[Effect of Rehmanniae Radix on depression-like behavior and hippocampal monoamine neurotransmitters of chronic unpredictable mild stress model rats].

To investigate the effect of Rehmanniae Radix on depression-like behavior and monoamine neurotransmitters of chronic unpredictable mild stress(CUMS) model rats. CUMS combined with isolated feeding was used to induce the depression model of rats. The depression-like behavior of rats was evaluated by sucrose preference test, open field test, and forced swim test. Hematoxylin-Eosin(HE) staining was used to investigate the pathological changes of neurons in the CA1 and CA3 area of hippocampus. Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS) was used to detect the contents of 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), 3,4-dihydroxyphenylacetic acid(DOPAC), homovanillic acid(HVA), norepinephrine(NE), and 3-methoxy-4-hydroxyphenyl glycol(MHPG) in rats. Western blot was used to detect the protein expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), and monoamine oxidase A(MAO-A) in the hippocampus of rats. Compared with the normal group, depressive-like behavior of rats was obvious in the model group. The arrangements of neurons in the CA1 and CA3 area of hippocampus were loose and disorderly. The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in the hippocampal area were decreased(P<0.01). The protein expression of TPH2 was decreased(P<0.01), but those of SERT and MAO-A were increased(P<0.01). In the Rehmanniae Radix groups with 1.8 g·kg~(-1) and 7.2 g·kg~(-1), the depression-like behavior of CUMS rats and pathological changes of neurons in CA1, CA3 area of hippocampus were improved. The protein expression of TPH2(P<0.05, P<0.01) was increased, and those of SERT and MAO-A were down-regulated(P<0.05, P<0.01). The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in hippocampus were increased(P<0.05, P<0.01). The changes in DA, DOPAC, HVA, DA/(DOPAC +HVA), NE, DHPG, and NE/DHPG were not statistically significant. The results suggested that Rehmanniae Radix improved depression-like behavior of CUMS rats, and the mechanism might be related to the regulation of synthesis, transportation, and metabolism of 5-HT neurotransmitter in the hippocampus.

Synaptic protein interaction networks encode experience by assuming stimulus-specific and brain-region-specific states.

A core network of widely expressed proteins within the glutamatergic post-synapse mediates activity-dependent synaptic plasticity throughout the brain, but the specific proteomic composition of synapses differs between brain regions. Here, we address the question, how does proteomic composition affect activity-dependent protein-protein interaction networks (PINs) downstream of synaptic activity? Using quantitative multiplex co-immunoprecipitation, we compare the PIN response of in vivo or ex vivo neurons derived from different brain regions to activation by different agonists or different forms of eyeblink conditioning. We report that PINs discriminate between incoming stimuli using differential kinetics of overlapping and non-overlapping PIN parameters. Further, these "molecular logic rules" differ by brain region. We conclude that although the PIN of the glutamatergic post-synapse is expressed widely throughout the brain, its activity-dependent dynamics show remarkable stimulus-specific and brain-region-specific diversity. This diversity may help explain the challenges in developing molecule-specific drug therapies for neurological disorders.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.

Reelin Regulates Neuronal Excitability through Striatal-Enriched Protein Tyrosine Phosphatase (STEP61) and Calcium Permeable AMPARs in an NMDAR-Dependent Manner.

Alzheimer's disease (AD) is a progressive neurodegenerative disease marked by the accumulation of amyloid-ß (Aß) plaques and neurofibrillary tangles. Aß oligomers cause synaptic dysfunction early in AD by enhancing long-term depression (LTD; a paradigm for forgetfulness) via metabotropic glutamate receptor (mGluR)-dependent regulation of striatal-enriched tyrosine phosphatase (STEP61). Reelin is a neuromodulator that signals through ApoE (apolipoprotein E) receptors to protect the synapse against Aß toxicity (Durakoglugil et al., 2009) Reelin signaling is impaired by ApoE4, the most important genetic risk factor for AD, and Aß-oligomers activate metabotropic glutamate receptors (Renner et al., 2010). We therefore asked whether Reelin might also affect mGluR-LTD. To this end, we induced chemical mGluR-LTD using DHPG (Dihydroxyphenylglycine), a selective mGluR5 agonist. We found that exogenous Reelin reduces the DHPG-induced increase in STEP61, prevents the dephosphorylation of GluA2, and concomitantly blocks mGluR-mediated LTD. By contrast, Reelin deficiency increased expression of Ca2+-permeable GluA2-lacking AMPA receptors along with higher STEP61 levels, resulting in occlusion of DHPG-induced LTD in hippocampal CA1 neurons. We propose a model in which Reelin modulates local protein synthesis as well as AMPA receptor subunit composition through modulation of mGluR-mediated signaling with implications for memory consolidation or neurodegeneration.SIGNIFICANCE STATEMENT Reelin is an important neuromodulator, which in the adult brain controls synaptic plasticity and protects against neurodegeneration. Amyloid-ß has been shown to use mGluRs to induce synaptic depression through endocytosis of NMDA and AMPA receptors, a mechanism referred to as LTD, a paradigm of forgetfulness. Our results show that Reelin regulates the phosphatase STEP, which plays an important role in neurodegeneration, as well as the expression of calcium-permeable AMPA receptors, which play a role in memory formation. These data suggest that Reelin uses mGluR LTD pathways to regulate memory formation as well as neurodegeneration.

Antioxidant effects of supplementation of 3,4-dihydroxyphenyl glycol on sperm parameters and oxidative markers following cryopreservation in canine semen.

Supplementing the extender with antioxidants with low molecular weight can enhance the quality of the post-thaw sperm during the freezing process. This study was aimed at determining the impacts of 3,4-dihydroxyphenyl glycol (DHPG) on the spermatozoa of the canine undergoing freeze-thawing process. In this study, 24 ejaculates were obtained from three mixed-breed dogs and were diluted in a Tris-based extender. The diluted semen was divided into aliquots for supplementation of 10, 30, 50 and 70 µg/ml of DHPG, control (without antioxidant) and control sham (DMSO). After being extended, the semen was equilibrated at a temperature of 4°C and then transferred to the straws and kept 4 cm above the liquid nitrogen for 20 min and was finally immersed in the liquid nitrogen. They were cryopreserved for seven days; then, sperm parameters including sperm motility evaluation, motility characteristics, viability, DNA and plasma membrane integrity, total antioxidant capacity (TAC), reduced glutathione content (GSH), antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT] and glutathione peroxidase [GPx]) activity malondialdehyde (MDA) levels were evaluated. This study showed that spermatozoa cryopreservation with 50, 30 and 70 µg/ml of DHPG concentrations had better progressive motility, Curvilinear Velocity, Linearity, viability, intact plasma membrane and the levels of TAC, GPx and GSH were higher than the control group. The 50, 30 and 70 µg/ml of DHPG concentrations led to the significant decrease of DNA damage compared to the control group. Total motility, average path velocity, straight-line velocity and CAT activity were significantly improved in 30 and 50 µg/ml of DHPG concentrations, compared to the control group. Also, the 50 and 30 µg/ml of DHPG concentrations, decreased MDA levels compared to the other groups, significantly. In conclusion, our study showed that the addition 50 µg/ml of DHPG to the canine semen extender improved the semen characteristics and oxidative markers in the cryopreservation process.

Regulation of mGluR1 on the Expression of PKC and NMDAR in Aluminum-Exposed PC12 Cells.

Aluminum demonstrates clear neurotoxicity and can cause Alzheimer's disease (AD)-like symptoms, including cognitive impairment. One toxic effect of aluminum is a decrease in synaptic plasticity, but the specific mechanism remains unclear. In this study, PC12 cells were treated with Al(mal)3 to construct a toxic cell model. (S)-3,5-Dihydroxyphenylglycine (DHPG), α-methyl-4-carboxyphenylglycine (MCPG), and mGluR1-siRNA were used to interfere with the expression of metabotropic glutamate receptor subtype 1 (mGluR1). Polymerase chain reaction and western blotting were used to investigate the expression of mGluR1, protein kinase C (PKC), and N-methyl-D-aspartate receptor (NMDAR) subunits. ELISA was used to detect PKC enzyme activity. In PC12 cells, mRNA and protein expressions of PKC and NMDAR subunits were inhibited by Al(mal)3. Aluminum may further regulate the expression of NMDAR1 and NMDAR2B through mGluR1 to regulate PKC enzyme activity, thereby affecting learning and memory functions. Furthermore, the results implied that the mGluR1-PKC-NMDAR signaling pathway may predominately involve positive regulation. These findings provide new targets for studying the neurotoxic mechanism of aluminum.

Ribosome profiling in mouse hippocampus: plasticity-induced regulation and bidirectional control by TSC2 and FMRP.

BACKGROUND: Mutations in TSC2 are the most common cause of tuberous sclerosis (TSC), a disorder with a high incidence of autism and intellectual disability. TSC2 regulates mRNA translation required for group 1 metabotropic glutamate receptor-dependent synaptic long-term depression (mGluR-LTD) and behavior, but the identity of mRNAs responsive to mGluR-LTD signaling is largely unknown. METHODS: We utilized Tsc2+/- mice as a mouse model of TSC and prepared hippocampal slices from these animals. We induced mGluR-LTD synaptic plasticity in slices and processed the samples for RNA-seq and ribosome profiling to identify differentially expressed genes in Tsc2+/- and following mGluR-LTD synaptic plasticity. RESULTS: Ribosome profiling reveals that in Tsc2+/- mouse hippocampal slices, the expression of several mRNAs was dysregulated: terminal oligopyrimidine (TOP)-containing mRNAs decreased, while FMRP-binding targets increased. Remarkably, we observed the opposite changes of FMRP binding targets in Fmr1-/y hippocampi. In wild-type hippocampus, induction of mGluR-LTD caused rapid changes in the steady-state levels of hundreds of mRNAs, many of which are FMRP targets. Moreover, mGluR-LTD failed to promote phosphorylation of eukaryotic elongation factor 2 (eEF2) in TSC mice, and chemically mimicking phospho-eEF2 with low cycloheximide enhances mGluR-LTD in TSC mice. CONCLUSION: These results suggest a molecular basis for bidirectional regulation of synaptic plasticity and behavior by TSC2 and FMRP. Our study also suggests that altered mGluR-regulated translation elongation contributes to impaired synaptic plasticity in Tsc2+/- mice.

Temporal Quantitative Proteomics of mGluR-induced Protein Translation and Phosphorylation in Neurons.

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses.

Endocannabinoid receptors contribute significantly to multiple forms of long-term depression in the rat dentate gyrus.

Cannabinoid receptors are widely expressed throughout the hippocampal formation, but are particularly dense in the dentate gyrus (DG) subregion. We, and others, have shown in mice that cannabinoid type 1 receptors (CB1Rs) are involved in a long-term depression (LTD) that can be induced by prolonged 10 Hz stimulation of the medial perforant path (MPP)-granule cell synaptic input to the DG. Here, we extend this work to examine the involvement of CB1Rs in other common forms of LTD in the hippocampus of juvenile male and female Sprague-Dawley rats (Rattus norvegicus). We found, as in mice, that prolonged 10 Hz stimulation (6000 pulses) could reliably induce a form of LTD that was dependent upon CB1R activation. In addition, we also discovered a role for both CB1R and mGluR proteins in LTD induced with 1 Hz low-frequency stimulation (1 Hz-LTD; 900 pulses) and in LTD induced by bath application of the group I mGluR agonist (RS)-3,5-Dihydroxyphenylglycine (DHPG; DHPG-LTD). This study elucidates an essential role for endocannabinoid receptors in a number of forms of LTD in the rat DG, and identifies a novel role for CB1Rs as potential therapeutic targets for conditions that involve impaired LTD in the DG.

Tumor necrosis factor-alpha aggravates gliosis and inflammation of activated retinal Müller cells.

Tumor necrosis factor-alpha (TNF-α), a major inflammatory factor released from activated retinal glial cells, is implicated in the pathogenesis of glaucoma. In this study, we investigated whether and how TNF-α may affect functional conditions of activated retinal Müller cells. Our results showed that in the group I metabotropic glutamate receptor (mGluR I) agonist DHPG-activated cultured Müller cells, TNF-α treatment aggravated cell gliosis, as evidenced by significantly increased expression of glial fibrillary acidic protein (GFAP). TNF-α treatment of the DHPG-activated Müller cells decreased cell proliferation and induced cell apoptosis. In normal Müller cells, TNF-α treatment increased the mRNA levels of leukocyte inhibitory factor (LIF), intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and chemokine C-C-motif ligand 2 (CCL2), which could be significantly attenuated when Müller cells were pre-activated. However, TNF-α-induced elevation in mRNA levels of inflammatory factors, such as TNF-α, inducible nitric oxide synthase (iNOS), and interleukin-6 (IL-6), in normal Müller cells still kept higher levels when Müller cells were pre-activated. Furthermore, the TNF-α-induced changes of cytokines were partially mediated by NF-κB signaling pathway. Our results suggest that TNF-α may promote gliosis and inflammatory response of activated Müller cells, thus aggravating RGC injury in glaucoma.

Differential contribution of metabotropic glutamate receptor 5 common allosteric binding site residues to biased allosteric agonism.

Allosteric modulators of metabotropic glutamate receptor subtype 5 (mGlu5) represent an attractive therapeutic strategy for multiple CNS disorders. Chemically distinct mGlu5 positive allosteric modulators (PAMs) that interact with a common binding site can demonstrate biased allosteric agonism relative to the orthosteric agonist, DHPG, when comparing activity in signaling assays such as IP1 accumulation, ERK1/2 phosphorylation (pERK1/2) and iCa2+ mobilization. However, the structural basis for such biased agonism is not well understood. Therefore, we evaluated biased allosteric agonism mediated by four mGlu5 PAM-agonists from diverse chemical scaffolds (i.e., DPFE, VU0409551, VU29, and VU0424465) for three measures of mGlu5 activation (i.e., iCa2+ mobilization, IP1 accumulation and ERK1/2 phosphorylation) at eight single-point mutations within the common allosteric binding pocket of mGlu5. In particular, mGlu5 allosteric site mutations had differential effects on the intrinsic efficacy of mGlu5 PAMs for multiple signaling pathways. Specifically, a loss of agonism for iCa2+ mobilization was evident for DPFE and VU0409551 for most mutants, whereas IP1 accumulation and ERK phosphorylation were retained, albeit with reduced maximal responses. Additionally, bias profiles between iCa2+ mobilization and IP1/ERK pathways remained similar to wild type for most mutants. However, W784A and A809G mutants lost bias between IP1 accumulation and ERK phosphorylation for VU0424465, whereas a loss of bias between iCa2+ mobilization and ERK1/2 phosphorylation was evident for F787A, S808A and A809G mutants. These data provide further insight into the structural requirements for allosteric agonism across multiple mGlu5-mediated signaling pathways. An understanding of mGlu5 biased agonism at a structural level may provide the foundation for rational structure-based design of biased allosteric ligands for the treatment of neurological disorders.

Probe dependence and biased potentiation of metabotropic glutamate receptor 5 is mediated by differential ligand interactions in the common allosteric binding site.

The metabotropic glutamate receptor 5 (mGlu5) is a promising therapeutic target for multiple CNS disorders. Recent mGlu5 drug discovery has focused on targeting binding sites within the mGlu5 7-transmembrane domain (7TM) that are topographically distinct from that of the endogenous ligand. mGlu5 primarily couples to Gq/11 proteins leading to mobilization of intracellular Ca2+ (iCa2+), but also activates iCa2+ independent signaling pathways, with biased agonism/modulation operative for multiple positive allosteric modulator (PAM) and PAM-agonist chemotypes. Although several residues within the common allosteric binding pocket are key determinants of PAM activity, how these residues affect biased modulation is unknown. The current study probed the molecular basis of mGlu5 PAM biased modulation. Modulation of mGlu5 activity by four chemically distinct mGlu5 PAMs (VU0424465, DPFE, VU29 and VU0409551) was assessed across two distinct receptor endpoints (iCa2+ mobilization and ERK1/2 phosphorylation) at mGlu5 receptors containing single-point mutations of allosteric binding pocket residues informed by computational modeling. Many mutations had differential effects on PAM affinity and cooperativity across signaling endpoints, resulting in gain or reversal of bias at the level of both affinity and functional cooperativity. Additionally, mutants had differential effects on functional cooperativity between the orthosteric ligands, DHPG and glutamate, and the PAMs, DPFE and VU29, but not VU0409551, indicating that probe dependence is linked to orthosteric agonists conferring activation states that differentially influence allosteric ligand-receptor interactions in a chemotype dependent fashion. Collectively, these data provide crucial insight into the residues that govern different activation states adopted by mGlu5 in order to signal via distinct intracellular pathways when co-bound by orthosteric agonists and PAMs.

TRPC1 Regulates the Activity of a Voltage-Dependent Nonselective Cation Current in Hippocampal CA1 Neurons.

The cation channel subunit TRPC1 is strongly expressed in central neurons including neurons in the CA1 region of the hippocampus where it forms complexes with TRPC4 and TRPC5. To investigate the functional role of TRPC1 in these neurons and in channel function, we compared current responses to group I metabotropic glutamate receptor (mGluR I) activation and looked for major differences in dendritic morphology in neurons from TRPC1+/+ and TRPC1-/- mice. mGluR I stimulation resulted in the activation of a voltage-dependent nonselective cation current in both genotypes. Deletion of TRPC1 resulted in a modification of the shape of the current-voltage relationship, leading to an inward current increase. In current clamp recordings, the percentage of neurons that responded to depolarization in the presence of an mGluR I agonist with a plateau potential was increased in TRPC1-/- mice. There was also a small increase in the minor population of CA1 neurons that have more than one apical dendrite in TRPC1-/- mice. We conclude that TRPC1 has an inhibitory effect on receptor-operated nonselective cation channels in hippocampal CA1 neurons probably as a result of heterotetramer formation with other TRPC isoforms, and that TRPC1 deletion has only minor effects on dendritic morphology.

Effect of olive-derived antioxidants (3,4-dihydroxyphenylethanol and 3,4 dihydroxyphenylglycol) on sperm motility and fertility in liquid ram sperm stored at 15°C or 5°C.

The aim of the present study was to evaluate the effect of the addition of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during liquid storage at 5°C and 15°C. Semen was collected, pooled, diluted and then divided into aliquots supplemented with different concentrations (5 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. Sperm motility characteristics were assessed in the different samples at 0, 6, 24, 48, 72 and 96 hr after cooling, and a fertility trial was also conducted. The results showed that the antioxidant addition did not significantly improve total and progressive motility in ovine cooled sperm maintained at 15° or 5°C. However, in samples stored at 5°C, LIN (48, 72, 96 hr), STR (0 hr) and WOB (0, 48, 72, 96 hr) values significantly decreased in comparison with control treatment when high antioxidant concentrations were added (MIX100 or HT100). When samples were maintained at 15°C, MIX50 showed significantly higher VCL values than the control treatment after 6 hr cooling, and MIX100 showed significantly lower VCL values at 96 hr after cooling. According to the artificial insemination trial, no significant differences were observed when antioxidants were added. In conclusion, the use of HT and DHPG showed small impact in sperm motility and fertility was not affected (nor detrimentally nor positively) when insemination was carried out using antioxidant-supplemented liquid sperm.

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons.

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.

Neuroprotective effects of mGluR5 activation through the PI3K/Akt pathway and the molecular switch of AMPA receptors.

Previous studies have demonstrated that antagonists of mGluR1, but not mGluR5, are neuroprotective in models of cerebral ischemia. To investigate the individual roles of mGlu1 and mGlu5 receptors in in vitro model of cerebral ischemia we used low doses of the non-selective group I agonist DHPG and mGlu1 and mGlu5 selective positive allosteric modulators (PAMs). In hippocampal slices subjected to 30 min oxygen-glucose deprivation (OGD), DHPG (1 µM) and the mGluR5 PAM (VU0092273) significantly reduced OGD-induced CA1 injury monitored by propidium iodide staining of the slices and quantitative analysis of CA1 neurons. In contrast, the mGluR1 PAM (VU0483605) showed no neuroprotection. These protective effects of DHPG and VU0092273 were prevented by inhibition of PI3K/Akt pathway by LY294002. The mGluR5 PAM (VU0092273) also prevented GluA2 down-regulation triggered by ischemic injury, via PI3K/Akt pathway, revealing a further contribution to its neuroprotective effects by reducing the excitotoxic effects of increased Ca2+ influx through GluA2-lacking AMPA receptors. Furthermore, immunohistochemical assays confirmed the neuroprotective effect of VU0092273 and revealed activation of glia, indicating the involvement reactive astrogliosis in the mechanisms of neuroprotection. Our data suggest that selective activation/potentiation of mGluR5 signalling represents a promising strategy for the development of new interventions to reduce or prevent ischemia-induced neuronal death.

Increased Homer1-mGluR5 mediates chronic stress-induced depressive-like behaviors and glutamatergic dysregulation via activation of PERK-eIF2α.

Glutamatergic dysregulation has served as one common pathophysiology of major depressive disorder (MDD) and a promising target for treatment intervention. Previous studies implicate neurotransmission via metabotropic glutamate receptors (mGluRs) and Homer1 in stress-induced anhedonia, but the mechanisms have not been well elucidated. In the present study, we used two different animal models of depression, chronic social defeat stress (CSDS) and chronic restraint stress (CRS), to investigate the expression of Homer1 isoforms and functional interaction with mGluRs. We found that chronic stress selectively upregulated the expression of Homer1b/c in the hippocampus, whereas the level of Homer1a was unchanged. Additionally, there was a significant negative correlation between the levels of Homer1-mGluR5 signaling and depressive-like behaviors. Both application of paired-pulse low-frequency stimulation (PP-LFS) and the selective group 1 mGluRs agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) significantly enhanced mGluR-dependent long-term depression (LTD) at CA3-CA1 pyramidal cell synapses in slices from susceptible mice, whereas there was no change in NMDAR-dependent LTD induced by LFS. Furthermore, these effects were associated with the internalization of surface AMPARs in hippocampal pyramidal neurons, including reduced the expression of AMPARs and amplitude of AMPARs-mediated mEPSC. Finally, we found that chronic stress activated the KR-like ER kinase-eukaryotic initiation factor 2α (PERK-eIF2α) signaling pathway, subsequently phosphorylated cAMP response element binding protein (CREB) at the S129 and reduced the BDNF level, eventually leading to the impairment of synaptic transmission and depressive-like behaviors. Therefore, our study suggests that PERK-eIF2α acts as a critical target downstream of Homer1-mGluR5 complex to mediate chronic stress-induced depressive-like behaviors, and highlights them as a potential target for the treatment of mood disorder.

Effect of different olive oil-derived antioxidants (hydroxytyrosol and 3,4-dihydroxyphenylglycol) on the quality of frozen-thawed ram sperm.

The aim of the present study was to evaluate the effect of the addition of different concentrations of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during the freezing-thawing process. Sperm was collected, pooled and diluted with commercial extenders and then divided into aliquots supplemented with different concentrations (10 µg/ml, 30 µg/ml, 50 µg/ml and 70 µg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. A control group, without antioxidant, was also prepared. Sperm motility, viability, acrosome integrity, mitochondrial membrane potential and lipid peroxidation (LPO) were assessed. The results showed that frozen-thawed ram spermatozoa exhibited lower values for motility, membrane integrity, acrosome and mitochondrial membrane potential than fresh samples (P ≤ 0.01). However, when antioxidants were added, thawed spermatozoa exhibited relatively low LPO, recording values similar to fresh spermatozoa; by contrast, the control group of frozen-thawed spermatozoa without antioxidants exhibited significantly higher LPO (P ≤ 0.01). The addition of a HT+DHPG mixture (MIX) had a negative impact on sperm membrane and acrosome integrity, suggesting that a pure antioxidant supplementation has the potential to offer superior results. In conclusion, HT and DHPG exhibited a positive effect on the frozen-thawed spermatozoa inasmuch as they reduced the LPO. These olive oil-derived antioxidants have the potential to improve frozen-thawed sperm quality, although further studies should be carried out to analyse the antioxidant effect at different times after thawing.

Single Neuron Imaging Reveals Metabotropic Glutamate Receptor-Mediated Bursting and Delay in Calcium Oscillation in Hippocampal Neurons.

Measurement of cytosolic calcium activity in single neuron and quantification of response to drug treatment over a longer period remain challenging. Especially, analysis of amplitude, frequency, and delay in calcium response in G-protein coupled receptors (GPCR) targeting drug-treated neurons is complicated due to inherent heterogeneity. To address this, we have utilized k- means clustering to identify various sub-populations. Here we focus on measuring drug dose response in the single neuron by means of high-resolution confocal imaging. Metabotropic glutamate receptor was activated using 3,5-dihydroxyphenylglycine (DHPG). The result reveals an unusual delay in calcium response for higher dose particularly in responding neurons. The proposed methodology can be used for selection of drug dose and optimization of the time window for neurodegenerative diseases.

Effect of edible pectin-fish gelatin films containing the olive antioxidants hydroxytyrosol and 3,4-dihydroxyphenylglycol on beef meat during refrigerated storage.

The objective of this research was to evaluate the effect of the addition of two antioxidants naturally present in olives, hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG), to a pectin-fish gelatin edible film on the preservation of raw beef meat during refrigerated storage. A new composite film that included beeswax was also prepared, resulting in a reduction in the film's oxygen permeability. Results showed that the meat samples wrapped with film containing antioxidants reduced the formation of oxidation products in the form of thiobarbituric acid reaction substances (TBARS) compared with control film without antioxidants. HT added at 0.5% to the film with beeswax suppressed the lipid oxidation of beef meat during 7 days of storage at 4 °C, possibly by the combined effect of acting as an oxygen barrier and the specific antioxidant activity. The interference of plasticizer agents (glycerol and sorbitol) incorporated to the film on the TBARS method was showed for the first time.