated anxiety-like behaviors. EA significantly inhibited the protein expression of p-PKMzeta (T560) in the ACC, and only a downward trend effect for other substances. Further, the microinjection of ZIP remarkably reversed PWT and anxiety-like behaviors. The present study provides direct evidence that the PKCzeta/PKMzeta-GluR1 pathway is related to pain and pain-induced anxiety-like behaviors. EA treatment both increases pain-related somatosensory behavior and decreases pain-induced anxiety-like behaviors by suppressing PKMzeta activity in the ACC.

The Role of Posttraumatic Hypothermia in Preventing Dendrite Degeneration and Spine Loss after Severe Traumatic Brain Injury

Posttraumatic hypothermia prevents cell death and promotes functional outcomes after traumatic brain injury (TBI). However, little is known regarding the effect of hypothermia on dendrite degeneration and spine loss after severe TBI. In the present study, we used thy1-GFP transgenic mice to investigate the effect of hypothermia on the dendrites and spines in layer V/VI of the ipsilateral cortex after severe TBI. We found that hypothermia (33 °C) dramatically prevented dendrite degeneration and spine loss 1 and 7 days after CCI. The Morris water maze test revealed that hypothermia preserved the learning and memory functions of mice after CCI. Hypothermia significantly increased the expression of the synaptic proteins GluR1 and PSD-95 at 1 and 7 days after CCI in the ipsilateral cortex and hippocampus compared with that of the normothermia TBI group. Hypothermia also increased cortical and hippocampal BDNF levels. These results suggest that posttraumatic hypothermia is an effective method to prevent dendrite degeneration and spine loss and preserve learning and memory function after severe TBI. Increasing cortical and hippocampal BDNF levels might be the mechanism through which hypothermia prevents dendrite degeneration and spine loss and preserves learning and memory function.

Chronic stress prior to pregnancy potentiated long-lasting postpartum depressive-like behavior, regulated by Akt-mTOR signaling in the hippocampus.

Postpartum depression (PPD) affects over 10% of new mothers and adversely impacts the health of offspring. One of the greatest risk factors for PPD is prepregnancy stress but the underlying biological mechanism is unknown. Here we constructed an animal model which recapitulated prepregnancy stress induced PPD and tested the role of Akt-mTOR signaling in the hippocampus. Female virgin Balb/c mice received chronic restraint stress, followed by co-housing with a normal male mouse. We found that the chronic stress led to a transient depressive-like condition that disappeared within two weeks. However, prepregnantly stressed females developed long-term postpartum depressive-like (PPD-like) symptoms as indicated by deficient performance in tests of sucrose preference, forced swim, and novelty-suppressed feeding. Chronic stress induced transient decrease in Akt-mTOR signaling and altered expressions of glutamate receptor subunits NR1 and GluR1, in contrast to long-term deficits in Akt-mTOR signaling, GluR1/NR1 ratio, and hippocampal neurogenesis in PPD-like mice. Acute ketamine improved the molecular signaling abnormality, and reversed the behavioral deficits in PPD-like mice in a rapid and persistent manner, in contrast to ineffectiveness by chronic fluoxetine treatment. Taken together, we find that chronic prepregnancy stress potentiates a long-term PPD, in which Akt-mTOR signaling may play a crucial role.

Roles of 5-HT1A receptor in the expression of AMPA receptor and BDNF in developing mouse cortical neurons

The possible interactions between serotonergic and glutamatergic systems during neural development and under the pathogenesis of depression remain unclear. We now investigated roles of 5-HT1A receptor in the mRNA expression of AMPA receptor subunits (GluR1 and GluR2) and brain-derived neurotrophic factor (BDNF) using primary culture of cerebral cortex of mouse embryos. Neurons at embryonic day 18 were cultured for 3days or 14days and then treated with 5-HT1A receptor agonist (8-OH-DPAT) for 3h or 24h. In neurons cultured for 3 days, 8-OH-DPAT treatment for both 3h and 24h increased the mRNA levels of BDNF and GluR1, but not GluR2. In neurons cultured for 14 days, however, 8-OH-DPAT had no effects on these mRNA levels. Next, we examined in vivo roles of 5-HT1A receptor by administration of 8-OH-DPAT to newborn mice. Twenty-four hours after the oral administration of 8-OH-DPAT, the mRNA expression of BDNF was decreased in the frontal cortex, but had no effects on the mRNA expression of GluR1 and GluR2. Taken together, the present study suggests that 5-HT1A receptor activation modulates mRNA expression of AMPA receptor subunit and BDNF in cortical neurons, and the effects are different between in vitro and in vivo.

Lentinan produces a robust antidepressant-like effect via enhancing the prefrontal Dectin-1/AMPA receptor signaling pathway

Lentinan (LNT) is an immune regulator and its potential and mechanism for the treatment of mood disorder is of our interest. Dectin-1 is a β-glucan (including LNT) receptor that regulates immune functions in many immune cell types. Cumulative evidence has suggested that the glutamatergic system seems to play an important role in the treatment of depression. Here, we studied the antidepressant-like effects of LNT and its therapeutical target in regulating the functions of AMPA receptors. We found that 60min treatment with LNT leads to a significant antidepressant-like effect in the tail suspension test (TST) and the forced swim test (FST) in mice. The antidepressant-like effects of LNT in TST and FST remained after 1day or 5days of injections. Additionally, LNT did not show a hyperactive effect in the open field test. Dectin-1 receptor levels were increased after LNT treatment for 5days and the specific Dectin-1 inhibitor laminarin was able to block the antidepressant-like effects of LNT. After 5days of treatment, LNT enhanced p-GluR1 (S845) in the prefrontal cortex (PFC); however, the total GluR1, GluR2, and GluR3 expression levels remained unchanged. We also found that the AMPA-specific blocker GYKI 52466 was able to block the antidepressant-like effects of LNT. This study identified LNT as a novel antidepressant with clinical potential and a new antidepressant mechanism for regulating prefrontal Dectin-1/AMPA receptor signaling.

PICK1 Regulates the Expression and Trafficking of AMPA Receptors in Remifentanil-Induced Hyperalgesia.

Remifentanil is used widely in clinical anesthesia because it induces more rapid and more common hyperalgesia than other opioid analgesics. Activation of N-methyl-D-aspartate (NMDA) receptors takes a pivotal part in remifentanil-induced hyperalgesia. Like NMDA receptors, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are excitatory ion glutamate receptors in postsynaptic membrane, which are involved in the transmission of both acute and chronic pain. Protein interacting with C kinase 1 (PICK1) plays an important role in NMDA receptor-mediated internalization of glutamate receptor 2 (GluR2)-containing AMPARs and contributes to the induction and maintenance of inflammation-induced pain. This study aimed to test the hypothesis that PICK1 contributes to remifentanil-induced hyperalgesia by regulating AMPAR expression and trafficking in the spinal cord. Using a rat model of remifentanil-induced hyperalgesia by intravenous infusion of remifentanil, we first measured changes in mechanical and thermal hyperalgesia at 24 hours before remifentanil infusion and 2, 6, 24, and 48 hours after infusion. PICK1 mRNA and protein expression and AMPAR subunit expression and trafficking in the spinal cord were then detected by reverse transcription-qualitative polymerase chain reaction, immunohistochemistry, and Western blot. In addition, we knocked down PICK1 expression by intrathecal administration of PICK1 antisense oligodeoxynucleotide to investigate the effects of PICK1 deficiency on remifentanil-induced hyperalgesia and the expression and trafficking of AMPARs. A significant time-group interaction was found for nociceptive thresholds (paw withdrawal threshold and paw withdrawal latency; all P < .0001). Remifentanil infusion induced distinct hyperalgesia at different time points (P < .0001), which was partly reversed by PICK1 knockdown (P < .007). Besides, remifentanil infusion increased the expression of PICK1 mRNA and protein (P < .0001) and the membrane GluR1 and GluR2 internalization in spinal dorsal horn neurons (P < .0011). More importantly, PICK1 deficiency could attenuate remifentanil-induced GluR2 internalization in the spinal cord dorsal horn (P < .01) but had no effect on remifentanil-induced membrane GluR1 expression (P ≥ .985). These results indicate that PICK1 deficiency might reverse remifentanil-induced hyperalgesia through regulating GluR2-containing AMPAR expression and trafficking in the spinal cord dorsal horn.

Learning and Memory Deficits in Male Adult Mice Treated with a Benzodiazepine Sleep-Inducing Drug during the Juvenile Period.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system, is also known to be important for brain development. Therefore, disturbances of GABA receptor (GABA-R) mediated signaling (GABA-R signal) during brain development may influence normal brain maturation and cause late-onset brain malfunctions. In this study, we examined whether the stimulation of the GABA-R signal during brain development induces late-onset adverse effects on the brain in adult male mice. To stimulate the GABA-R signal, we used either the benzodiazepine sleep-inducing drug triazolam (TZ) or the non-benzodiazepine drug zolpidem (ZP). We detected learning and memory deficits in mice treated with TZ during the juvenile period, as seen in the fear conditioning test. On the other hand, ZP administration during the juvenile period had little effect. In addition, decreased protein expression of GluR1 and GluR4, which are excitatory neurotransmitter receptors, was detected in the hippocampi of mice treated with TZ during the juvenile period. We measured mRNA expression of the immediate early genes (IEGs), which are neuronal activity markers, in the hippocampus shortly after the administration of TZ or ZP to juvenile mice. Decreased IEG expression was detected in mice with juvenile TZ administration, but not in mice with juvenile ZP administration. Our findings demonstrate that TZ administration during the juvenile period can induce irreversible learning and memory deficits in adult mice. It may need to take an extra care for the prescription of benzodiazepine sleep-inducing drugs to juveniles because it might cause learning and memory deficits.

Sex-Specific Effects of Prenatal Stress on Memory and Markers of Neuronal Activity in Juvenile Rats

Stress during pregnancy can increase the incidence of emotional problems, learning and language difficulties in human infants and pre-adolescents. Most preclinical studies in rats that attempted to find experimental support for these observations were performed in adult male offspring, but the results are inconsistent. The aim of the current study was to examine the effect of prenatal stress on novel object recognition (NOR) and spatial learning and memory in the Morris water maze (MWM) of juvenile rats of both sexes. By the use of fluorescence immunohistochemistry and protein measurements by Western blot, we measured the expression of markers of neurogenesis (doublecortin, DCX) and neuronal activity that are important for synaptic plasticity and learning (c-fos, GluR1, nNOS). Since neuronal activity in the developing and adult brain can be regulated by astrocytes, we also measured the number of astrocytes and the expression of two astroglial proteins (GFAP and S100B) in the stress-responsive hippocampal dentate gyrus (DG). Experiments were performed on littermates of rats in which its effects on behavior were measured. We found for the first time that juvenile females performed better than males in the NOR and MWM tests. They also had higher densities of DCX and c-fos in the DG, together with the expression of nNOS and GluR1 in the subgranular zone (SGZ) of the DG. There were no sex differences in the expression of GFAP and S100B in astrocytes. Prenatal stress did not affect NOR in females, but improved it in males, together with an increase in DCX+ and c-fos, the number of GFAP-expressing astrocytes and the intensity of GFAP and S100B immunofluorescence in the DG. Staining intensity of GluR1 and nNOS in the hilus and SGZ of the DG, and protein expression in the whole DG, was unchanged in prenatally stressed males. Thus, prenatal stress changed the behavior and expression of key proteins in the DG to resemble that in females. A reduction in plasma testosterone, which although not attaining statistical significance was associated with that in anogenital distance, may contribute to the effect of prenatal stress in males. In females, prenatal stress had no effect on c-fos, DCX or the number of astrocytes but reduced the staining intensity of GluR1 and nNOS. Protein expression of nNOS was also significantly lower than that in prenatally stressed males. The differential effects of prenatal stress on hippocampal neuronal and glial markers may help to explain the sex-dependent effect on spatial learning in prepubertal rats.