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Social isolation in adolescence leads to lasting deficits, including emotional and cognitive dysregulation. It remains unclear, however, how social isolation affects certain processes of memory and what molecular mechanisms are involved. In this study, we found that social isolation during the post-weaning period resulted in forgetting of the long-term fear memory, which was attributable to the downregulation of synaptic function in the hippocampal CA1 region mediated by EphB2, a receptor tyrosine kinase which involves in the glutamate receptor multiprotein complex. Viral-mediated EphB2 knockdown in CA1 mimicked the memory defects in group-housed mice, whereas restoration of EphB2 by either viral overexpression or resocialization reversed the memory decline in isolated mice. Taken together, our finding indicates that social isolation gives rise to memory forgetting by disrupting EphB2-mediated synaptic plasticity, which may provide a potential target for preventing memory loss caused by social isolation or loneliness.

Soil microbial communities and enzyme activities play key roles in soil ecosystems. Both are sensitive to changes in environmental factors, including seasonal temperature, precipitation variations and soil properties. To understand the interactive mechanisms of seasonal changes that affect soil microbial communities and enzyme activities in a subtropical masson pine (Pinus massoniana) forest, we investigated the soil microbial community structure and enzyme activities to identify the effect of seasonal changes on the soil microbial community for two years in Jinyun Mountain National Nature Reserve, Chongqing, China. The soil microbial community structure was investigated using phospholipid fatty acids (PLFAs). The results indicated that a total of 36 different PLFAs were identified, and 16:0 was found in the highest proportions in the four seasons, moreover, the total PLFAs abundance were highest in spring and lowest in winter. Bacteria and actinomycetes were the dominant types in the study area. Seasonal changes also had a significant (P < 0.05) influence on the soil enzyme activity. The maximum and minimum values of the invertase and catalase activities were observed in autumn and winter, respectively. However, the maximum and minimum values of the urease and phosphatase acid enzymatic activities were found in spring and winter, respectively. Canonical correspondence analysis (CCA) analysis revealed that the seasonal shifts in soil community composition and enzyme activities were relatively more sensitive to soil moisture and temperature, but the microbial community structure and enzyme activity were not correlated with soil pH in the study region. This study highlights how the seasonal variations affect the microbial community and function (enzyme activity) to better understand and predict microbial responses to future climate regimes in subtropical area.

Social interaction and communication are evolutionary conserved behaviours that are developed in mammals to establish partner cognition. Deficit in sociability has been represented in human patients and animal models of neurodevelopmental disorders, which are connected with genetic variants of synaptic glutamate receptors and associated PDZ-binding proteins. However, it remains elusive how these key proteins are specialized in the cellular level for the initial social behaviour during postnatal developmental stage. Here we identify a hippocampal CA3 specifically expressed PDZ scaffold protein Lnx1 required for initial social behaviour. Through gene targeting we find that Lnx1 deficiency led to a hippocampal subregional disorder in neuronal activity and social memory impairments for partner discrimination observed in juvenile mice which also show cognitive defects in adult stage. We further demonstrate that Lnx1 deletion causes NMDA receptor (NMDAR) hypofunction and this is attributable to decreased GluN2B expression in PSD compartment and disruption of the Lnx1–NMDAR–EphB2 complex. Specific restoration of Lnx1 or EphB2 protein in the CA3 area of Lnx1−/− mice rescues the defective synaptic function and social memory. These findings thus reveal crucial roles of postsynaptic NMDAR multiprotein complex that regulates the formation of initial social memory during the adolescent period.

Objectives To probe microstructural changes that are associated with subconcussive head impact exposure in deep and cortical gray matter of high school football players over a single season. Methods Players underwent diffusion kurtosis imaging (DKI) and quantitative susceptibility mapping (QSM) scans. Head impact data was recorded. Association between parametric changes and frequency of frontal head impact was assessed. Results In deep gray matter, significant decreases in mean kurtosis (MK) and increases in mean diffusivity (MD) over the season were observed in the thalamus and putamen. Correlations between changes in DKI metrics and frequency of frontal impacts were observed in the putamen and caudate. In cortical gray matter, decreases in MK were observed in regions including the pars triangularis and inferior parietal. In addition, increases in MD were observed in the rostral middle frontal cortices. Negative correlations between MK and frequency of frontal impacts were observed in the posterior part of the brain including the pericalcarine, lingual and middle temporal cortices. Magnetic susceptibility values exhibited no significant difference or correlation, suggesting these diffusion changes common within the group may not be associated with iron-related mechanisms. Conclusion Microstructural alterations over the season and correlations with head impacts were captured by DKI metrics, which suggested that DKI imaging of gray matter may yield valuable biomarkers for evaluating brain injuries associated with subconcussive head impact. Findings of associations between frontal impacts and changes in posterior cortical gray matter also indicated that contrecoup injury rather than coup injury might be the dominant mechanism underlying the observed microstructural alterations. Advances in knowledge Significant microstructural changes, as reflected by DKI metrics, in cortical gray matter such as the rostral middle frontal cortices, and in deep gray matter such as the thalamus were observed in high school football players over the course of a single season without clinically diagnosed concussion. QSM showed no evidence of iron-related changes in the observed subconcussive brain injuries. The detected microstructural changes in cortical and deep gray matter correlated with frequency of subconcussive head impacts. Implications for patient care DKI may yield valuable biomarkers for evaluating the severity of brain injuries associated with subconcussive head impacts in contact sport athletes.

Purpose To probe cerebral microstructural abnormalities and assess changes of neuronal density in Disrupted-in-Schizophrenia-1 (DISC1) mice using non-Gaussian diffusion and quantitative susceptibility mapping (QSM). Materials and methods Brain specimens of transgenic DISC1 mice (n = 8) and control mice (n = 7) were scanned. Metrics of neurite orientation dispersion and density imaging (NODDI) and diffusion kurtosis imaging (DKI), as well as QSM, were acquired. Cell counting was performed on Nissl-stained sections. Group differences of imaging metrics and cell density were assessed. Pearson correlations between imaging metrics and cell densities were also examined. Results Significant increases of neuronal density were observed in the hippocampus of DISC1 mice. DKI metrics such as mean kurtosis exhibited significant group differences in the caudate putamen (P = 0.015), cerebral cortex (P = 0.021), and hippocampus (P = 0.011). However, DKI metrics did not correlate with cell density. In contrast, significant positive correlation between density of neurons and the neurite density index of NODDI in the hippocampus was observed (r = 0.783, P = 0.007). Significant correlation between density of neurons and susceptibility (r = 0.657, P = 0.039), as well as between density of neuroglia and susceptibility (r = 0.750, P = 0.013), was also observed in the hippocampus. Conclusion The imaging metrics derived from DKI were not sensitive specifically to cell density, while NODDI could provide diffusion metrics sensitive to density of neurons. The magnetic susceptibility values derived from the QSM method can serve as a sensitive biomarker for quantifying neuronal density.

Astrocytes are fundamental for maintaining brain homeostasis and are commonly involved in the progression of neurodegenerative diseases including Alzheimer's disease (AD). In response to injury or toxic material, astrocytes undergo activation that results in hypertrophy and process ramification. Although numerous studies have shown that reactive astrocytes are intimately related to the pathogenesis of AD, their characteristic features including morphological and molecular alterations that occur during different stages of AD progression remain to be elucidated. Here, we crossed astrocyte-specific reporter mice hGFAP-CreERT2;Rosa-tdTomato with APP/PS1 mice, and then used genetic tracing to characterize the morphological profiles and expression of molecular biomarkers associated with progressive β-amyloid deposits in the cortical region of AD mice. Expression of glutamine synthetase (GS) was lower in cortical reactive astrocytes, in contrast to the higher expression of glial fibrillary acidic protein, of APP/PS1 mice and AD patients relative to that in cortical astrocytes of wild-type mice and age-matched controls, respectively. GS activity was also decreased obviously in the cortex of APP/PS1 mice at 6 and 12 months of age relative to that in the wild-type mice of the same ages. Furthermore, cortical reactive astrocytes in APP/PS1 mice and AD patients did not undergo proliferation. Finally, based on RNA-sequencing analysis, we identified differentially expressed transcripts of signal transduction molecules involved in early induction of reactive astrocytes in the cortex of APP/PS1 mice. These findings provide a morphological and molecular basis with which to understand the function and mechanism of reactive astrocytes in the progression of AD.

Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signaling clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells.

Learning to extinguish traumatic memory depends on fine-tuning of hippocampal-prefrontal connections via the ASIC1a channel., Extinction of conditioned fear necessitates the dynamic involvement of hippocampus, medial prefrontal cortex (mPFC), and basolateral amygdala (BLA), but key molecular players that regulate these circuits to achieve fear extinction remain largely unknown. Here, we report that acid-sensing ion channel 1a (ASIC1a) is a crucial molecular regulator of fear extinction, and that this function requires ASIC1a in ventral hippocampus (vHPC), but not dorsal hippocampus, mPFC, or BLA. While genetic disruption or pharmacological inhibition of ASIC1a in vHPC attenuated the extinction of conditioned fear, overexpression of the channel in this area promoted fear extinction. Channelrhodopsin-2–assisted circuit mapping revealed that fear extinction involved an ASIC1a-dependent modification of the long-range hippocampal-prefrontal correlates in a projection-specific manner. Gene expression profiling analysis and validating experiments identified several neuronal activity–regulated and memory-related genes, including Fos, Npas4, and Bdnf, as the potential mediators of ASIC1a regulation of fear extinction. Mechanistically, genetic overexpression of brain-derived neurotrophic factor (BDNF) in vHPC or supplement of BDNF protein in mPFC both rescued the deficiency in fear extinction and the deficits on extinction-driven adaptations of hippocampal-prefrontal correlates caused by the Asic1a gene inactivation in vHPC. Together, these results establish ASIC1a as a critical constituent in fear extinction circuits and thus a promising target for managing adaptive behaviors.

Synapse formation relies on the coordination of dynamic pre- and postsynaptic structures during brain development. Liu et al. reveal that presynaptic terminal maturation of mossy fiber axons is retrogradely regulated by postsynaptic scaffold protein Lnx1 via stabilizing EphB receptor kinases., Neuronal connections are initiated by axon targeting to form synapses. However, how the maturation of axon terminals is modulated through interacting with postsynaptic elements remains elusive. In this study, we find that ligand of Numb protein X 1 (Lnx1), a postsynaptic PDZ protein expressed in hippocampal CA3 pyramidal neurons, is essential for mossy fiber (MF) axon targeting during the postnatal period. Lnx1 deletion causes defective synaptic arrangement that leads to aberrant presynaptic terminals. We further identify EphB receptors as novel Lnx1-binding proteins to form a multiprotein complex that is stabilized on the CA3 neuron membrane through preventing proteasome activity. EphB1 and EphB2 are independently required to transduce distinct signals controlling MF pruning and targeting for precise DG-CA3 synapse formation. Furthermore, constitutively active EphB2 kinase rescues structure of the wired MF terminals in Lnx1 mutant mice. Our data thus define a retrograde trans-synaptic regulation required for integration of post- and presynaptic structure that participates in building hippocampal neural circuits during the adolescence period., Graphical Abstract

The molecular and cellular mechanisms underlying the anti-proliferative effects of preoptic regulator factor 2 (Porf-2) on neural stem cells (NSCs) remain largely unknown. Here, we found that Porf-2 inhibits the activity of ras-related C3 botulinum toxin substrate 1 (Rac1) protein in hippocampus-derived rat NSCs. Reduced Rac1 activity impaired the nuclear translocation of β-catenin, ultimately causing a repression of NSCs proliferation. Porf-2 knockdown enhanced NSCs proliferation but not in the presence of small molecule inhibitors of Rac1 or Wnt. At the same time, the repression of NSCs proliferation caused by Porf-2 overexpression was counteracted by small molecule activators of Rac1 or Wnt. By using a rat optic nerve crush model, we observed that Porf-2 knockdown enhanced the recovery of visual function. In particular, optic nerve injury in rats led to increased Wnt family member 3a (Wnt3a) protein expression, which we found responsible for enhancing Porf-2 knockdown-induced NSCs proliferation. These findings suggest that Porf-2 exerts its inhibitory effect on NSCs proliferation via Rac1-Wnt/β-catenin pathway. Porf-2 may therefore represent and interesting target for optic nerve injury recovery and therapy.

BACKGROUND: The study aimed to compare the tolerability and efficacy of gefitinib combined with chemotherapy agents versus chemotherapy alone for the treatment of epidermal growth factor receptor (EGFR)–mutated lung adenocarcinoma in heavily pretreated patients. METHODS: The study was designed as a matched-pair case-control investigation to minimize intergroup heterogeneity. Patients were stratified into gefitinib plus chemotherapy and chemotherapy alone groups with matching for sex, age, ECOG performance status, progress-free survival (PFS) from previous EGFR tyrosine kinase inhibitor treatment, EGFR mutation types, and tumor metastasis status. RESULTS: Sixty-six patients were selected from our database using the matched-pair method. The median age was 61 years (95% confidence interval, 57-65 years). During a follow-up period of 14.5 months on average, the overall response rates of the gefitinib-integrated and chemotherapy alone groups were 9.1% and 6.5%, respectively (P > .05), whereas the corresponding disease-control rates were 39.4% and 30.3%, respectively (P > .05). No statistically significant differences in PFS (median, 4.2 vs 3.3 months; P = .06) and overall survival (median, 10.4 vs 7.9 months; P = .44) were observed between two groups. The 6-month survival rates of the gefitinib-integrated and chemotherapy alone groups were 21.2% and 12.1%, respectively (P < .05). Side effects were mild, and all treatments were well tolerated. CONCLUSIONS: Our results indicated that gefitinib-integrated therapy offered a trend to better PFS and an improved 6-month survival rate in heavily pretreated patients with metastatic EGFR-mutated lung adenocarcinoma. All treatments were well tolerated. Future prospective studies are warranted to confirm our findings.

As the neural network becomes wired, postsynaptic signaling molecules are thought to control the growth of dendrites and synapses. However, how these molecules are coordinated to sculpt postsynaptic structures is less well understood. We find that ephrin-B3, a transmembrane ligand for Eph receptors, functions postsynaptically as a receptor to transduce reverse signals into developing dendrites of mouse hippocampal neurons. Both tyrosine phosphorylation-dependent GRB4 SH2/SH3 adaptor-mediated signals and PSD-95-discs large-zona occludens-1 (PDZ) domain-dependent signals are required for inhibition of dendrite branching, whereas only PDZ interactions are necessary for spine formation and excitatory synaptic function. PICK1 and syntenin, two PDZ domain proteins, participate with ephrin-B3 in these postsynaptic activities. PICK1 has a specific role in spine and synapse formation, and syntenin promotes both dendrite pruning and synapse formation to build postsynaptic structures that are essential for neural circuits. The study thus dissects ephrin-B reverse signaling into three distinct intracellular pathways and protein-protein interactions that mediate the maturation of postsynaptic neurons.

It has been suggested that ephrin-B proteins have receptor-like roles in the control of axon pathfinding by repulsion, although it is largely unknown how the reverse signals are coupled to downstream intracellular molecules and how they induce cytoskeletal reorganization at the axon terminal. We found that ephrin-B3 (EB3) was able to function as a repulsive guidance receptor and mediate stereotyped pruning of murine hippocampal mossy fiber axons during postnatal development. Targeted intracellular point mutants showed that axon pruning requires tyrosine phosphorylation-dependent reverse signaling and coupling to the SH2/SH3 adaptor protein Grb4 (also known as Nckbeta/Nck2). Furthermore, we found that the second SH3 domain of Grb4 is required and sufficient for axon pruning/retraction by mediating interactions with Dock180 and PAK to bring about guanine nucleotide exchange and signaling downstream of Rac, respectively. Our results reveal a previously unknown pathway that controls axon pruning and elucidate the biochemical mechanism by which ephrin-B reverse signals regulate actin dynamics to bring about the retraction of growth cones.

Innate emotion response to environmental stimuli is a fundamental brain function that is controlled by specific neural circuits. Dysfunction of early emotional circuits may lead to neurodevelopmental disorders such as autism and schizophrenia. However, how the functional circuits are formed to prime initial emotional behaviours remain elusive. We reveal here using gene-targeted mutations an essential role for ephrin-B3 ligand-like activity in the development of innate fear in the neonatal brain. We further demonstrate that ephrin-B3 controls axon targeting and coordinates spinogenesis and neuronal activity within the amygdala. The morphological and behavioural abnormalities in ephrin-B3 mutant mice are rescued by conditional knock-in of wild-type ephrin-B3 during the critical period when axon targeting and fear responses are initiated. Our results thus define a key axonal molecule that participates in the wiring of amygdala circuits and helps bring about fear emotion during the important adolescence period., The molecular mechanism underlying initial circuit wiring in amygdala is poorly understood. Here the authors show that ephrin-B3 is required for axon targeting and amygdala spinogenesis during a critical period in development, and plays an important role in amygdala mediated fear responses.

Opiate abuse is associated with long-lasting neural adaptative changes in the brain. Increasing evidence demonstrates that opiates significantly alter the function of the glutamatergic system, while how the system is regulated still remains elusive. In the present study, we studied the effect of morphine on extracellular glutamate concentration in the hippocampus, a nucleus rich of the glutamatergic neurons. The results showed that glutamate concentration in the extracellular fluid of the hippocampus was decreased following either acute or chronic treatment of morphine. However, naloxone-induced withdrawal increased glutamate concentration significantly. These results suggest an adaptation of the glutamatergic neuronal transmission in the hippocampus after morphine treatment.

Opiate abuse has been shown to cause adaptive changes in presynaptic release and protein phosphorylation-mediated synaptic plasticity, but the underlying mechanisms remain unclear. Neuronal SNARE proteins serve as important regulatory molecules underlying neural plasticity in view of their major role in the process of neurotransmitter release. In the present study, the expression of SNAP-25, a t-SNARE protein essential for vesicle release, was found to be dramatically regulated in hippocampus after chronic morphine treatment, which was visualized with two-dimensional gel electrophoresis. The spots of SNAP-25 in the gel were shifted along the dimension of isoelectric point, indicating a likely change of the post-transcriptional modification. Immunoblotting analysis with specific antibody to Ser187, a protein kinase C (PKC) phosphorylation site of SNAP-25, revealed that the specific phosphorylation was correspondingly decreased, which was correlated with morphine-induced inhibition of PKC activity. Moreover, the level of ternary complex of SNARE proteins in either synaptosomes or PC12 cells was significantly reduced after chronic morphine treatment. This suggests a causal relationship between the inhibition of PKC-dependent SNAP-25 phosphorylation and the down-regulation of SNARE complex formation after chronic morphine treatment. Further analysis of SNARE complex formed by transfection of the wild-type or Ser187 mutants of SNAP-25 showed that only wild-type-formed complex was inhibited by morphine treatment. Thus, these results indicate that chronic morphine treatment inhibits phosphorylation of SNAP-25 at Ser187 and leads to a down-regulation of SNARE complex formation, which presents a potential molecular mechanism for the alteration of exocytotic process and neural plasticity during opiate abuse.

Ginsenosides, the main effective components of the root of Panax ginseng, have been reported to modulate morphine action. In the present study, ginsenosides Rd, Rb2, Rg1 and Re were divided into two groups according to their effects in mice on morphine-induced hyperactivity and conditioned place preference (CPP). Ginsenosides Rd, Rb2, Rg1 had no effect on morphine-induced hyperactivity, but antagonized morphine-induced CPP. On the contrary, ginsenoside Re increased morphine-induced hyperactivity whereas it showed no effect on morphine-induced CPP. Furthermore, Re antagonized the inhibitory effect of the mixture involving Rd, Rb2 and Rg1 on the morphine action. These results suggest that ginsenosides with different structures have antagonizing properties in the regulation of morphine-induced reinforcement.

Analgesia is a well-documented effect of acupuncture. A critical role in pain sensation plays the nervous system, including the GABAergic system and opioid receptor (OR) activation. Here we investigated regulation of GABA transporter GAT1 byδOR in rats and inXenopusoocytes. Synaptosomes of brain from rats chronically exposed to opiates exhibited reduced GABA uptake, indicating that GABA transport might be regulated by opioid receptors. For further investigation we have expressed GAT1 of mouse brain together with mouseδOR andμOR inXenopusoocytes. The function of GAT1 was analyzed in terms of Na+-dependent [3H]GABA uptake as well as GAT1-mediated currents. Coexpression ofδOR led to reduced number of fully functional GAT1 transporters, reduced substrate translocation, and GAT1-mediated current. Activation ofδOR further reduced the rate of GABA uptake as well as GAT1-mediated current. Coexpression ofμOR, as well asμOR activation, affected neither the number of transporters, nor rate of GABA uptake, nor GAT1-mediated current. Inhibition of GAT1-mediated current by activation ofδOR was confirmed in whole-cell patch-clamp experiments on rat brain slices of periaqueductal gray. We conclude that inhibition of GAT1 function will strengthen the inhibitory action of the GABAergic system and hence may contribute to acupuncture-induced analgesia.

Axon-cell and axon-dendrite contact is a highly regulated process necessary for the formation of precise neural circuits and a functional neural network. Eph-ephrin interacting molecules on the membranes of axon nerve terminals and target dendrites act as bidirectional ligands/receptors to transduce signals into both the Eph-expressing and ephrin-expressing cells to regulate cytoskeletal dynamics. In particular, recent evidence indicates that ephrin reverse signal transduction events are important in controlling both axonal and dendritic elaborations of neurons in the developing nervous system. Here we review how ephrin reverse signals are transduced into neurons to control maturation of axonal pre-synaptic and dendritic post-synaptic structures.

The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor (AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of beta-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or beta-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/beta-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or beta-catenin into mouse limb muscles, where ectopic Wnt3a or beta-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/beta-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.

Excitatory synapses in the CNS are formed on both dendritic spines and shafts. Recent studies show that the density of shaft synapses may be independently regulated by behavioral learning and the induction of synaptic plasticity, suggesting that distinct mechanisms are involved in regulating these two types of synapses. Although the molecular mechanisms underlying spinogenesis and spine synapse formation are being delineated, those regulating shaft synapses are still unknown. Here, we show that postsynaptic ephrinB3 expression promotes the formation of glutamatergic synapses specifically on the shafts, not on spines. Reducing or increasing postsynaptic ephrinB3 expression selectively decreases or increases shaft synapse density, respectively. In the ephrinB3 knock-out mouse, although spine synapses are normal, shaft synapse formation is reduced in the hippocampus. Overexpression of glutamate receptor-interacting protein 1 (GRIP1) rescues ephrinB3 knockdown phenotype by restoring shaft synapse density. GRIP1 knockdown prevents the increase in shaft synapse density induced by ephrinB3 overexpression. Together, our results reveal a novel mechanism for independent modulation of shaft synapses through ephrinB3 reverse signaling.

Opiate abuse causes adaptive changes in several processes of synaptic transmission in which the glutamatergic system appears a critical element involved in opiate tolerance and dependence, but the underlying mechanisms remain unclear. In the present study, we found that glutamate uptake in hippocampal synaptosomes was significantly increased (by 70% in chronic morphine-treated rats) during the morphine withdrawal period, likely attributable to an increase in the number of functional glutamate transporters. Immunoblot analysis showed that expression of GLT1 (glutamate transporter subtype 1) was identified to be upregulated in synaptosomes but not in total tissues, suggesting a redistribution of glutamate transporter expression. Moreover, the increase in glutamate uptake was reproduced in cultured neurons during morphine withdrawal, and the increase of uptake in neurons could be blocked by dihydrokainate, a specific inhibitor of GLT1. Cell surface biotinylation and immunoblot analysis showed that morphine withdrawal produced an increase in GLT1 expression rather than EAAC1 (excitatory amino acids carrier 1), a neuronal subtype, at the cultured neuronal cell surface, whereas no significant change was observed in that of cultured astrocytes. Electron microscopy also revealed that GLT1 expression was markedly increased in the nerve terminals of hippocampus and associated with the plasma membranein vivo. These results suggest that GLT1 in hippocampal neurons can be induced to translocate to the nerve terminals and express on the cell surface during morphine withdrawal. The translocation of GLT1 at synapses during morphine withdrawal provides a neuronal mechanism for modulation of excitatory neurotransmission during opiate abuse.

The effect of oleamide on apoptosis was investigated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay, DNA staining assay with propidium iodide and caspase-3 activity analyses. The present results showed that oleamide significantly attenuated the cell death and nuclear condensation of cultured rat cerebellar granule neurons induced by K(+) deprivation in a dose-dependent manner. The oleamide actions were well parallel with the attenuation of caspase-3 activity in the process of apoptotic death. Moreover, neither elaidic acid nor stearic acid, two fatty acids structurally related to oleamide without the Delta(9)-cis double bond, had similar effects on the cell death, suggesting the selectively structural features of oleamide required for this action. These data provided the first evidence of a protective effect of oleamide against apoptosis in a structurally specific manner.

Pseudoginsenoside-F11 (PF11), an ocotillol type saponin isolated from Panax quinquefolium L., has been shown to antagonize the behavioral actions of morphine. Biochemical experiments revealed that PF11 could inhibit diprenorphine (DIP) binding with an IC50 of approximately 6.1 microM and reduced the binding potency of morphine in Chinese hamster ovary (CHO)-mu cells. Furthermore, PF11 significantly attenuated morphine-stimulated [35S]GTPgammaS binding in a dose dependent manner, and strongly decreased the efficacy of morphine to inhibit intracellular cAMP production. In addition, PF11 pretreatment could also significantly inhibit naloxone induced cAMP overshoot in the morphine-pretreated cells. However, PF11 per se had no effect on either [35S]GTPgammaS binding or intracellular cAMP accumulation. These data suggested that PF11 antagonized the morphine stimulated opioid receptor signalling directly at the cellular level.

The effects of morphine and morphine withdrawal on memory performance were examined in mice by using Morris water maze task. Morphine-induced memory impairment at the doses of 5 and 10 mg/kg recovered after repeated administration. Oxotremorine, a muscarinic receptor agonist, at the dose of 0.1 mg/kg ip, and physostigmine, a cholinesterase inhibitor, at the dose of 0.1 mg/kg ip, significantly antagonized morphine (10 mg/kg sc)-induced memory impairment in mice. Furthermore, repeated naloxone (0.5 mg/kg ip) attenuated scopolamine (0.2 mg/kg ip)-induced memory impairment. By using escalating doses of morphine for 13 days, morphine-induced memory impairment was continuously maintained. When withdrawal was precipitated by naloxone (5 mg/kg ip), or administration of oxotremorine (0.1 and 0.2 mg/kg ip) or physostigmine (0.05 and 0.1 mg/kg ip), the impairment was completely reversed. These results suggest that morphine-induced memory impairment could be partially due to the inhibition of the central cholinergic activity.

SummaryAgrin induces, whereas acetylcholine (ACh) disperses, ACh receptor (AChR) clusters during neuromuscular synaptogenesis. Such counteractive interaction leads to eventual dispersal of nonsynaptic AChR-rich sites and formation of receptor clusters at the postjunctional membrane. However, the underlying mechanisms are not well understood. Here we show that calpain, a calcium-dependent protease, is activated by the cholinergic stimulation and is required for induced dispersion of AChR clusters. Interestingly, the AChR-associated protein rapsyn interacted with calpain in an agrin-dependent manner, and this interaction inhibited the protease activity of calpain. Disrupting the endogenous rapsyn/calpain interaction enhanced CCh-induced dispersion of AChR clusters. Moreover, the loss of AChR clusters in agrin mutant mice was partially rescued by the inhibition of calpain via overexpressing calpastatin, an endogenous calpain inhibitor, or injecting calpeptin, a cell-permeable calpain inhibitor. These results demonstrate that calpain participates in ACh-induced dispersion of AChR clusters, and rapsyn stabilizes AChR clusters by suppressing calpain activity.

SummarySignaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth.