DHPG Research Articles

A Nanobody-Based Proximity Ligation Assay Detects Constitutive and Stimulus-Regulated Native Arc/Arg3.1 Oligomers in Hippocampal Neuronal Dendrites.

Activity-regulated cytoskeleton-associatedprotein (Arc), the product of an immediate early gene, plays critical roles in synaptic plasticity and memory. Evidence suggests that Arc function is determined by its oligomeric state; however, methods for localization of native Arc oligomers are lacking. Here, we developed a nanobody-based proximity ligation assay (PLA) for detection, localization, and quantification of Arc-Arc complexes in primary rat hippocampal neuronal cultures. We used nanobodies with single, structurally defined epitopes in the bilobar Arc capsid domain. Nanobody H11 binds inside the N-lobe ligand pocket, while nanobody C11 binds to the C-lobe surface. For each nanobody, ALFA- and FLAG-epitope tags created a platform for antibody binding and PLA. Surprisingly, PLA puncta in neuronal dendrites revealed widespread constitutive Arc-Arc complexes. Treatment of cultures with tetrodotoxin or cycloheximide had no effect, suggesting stable complexes that are independent of recent neuronal activity and protein synthesis. To assess detection of oligomers, cultures were exposed to a cell-penetrating peptide inhibitor of the Arc oligomerization motif (OligoOFF). Arc-Arc complexes detected by H11 PLA were inhibited by OligoOff but not by control peptide. Notably, Arc complexes detected by C11 were unaffected by OligoOFF. Furthermore, we evaluated Arc complex formation after chemical stimuli that increase Arc synthesis. Brain-derived neurotrophic factor increased Arc-Arc signal detected by C11, but not H11. Conversely, dihydroxyphenylglycine (DHPG) treatment selectively enhanced H11 PLA signals. In sum, nanobody-based PLA reveals constitutive and stimulus-regulated Arc oligomers in hippocampal neuronal dendrites. A model is proposed based on detection of Arc dimer by C11 and higher-order oligomer by H11 nanobody.

Dynamics of astrocytes Ca2+ signaling: a low-cost fluorescence customized system for 2D cultures.

In an effort to help reduce the costs of fluorescence microscopy and expand the use of this valuable technique, we developed a low-cost platform capable of visualising and analysing the spatio-temporal dynamics of intracellular Ca2+ signalling in astrocytes. The created platform, consisting of a specially adapted fluorescence microscope and a data analysis procedure performed with Imagej Fiji software and custom scripts, allowed us to detect relative changes of intracellular Ca2+ ions in astrocytes. To demonstrate the usefulness of the workflow, we applied the methodology to several in vitro astrocyte preparations, specifically immortalised human astrocyte cells and wild-type mouse cells. To demonstrate the reliability of the procedure, analyses were conducted by stimulating astrocyte activity with the agonist dihydroxyphenylglycine (DHPG), alone or in the presence of the antagonist 2-methyl-6-phenylethyl-pyridine (MPEP).

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-β (Aβ) plaques and neurofibrillary tangles. Aβ 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 Aβ toxicity (Durakoglugil et al., 2009) Reelin signaling is impaired by ApoE4, the most important genetic risk factor for AD, and Aβ-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-β 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.

Orai1 is a crucial downstream partner of group I metabotropic glutamate receptor signaling in dorsal horn neurons.

Group I metabotropic glutamate receptors (group I mGluRs) have been implicated in several central nervous system diseases including chronic pain. It is known that activation of group I mGluRs results in the production of inositol triphosphate (IP3) and diacylglycerol that leads to activation of extracellular signal-regulated kinases (ERKs) and an increase in neuronal excitability, but how group I mGluRs mediate this process remains unclear. We previously reported that Orai1 is responsible for store-operated calcium entry and plays a key role in central sensitization. However, how Orai1 is activated under physiological conditions is unknown. Here, we tested the hypothesis that group I mGluRs recruit Orai1 as part of its downstream signaling pathway in dorsal horn neurons. We demonstrate that neurotransmitter glutamate induces STIM1 puncta formation, which is not mediated by N-Methyl-D-aspartate (NMDA) or α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Glutamate-induced Ca2+ entry in the presence of NMDA or AMPA receptor antagonists is eliminated in Orai1-deficient neurons. Dihydroxyphenylglycine (DHPG) (an agonist of group I mGluRs)-induced Ca2+ entry is abolished by Orai1 deficiency, but not affected by knocking down of transient receptor potential cation channel 1 (TRPC1) or TRPC3. Dihydroxyphenylglycine-induced activation of ERKs and modulation of neuronal excitability are abolished in cultured Orai1-deficient neurons. Moreover, DHPG-induced nociceptive behavior is markedly reduced in Orai1-deficient mice. Our findings reveal previously unknown functional coupling between Orai1 and group I mGluRs and shed light on the mechanism underlying group I mGluRs-mediated neuronal plasticity.

Impaired cerebellar plasticity and eye-blink conditioning in calpain-1 knock-out mice.

Calpain-1 and calpain-2 are involved in the regulation of several signaling pathways and neuronal functions in the brain. Our recent studies indicate that calpain-1 is required for hippocampal synaptic plasticity, including long-term depression (LTD) and long-term potentiation (LTP) in field CA1. However, little is known regarding the contributions of calpain-1 to cerebellar synaptic plasticity. Low frequency stimulation (LFS, 5 Hz, 5 min)-induced LTP at parallel fibers to Purkinje cell synapses was markedly impaired in cerebellar slices from calpain-1 knock-out (KO) mice. Application of a selective calpain-2 inhibitor enhanced LFS-induced LTP in both wild-type (WT) and calpain-1 KO mice. Three protocols were used to induce LTD at these synapses: LFS (1 Hz, 15 min), perfusion with high potassium and glutamate (K-Glu) or dihydroxyphenylglycine (DHPG), a mGluR1 agonist. All three forms of LTD were impaired in calpain-1 KO mice. DHPG application stimulated calpain-1 but not calpain-2 in cerebellar slices, and DHPG-induced LTD impairment was reversed by application of a protein phosphatase 2A (PP2A) inhibitor, okadaic acid. As in hippocampus, BDNF induced calpain-1 activation and PH domain and Leucine-rich repeat Protein Phosphatase 1/suprachiasmatic nucleus oscillatory protein (PHLPP1/SCOP) degradation followed by extracellular signal-regulated kinase (ERK) activation, as well as calpain-2 activation leading to degradation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in cerebellar slices. The role of calpain-1 in associative learning was evaluated in the delay eyeblink conditioning (EBC). Calpain-1 KO mice exhibited significant learning impairment in EBC during the first 2 days of acquisition training. However, after 5 days of training, the percentage of conditioned responses (CRs) between calpain-1 KO and WT mice was identical. Both calpain-1 KO and WT mice exhibited typical extinction patterns. Our results indicate that calpain-1 plays critical roles in multiple forms of synaptic plasticity and associative learning in both hippocampus and cerebellum.

The sigma1 receptor agonist enhances long-term depression caused by activation of metabotropic glutamate receptors in rat hippocampal neurons

Introduction. Metabotropic glutamate receptors of group 1 (mGluR1/5) are an important component of the hippocampal inhibitory system. Disturbances of their work lead to manifestation of various pathologies of the nervous system. The search for effective ways of regulating the work of mGluR1/5 is an urgent problem of neuropharmacology. In the present study, we assessed for the first time the possibility of modulating mGluR1/5 by activation of sigma1 receptors (Sig1-R), chaperone proteins capable of directly to directly interact with other proteins and to enhance their activity. Objective. To study the effect of a specific Sig1-R agonist, PRE-084, on long-term depression of glutamate inputs in the hippocampus caused by activation of mGluR1/5. Materials and methods. On slices of the hippocampus of rat brain in the CA1 region, population spikes (PS) caused by stimulation of Shaffer collaterals were registered. Activation of mGluR1/5 was induced by the application of 50 μM of specific agonist of these receptors, dihydroxyphenylglycine (DHPG), for 10 min. Activation of Sig1-R was induced by the application of 10 μM of specific agonist PRE-084 for 20 min. Results. A short-term incubation of a rat hippocampal slice in a solution containing DHPG, caused a long-term depression of PS, so that within 30 min after the slice was washed away from the preparation, its amplitude was 60.0±14.4% of the control value (n=5). In the second series of experiments DHPG was applied against the background of the agonist Sig1-R PRE-084. PRE-084 was found to cause an increase in the inhibitory aftereffect of DHPG, so that the amplitude of the PS measured after 30 min after removal of the preparations from the surrounding solution was 16.0±1.7% (р<0.05). Conclusion. It was shown for the first time that activation of Sig1-R enhances the functional activity of mGluR1/5.

Emergence of Endocytosis-Dependent mGlu1 LTD at Nucleus Accumbens Synapses After Withdrawal From Cocaine Self-Administration.

Extended-access cocaine self-administration induces a progressive intensification of cue-induced drug craving during withdrawal termed “incubation of cocaine craving”. Rats evaluated after >1 month of withdrawal (when incubation of craving is robust) display alterations in excitatory synapses onto medium spiny neurons (MSNs) of the nucleus accumbens (NAc), including elevated levels of Ca2+-permeable AMPA receptors (CP-AMPAR) and a transition from group I metabotropic glutamate receptor (mGluR) mGlu5- to mGlu1-mediated synaptic depression. It is important to further characterize the emergent form of mGlu1-mediated synaptic depression because it has been demonstrated that mGlu1 stimulation, by normalizing CP-AMPAR transmission, reduces cue-induced cocaine craving. In the present study, we conducted whole-cell patch-clamp recordings in NAc core MSNs, comparing rats that underwent >35 days of withdrawal from cocaine self-administration to control rats that had self-administered saline. Bath application of the nonselective group I mGluR agonist dihydroxyphenylglycine (DHPG) produced a transient mGlu5-mediated synaptic depression in saline controls, whereas a persistent mGlu1-mediated synaptic depression emerged in cocaine rats. This form of long-term depression (LTD) was abolished by the inclusion of dynamin inhibitory peptide (DIP) in the recording electrode, indicating that it is mediated by removal of CP-AMPARs through a dynamin-dependent endocytosis mechanism. We further showed that CP-AMPAR endocytosis is normally coupled to the PICK1-mediated insertion of Ca2+-impermeable AMPARs (CI-AMPAR). Interestingly, this coupling is not obligatory because disruption of PICK1-mediated CI-AMPAR insertion with pep2-EVKI spared mGlu1-mediated CP-AMPAR endocytosis. Collectively, these results reveal similarities but also differences from mGlu1-LTD observed in other brain regions, and further our understanding of a form of plasticity that may be targeted to reduce cue-induced craving for cocaine and methamphetamine.

AgRP/NPY Neuron Excitability Is Modulated by Metabotropic Glutamate Receptor 1 During Fasting.

The potential to control feeding behavior via hypothalamic AgRP/NPY neurons has led to many approaches to modulate their excitability—particularly by glutamatergic input. In the present study using NPY-hrGFP reporter mice, we visualize AgRP/NPY neuronal metabotropic glutamate receptor 1 (mGluR1) expression and test the effect of fasting on mGluR1 function. Using the pharmacological agonist dihydroxyphenylglycine (DHPG), we demonstrate the enhanced capacity of mGluR1 to drive firing of AgRP/NPY neurons after overnight fasting, while antagonist 3-MATIDA reduces firing. Further, under synaptic blockade we demonstrate that DHPG acts directly on AgRP/NPY neurons to create a slow inward current. Using an in vitro approach, we show that emulation of intracellular signals associated with fasting by forskolin enhances DHPG induced phosphorylation of extracellularly regulated-signal kinase (1/2) in GT1-7 cell culture. We show in vivo that blocking mGluR1 by antagonist 3-MATIDA lowers fasting induced refeeding. In summary, this study identifies a novel layer of regulation on AgRP/NPY neurons integrated with whole body energy balance.

Pinetonina&amp;lt;sup&amp;gt;TM&amp;lt;/sup&amp;gt;, an Intranasally Administered Essential Oil Preparation, Is Effective in Decrease of Cortisol Levels and on the Glutamate Release Modulation

Background: Most anxiety complaints are treated with pharmacological measures involving barbiturates and benzodiazepines, in which they may end up causing tolerance and pharmacological dependence. Integrative approaches such as aromatherapy are used in addition to medications to improve sleep quality and reduce anxiety. Thus, PinetoninTM, a phytocomplex obtained from a blend of essential oils aims to aid in the symptoms of stress and anxiety. Methods: The cytotoxicity of PinetoninTM was evaluated MTT assay using fibroblasts and astrocytes showed reduction in the cell viability only at high concentrations. Evaluation of intracellular calcium and determination of residual glutamate in the supernatant of astrocyte cultures showed agonist action of dihydroxyphenylglycine (DHPG) increasing linearly the concentration of intracellular calcium and the glutamate levels in the supernatants of the cultures. On the other hand, cultures of astrocytes treated with PinetoninTM showed residual glutamate levels in the supernatants reducing proportionally, as well as, intracellular calcium reduction. The determination of salivary cortisol showed a significant reduction in salivary cortisol levels in the group that received PinetoninTM. The evaluation of the electroencephalogram in patients treated with PinetoninTM had a significant increase (P TM treated fibroblasts with control culture. When PinetoninTM and linole are administered in astrocytic cells, there was a reduction of the intracellular concentration of Ca2+ against a control group treated with DHPG agonist. The evaluation of salivary cortisol demonstrated a reduction when the patient group was treated with PinetoninTM by purchasing the results against the group of patients treated with saline. Reinforcing the relaxed state of that group, alpha waves have been increased and reductions in beta waves. Conclusion: The results obtained after intranasal administration of PinetoninTM suggest that this phytocomplex can help reduce the symptoms of stress and the better quality of life.

Role of TRPC1 ion channel in hippocampal neurons

Event Abstract Back to Event Role of TRPC1 ion channel in hippocampal neurons Sophie Lepannetier1*, Olivier Schakman1, Francois Seghers1 and Philippe Gailly1 1 Université catholique de Louvain, Institute of Neuroscience, Belgium Transient receptor potential canonical 1 (TRPC1) is a non selective cation channel. It is involved in stored-operated Ca2+ entry in cooperation with Orai channel and activated by STIM1, a sensor of Ca2+ contents in the endoplamic reticulum. However, several pieces of evidence suggest that TRPC1 can be activated independently of store depletion in response to agonists. Using LacZ reporter mouse, we found that TRPC1 is abundantly expressed in neurons from CA1-CA3 regions and to a lesser extent from the dentate gyrus region of the hippocampus. We compared cellular culture of hippocampal neurons from Trpc1-/- mice to wild type mice. Interestingly, stimulation with dihydroxyphenylglycine (DHPG), an agonist of group I metabotropic glutamate receptors (consisting of mGluR1 and mGluR5) induced a store-independent entry of Ca2+ that was very much reduced in hippocampal neurons from Trpc1-/- mice. Studying brains slices, we observed that LTP in Schaffer collaterals-CA1 synpases was diminished in Trpc1-/- mice when induced with a mild stimulus (theta burst consisting of 4 stimuli at 100Hz repeated at 5 Hz during 1s). Cognition tests such as Modified Y maze and contextual fear conditioning, realized on 4-8 months old mice revealed a deficit memory. Moreover, we noted that the specific hippocampal overexpression of the immediate early gene zif268 related to new environment learning was blunted in Trpc1-/- mice. We conclude that TRPC1 channels are activated by mGluR5 stimulation and play a role in neuronal plasticity. Keywords: TRPC1, mGluR, Hippocampus, Neuronal Plasticity, Memory Conference: 12th National Congress of the Belgian Society for Neuroscience, Gent, Belgium, 22 May - 22 May, 2017. Presentation Type: Poster Presentation Topic: Neural Excitability, Synapses, and Glia: Cellular Mechanisms Citation: Lepannetier S, Schakman O, Seghers F and Gailly P (2019). Role of TRPC1 ion channel in hippocampal neurons. Front. Neurosci. Conference Abstract: 12th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2017.94.00031 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 Apr 2017; Published Online: 25 Jan 2019. * Correspondence: Mrs. Sophie Lepannetier, Université catholique de Louvain, Institute of Neuroscience, Brussels, 1150, Belgium, slepannetier@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sophie Lepannetier Olivier Schakman Francois Seghers Philippe Gailly Google Sophie Lepannetier Olivier Schakman Francois Seghers Philippe Gailly Google Scholar Sophie Lepannetier Olivier Schakman Francois Seghers Philippe Gailly PubMed Sophie Lepannetier Olivier Schakman Francois Seghers Philippe Gailly Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Metabotropic glutamate receptor 1 promotes cementoblast proliferation via MAP kinase signaling pathways

ABSTRACTPurpose/aim: Glutamate is one of the signaling molecules responsible for transmission in the central nervous system. Periodontal ligament (PDL) cells were recently reported to express metabotropic glutamate receptors (mGluRs). However, the functions of mGluR signaling in PDL cells or PDL-related cells remain largely unknown. The aim of this study was to investigate the expression and function of mGluRs in PDL-related cells. Materials and methods: OCCM-30 cells, immortalized murine cementoblasts, were stimulated with l-glutamate or mGluRs antagonists. The cells’ proliferative response was evaluated using a colorimetric assay and gene expression was assessed using real-time polymerase chain reaction. The nuclear translocation of cyclin D1 was evaluated by immunohistochemistry. Results: l-Glutamate promoted the proliferation of OCCM-30 cells, which expressed mGluR1, but not mGluR5. Dihydroxyphenylglycine (DHPG), an agonist of group I mGluRs (mGluR1 and mGluR5), also promoted cell proliferation, and this was inhibited by LY456236, an mGluR1 antagonist. DHPG increased the expression of cyclin D1, a key regulator of cell proliferation, and its nuclear translocation. DHPG also increased the expression of Bcl2A1, an antiapoptotic oncogene and simultaneously reduced the expression of Bax, a pro-apoptotic marker. Furthermore, the DHPG-induced proliferation of OCCM-30 cells was reduced by pretreatment with SB203580, SP600125, and PD98059, inhibitors of p38, JNK, and ERK1/2, respectively. Conclusions: These findings indicate that activation of mGluR1 expressed by OCCM-30 cells induces cell proliferation in a manner that is dependent on mitogen-activated protein kinase pathways and that cyclin D1 and Bcl2A1/Bax may be involved. Our results provide useful information for elucidating the mechanisms underlying cementum homeostasis and regeneration.

Effects of groupImetabotropic glutamate receptor on the synaptic transmission efficiency of the neurons in primary visual cortex of monocular form deprivated rats

Background The formation of amblyopia is closely associated with glutamate receptors of visual cortex, and researches determined that the expression of metabolic glutamate receptor 1 (mGluR1) in the visual cortex of amblyopic rats is down-regulated.However, the effect of mGluR1 on synaptic transmission of neurons in the visual cortex is unclear. Objective This study was to investigate the role of mGluR1 in the synaptic transmission efficiency of neurons in the primary visual cortex of monocular form-deprivated rats. Methods Sixteen 14-day-old SD rats were randomly divided into normal control group and form-deprivation group, with 8 rats in each group.The eyelids of the left eyes were sutured to establish the monocular form-deprivation models in the rats of the form-deprivation group, and the visual cortex slices were prepared until 31 days after modeling and incubated in artificial cerebrospinal fluid with 3, 5-dihydroxyphenylglycine (DHPG), LY367385+ DHPG, 2-methyl-6-(phenyl acetylene) pyridine hydrochloride(MPEP)+ DHPG and LY367385+ MPEP+ DHPG, respectively.The field excitatory postsynaptic potential (fEPSP) of neurons in the visual cortex of V1 area was detected by extracellular microelectrode recording.The use and care of the animals followed the statement for the Use of Animals in Ophthalmic and Vision Research. Results The slope value before drug treatment was set as 100%, the fEPSP-slopes of the normal control group and the form-deprivation group were increased to (136.4±17.3)% and (120.7±12.8)% after DHPG induction with significant difference between the two groups (t=2.280; P<0.05). The fEPSP-slopes of the normal control group were (114.9±9.3)% and (112.6±15.3)% after LY367385 and MPEP treatment, and those of the form-deprivation group were (107.3±6.0)% and (110.1±4.1)%, which were significantly lower than those after DHPG induction (normal control group: t=2.641, 2.915; both at P<0.05; form-deprivation group: t=2.410, 2.372; both at P<0.05). The fEPSP-slopes of the normal control group and the form-deprivation group were (104.5±2.2)% and (102.8±14.9)% after LY367385+ MPEP treatment, which were lower than that after DHPG induction (t=3.080, 2.306; both at P<0.05). Conclusions The effect of mGluR1 on the synaptic transmission of neurons in the visual cortex weakens after monocular form-deprivation in rats.Both mGluR1 and mGluR5 participate in the neuronic synaptic transmission on visual cortex area and appear to be equivalent contribution. Key words: Electrophysiology; Excitatory amino acid agonists/pharmacology; Excitatory amino acid antagonists/pharmacology; Phenylacetates/pharmacology; Receptors, metabotropic glutamate/physiology; Visual cortex/growth & development; Neuronic synaptic transmission/drug effect; Form deprivation; Rats, SD

Antagonists reversibly reverse chemical LTD induced by group I, group II and group III metabotropic glutamate receptors

Metabotropic glutamate (mGlu) receptors are implicated in many neurological and psychiatric diseases and are the targets of therapeutic agents currently in clinical development. Their activation has diverse effects in the central nervous system (CNS) that includes an involvement in synaptic plasticity. We previously reported that the brief exposure of hippocampal slices to dihydroxyphenylglycine (DHPG) can result in a long-term depression (LTD) of excitatory synaptic transmission. Surprisingly, this LTD could be fully reversed by mGlu receptor antagonists in a manner that was itself fully reversible upon washout of the antagonist. Here, 15 years after the discovery of DHPG-LTD and its reversible reversibility, we summarise these initial findings. We then present new data on DHPG-LTD, which demonstrates that evoked epileptiform activity triggered by activation of group I mGlu receptors can also be reversibly reversed by mGlu receptor antagonists. Furthermore, we show that the phenomenon of reversible reversibility is not specific to group I mGlu receptors. We report that activation of group II mGlu receptors in the temporo-ammonic pathway (TAP) and mossy fibre pathway within the hippocampus and in the cortical input to neurons of the lateral amygdala induces an LTD that is reversed by LY341495, a group II mGlu receptor antagonist. We also show that activation of group III mGlu8 receptors induces an LTD at lateral perforant path inputs to the dentate gyrus and that this LTD is reversed by MDCPG, an mGlu8 receptor antagonist. In conclusion, we have shown that activation of representative members of each of the three groups of mGlu receptors can induce forms of LTD than can be reversed by antagonists, and that in each case washout of the antagonist is associated with the re-establishment of the LTD.This article is part of the Special Issue entitled ‘Glutamate Receptor-Dependent Synaptic Plasticity’.

Norepinephrine transporter variant A457P knock-in mice display key features of human postural orthostatic tachycardia syndrome

SUMMARYPostural orthostatic tachycardia syndrome (POTS) is a common autonomic disorder of largely unknown etiology that presents with sustained tachycardia on standing, syncope and elevated norepinephrine spillover. Some individuals with POTS experience anxiety, depression and cognitive dysfunction. Previously, we identified a mutation, A457P, in the norepinephrine (NE; also known as noradrenaline) transporter (NET; encoded by SLC6A2) in POTS patients. NET is expressed at presynaptic sites in NE neurons and plays a crucial role in regulating NE signaling and homeostasis through NE reuptake into noradrenergic nerve terminals. Our in vitro studies demonstrate that A457P reduces both NET surface trafficking and NE transport and exerts a dominant-negative impact on wild-type NET proteins. Here we report the generation and characterization of NET A457P mice, demonstrating the ability of A457P to drive the POTS phenotype and behaviors that are consistent with reported comorbidities. Mice carrying one A457P allele (NET+/P) exhibited reduced brain and sympathetic NE transport levels compared with wild-type (NET+/+) mice, whereas transport activity in mice carrying two A457P alleles (NETP/P) was nearly abolished. NET+/P and NETP/P mice exhibited elevations in plasma and urine NE levels, reduced 3,4-dihydroxyphenylglycol (DHPG), and reduced DHPG:NE ratios, consistent with a decrease in sympathetic nerve terminal NE reuptake. Radiotelemetry in unanesthetized mice revealed tachycardia in NET+/P mice without a change in blood pressure or baroreceptor sensitivity, consistent with studies of human NET A457P carriers. NET+/P mice also demonstrated behavioral changes consistent with CNS NET dysfunction. Our findings support that NET dysfunction is sufficient to produce a POTS phenotype and introduces the first genetic model suitable for more detailed mechanistic studies of the disorder and its comorbidities.

Dual Effect of 17β-Estradiol on NMDA-Induced Neuronal Death: Involvement of Metabotropic Glutamate Receptor 1

Pretreatment with 10 nm 17β-estradiol (17βE2) or 100 μm of the metabotropic glutamate 1 receptor (mGlu1R) agonist, dihydroxyphenylglycine (DHPG), protected neurons against N-methyl-d-aspartate (NMDA) toxicity. This effect was sensitive to blockade of both estrogen receptors and mGlu1R by their respective antagonists. In contrast, 17βE2 and/or DHPG, added after a low-concentration NMDA pulse (45 μm), produced an opposite effect, i.e. an exacerbation of NMDA toxicity. Again this effect was prevented by both receptor antagonists. In support of an interaction of estrogen receptors and mGlu1R in mediating a neurotoxic response, exacerbation of NMDA toxicity by 17βE2 disappeared when cultures were treated with DHPG prior to NMDA challenge, and conversely, potentiation of NMDA-induced cell death by DHPG was prevented by pretreatment with 17βE2. Addition of calpain III inhibitor (10 μm), 2 h before NMDA, prevented the increased damage induced by the two agonists, an affect that can be secondary to cleavage of mGlu1R by calpain. Accordingly, NMDA stimulation reduced expression of the full-length (140 kDa) mGluR1, an effect partially reversed by calpain inhibitor. Finally, in the presence of NMDA, the ability of 17βE2 to stimulate phosphorylation of AKT and ERK was impaired. Pretreatment with calpain inhibitor prevented the reduction of phosphorylated ERK but had no significant effect on phosphorylated AKT. Accordingly, the inhibition of ERK signaling by U0126 (1 μm) counteracted the effect of calpain inhibition on 17βE2-induced exacerbation of NMDA toxicity. The present data confirm the dual role of estrogens in neurotoxicity/neuroprotection and highlight the role of the timing of exposure to estrogens.

Single-molecule imaging of translational output from individual RNA granules in neurons.

Dendritic RNAs are localized and translated in RNA granules. Here we use single-molecule imaging to count the number of RNA molecules in each granule and to record translation output from each granule using Venus fluorescent protein as a reporter. For RNAs encoding activity-regulated cytoskeletal-associated protein (ARC) or fragile X mental retardation protein (FMRP), translation events are spatially clustered near individual granules, and translational output from individual granules is either sporadic or bursty. The probability of bursty translation is greater for Venus-FMRP RNA than for Venus-ARC RNA and is increased in Fmr1-knockout neurons compared to wild-type neurons. Dihydroxyphenylglycine (DHPG) increases the rate of sporadic translation and decreases bursty translation for Venus-FMRP and Venus-ARC RNAs. Single-molecule imaging of translation in individual granules provides new insight into molecular, spatial, and temporal regulation of translation in granules.

Examination of a role for metabotropic glutamate receptor 5 in the medial prefrontal cortex in cocaine sensitization in rats

Glutamatergic projection neurons in the medial prefrontal cortex (mPFC) are hyperexcitable in cocaine-sensitized animals, resulting in increased excitatory output to addiction-associated regions such as the ventral tegmental area (VTA) and nucleus accumbens. Evidence suggests that Group I metabotropic glutamate receptor 5 (mGluR5) is necessary for cocaine sensitization, and stimulation of this receptor in the mPFC potentially alters cell excitability directly through glutamate release or indirectly through downstream signaling cascades. Experiments in this report examined the role of mPFC mGluR5 in behavioral sensitization to cocaine. Group I mGluR agonist dihydroxyphenylglycine (DHPG) (15 nmol/side), mGluR5 antagonist 3((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP) (15 nmol/side), mGluR1 antagonist YM298198 (15 nmol/side), AMPA receptor antagonist CNQX (1 nmol/side), and/or saline were administered through cannulae implanted 1 mm above the mPFC and/or VTA in male rats. Cocaine (15 mg/kg, i.p.) was systemically administered for four consecutive days to induce sensitization and/or once on test day immediately preceding locomotor monitoring. Intra-mPFC DHPG induced an mGluR5-mediated cross-sensitization to cocaine preventable through the prior administration of an AMPA receptor antagonist in the VTA. Furthermore, mGluR5 blockade in the mPFC failed to prevent the initiation of sensitization. However, intra-mPFC injections of the mGluR5 antagonist MTEP prevented the expression of cocaine sensitization at 21, but not 7, days following daily cocaine injections suggesting a possible role for mPFC mGluR5 in the persistence of the cocaine-sensitized state. These data suggest that stimulation of mGluR5s in the mPFC is sufficient to induce cocaine sensitization and is necessary for the expression of this sensitized response.

Excitatory Modulation in the Cochlear Nucleus through Group I Metabotropic Glutamate Receptor Activation

Activation of group I metabotropic glutamate receptors (mGluRs) has been suggested to modulate development of auditory neurons. However, the acute effects of mGluR activation on physiological response properties are unclear. To address this, we studied the effects of mGluRs in bushy cells (BCs) of the mammalian anteroventral cochlear nucleus (AVCN). Activation of mGluRs with dihydroxyphenylglycine (DHPG) caused depolarization of BCs in mice as old as P42, but did not affect neurotransmitter release by presynaptic auditory nerve (AN) fibers. Application of mGluR antagonists indicated that mGluRs are tonically active, and are highly sensitive to small elevations in ambient glutamate by the glutamate reuptake blocker threo-β-benzyloxyaspartic acid (TBOA). mGluR-mediated depolarization enhanced the firing probability in response to AN stimulation, and reduced the latency and jitter. Furthermore, excitation through postsynaptic mGluRs can significantly counterbalance the inhibitory effects of presynaptic GABA(B) receptors. Thus, interaction between these two modulatory pathways may provide additional flexibility for fine-tuning the BC relay.

Preclinical profile of a novel metabotropic glutamate receptor 5 positive allosteric modulator

Recent reports have indicated that patients with schizophrenia have a profound hypo-functionality of glutamatergic signaling pathways. Positive allosteric modulation of mGlu 5 receptor has been postulated to augment NMDA function and thereby alleviate the glutamatergic hypo-function observed in schizophrenic patients. Here we report the in vitro and in vivo characterization of CPPZ (1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone), a structurally novel positive allosteric modulator selective for mGlu 5 receptor. In HEK293 cells stably over-expressing human mGlu 5 receptor, CPPZ potentiates the intracellular calcium response elicited by a suboptimal concentration of the endogenous agonist glutamate. CPPZ does not have any intrinsic agonist activity and behaves functionally as a positive allosteric modulator. This is further supported by binding data, which demonstrate that CPPZ is able to displace the negative allosteric modulator MPEP but does not compete with the orthosteric ligand quisqualic acid. Instead, CPPZ enhances the binding of the orthosteric ligand. In native preparations, CPPZ potentiates calcium flux in rat cortical neurons stimulated with the group I agonist dihydroxyphenylglycine (DHPG). In addition, CPPZ modulates long-term potentiation in rat hippocampal slices, a process known to be NMDA dependent. In vivo, CPPZ reverses hyper locomotion triggered by the NMDA open channel blocker MK801 in CD1 mice. CPPZ was also able to reduce rat conditioned avoidance responding to electric shock. Both in vitro and in vivo data demonstrate that this novel compound acts as an mGlu 5 receptor positive allosteric modulator, which modulates NMDA dependent responses and suggests that the enhancement of mGlu 5 receptor activity may prove useful in the treatment of schizophrenia.

CB1 receptor antagonism impairs the induction of epileptiform activity by group I metabotropic glutamate receptor activation

Exposure to the group I metabotropic glutamate receptor (mGluR) agonist dihydroxy phenylglycine (DHPG) induces epileptiform activity in the CA3 region of the hippocampus that persists following washout of DHPG. DHPG also can cause long-term depression of synaptic transmission, and at some synapses this may be mediated by endocannabinoids. We evaluated whether the selective cannabinoid type 1 (CB1) receptor antagonists SR 141716 or AM 251 could modify induction of epileptiform activity produced by DHPG exposure. The induction of epileptiform activity by DHPG exposure was significantly reduced by CB1 receptor antagonists, SR 141716 or AM 251. Minimal effects on epileptiform activity were noted once the activity had been induced. In control slices, exposure to DHPG for 30 min produced long-term depression (LTD) of synaptic transmission, on average about a 70% reduction in slope of the field excitatory postsynaptic potential (EPSP). When slices were exposed to both DHPG and SR 141716 (3 microm), LTD did not occur and the population EPSP remained at control values or greater. These results suggest that CB1 receptors mediate some of DHPG effects that result in persistent epileptiform activity, and antagonism of CB1 receptors has antiepileptogenic properties. Paradoxically DHPG also caused LTD of excitatory synaptic transmission in the CA3 region and CB1 receptor antagonism prevents the depression. We hypothesize that the ictal activity induced by DHPG requires depression of synaptic strength and CB1 receptor antagonism prevents this depression and the induction of ictal activity.