Evidence for Glutamate as a Neuroglial Transmitter within Sensory Ganglia

This study examines key elements of glutamatergic transmission within sensory ganglia of the rat. We show that the soma of primary sensory neurons release glutamate when depolarized. Using acute dissociated mixed neuronal/glia cultures of dorsal root ganglia (DRG) or trigeminal ganglia and a colorimetric assay, we show that when glutamate uptake by satellite glial cells (SGCs) is inhibited, KCl stimulation leads to simultaneous increase of glutamate in the culture medium. With calcium imaging we see that the soma of primary sensory neurons and SGCs respond to AMPA, NMDA, kainate and mGluR agonists, and selective antagonists block this response. Using whole cell patch-clamp technique, inward currents were recorded from small diameter (<30 µm) DRG neurons from intact DRGs (ex-vivo whole ganglion preparation) in response to local application of the above glutamate receptor agonists. Following a chronic constriction injury (CCI) of either the inferior orbital nerve or the sciatic nerve, glutamate expression increases in the trigeminal ganglia and DRG respectively. This increase occurs in neurons of all diameters and is present in the somata of neurons with injured axons as well as in somata of neighboring uninjured neurons. These data provides additional evidence that glutamate can be released within the sensory ganglion, and that the somata of primary sensory neurons as well as SGCs express functional glutamate receptors at their surface. These findings, together with our previous gene knockdown data, suggest that glutamatergic transmission within the ganglion could impact nociceptive threshold.     

Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)

Voltage-gated sodium channels play important roles in modulating dorsal root ganglion (DRG) neuron hyperexcitability and hyperalgesia after peripheral nerve injury or inflammation. We report that chronic compression of DRG (CCD) produces profound effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents, which are different from that by chronic constriction injury (CCI) of the sciatic nerve in small DRG neurons. Whole cell patch-clamp recordings were obtained in vitro from L₄ and/or L₅ dissociated, small DRG neurons following in vivo DRG compression or nerve injury. The small DRG neurons were classified into slow and fast subtype neurons based on expression of the slow-inactivating TTX-R and fast-inactivating TTX-S Na⁺ currents. CCD treatment significantly reduced TTX-R and TTX-S current densities in the slow and fast neurons, but CCI selectively reduced the TTX-R and TTX-S current densities in the slow neurons. Changes in half-maximal potential (V_1/2) and curve slope (k) of steady-state inactivation of Na⁺ currents were different in the slow and fast neurons after CCD and CCI treatment. The window current of TTX-R and TTX-S currents in fast neurons were enlarged by CCD and CCI, while only that of TTX-S currents in slow neurons was increased by CCI. The decay rate of TTX-S and both TTX-R and TTX-S currents in fast neurons were reduced by CCD and CCI, respectively. These findings provide a possible sodium channel mechanism underlying CCD-induced DRG neuron hyperexcitability and hyperalgesia and demonstrate a differential effect in the Na⁺ currents of small DRG neurons after somata compression and peripheral nerve injury. This study also points to a complexity of hyperexcitability mechanisms contributing to CCD and CCI hyperexcitability in small DRG neurons.     

mGluR long‐term depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum

mGluR long‐term depression (mGluR‐LTD) is a form of synaptic plasticity induced at excitatory synapses by metabotropic glutamate receptors (mGluRs). mGluR‐LTD reduces synaptic strength and is relevant to learning and memory, autism, and sensitization to cocaine; however, the mechanism is not known. Here we show that activation of Group I mGluRs in medium spiny neurons induces trafficking of GluA2 from the endoplasmic reticulum (ER) to the synapse by enhancing GluA2 binding to essential COPII vesicle proteins, Sec23 and Sec13. GluA2 exit from the ER further depends on IP3 and Ryanodine receptor‐controlled Ca²⁺ release as well as active translation. Synaptic insertion of GluA2 is coupled to removal of high‐conducting Ca²⁺‐permeable AMPA receptors from synapses, resulting in synaptic depression. This work demonstrates a novel mechanism in which mGluR signals release AMPA receptors rapidly from the ER and couple ER release to GluA2 synaptic insertion and GluA1 removal.     

Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X(3) receptor

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X(3) receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X(3) receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X(3) agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X(3) mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X(3) agonist-activated currents and P2X(3) mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X(3) receptor during CCI.     

Cell-Specific Cre Recombinase Expression Allows Selective Ablation of Glutamate Receptors from Mouse Horizontal Cells

In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input, can thus be used to analyze the contribution of horizontal cells to retinal processing.     

Roles of ionotropic glutamate receptors in early developing neurons derived from the P19 mouse cell line

We cultured a P19 mouse teratocarcinoma cell line and induced its neuronal differentiation to study the function of ionotropic glutamate receptors (GluRs) in early neuronal development. Immunocytochemical studies showed 85% neuronal population at 5 days in vitro (DIV) with microtubule-associated protein 2-positive staining. Thirty percent and 50% of the cells expressed the alpha-amino-3-hydroxy-5-methyl-4-isopropinonate (AMPA) receptor subunit, GluR2/3, and the kainate (kainic acid; KA) receptor subunit, GluR5/6/7, respectively. In Western blot analysis, the temporal expression of GluR2/3 began to appear at 3 DIV, whereas GluR5/6/7 was already expressed in the undifferentiated cells. P19-derived neurons began to respond to glutamate, AMPA and KA, but not to the metabotropic GluR agonist trans-1-aminocyclopentane-1,3-decarboxylic acid, by 5 DIV in terms of increases in intracellular calcium and phospholipase C-mediated poly-phosphoinositide turnover. Furthermore, KA reduced cell death of P19-derived neurons in both atmospheric and hypobaric conditions in a phospholipase C-dependent manner. The common AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, but not the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium, profoundly increased hypobaric insult-induced neurotoxicity. In a flow cytometry study, the nerve growth factor-mediated antiapoptotic effect was facilitated by AMPA, with an induction of TrkA, but not p75(NTR) expression. Therefore, AMPA and KA receptors might mediate neurotrophic functions to facilitate neurotrophic factor signaling to protect neurons against hypoxic insult in early neuronal development.     

The mechanism of presynaptic long-term depression mediated by group I metabotropic glutamate receptors

1. Metabotropic glutamate receptors (mGluRs) are known to play a role in synaptic plasticity. In a study of rat hippocampal brain slices, we find that a brief perfusion of a group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), induced a robust long-term depression (DHPG-LTD) in area CA1.2. The action was accompanied by an enhancement of the paired-pulse facilitation (PPF) ratio.3. At the same time DHPG enhanced ionophoretic responses to alpha-amino-3- hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), kainic acid (KA), and N-methyl-D-aspartate (NMDA) in CA1 pyramidal neurons. This was only partially reversed by washing.4. These observations indicate that DHPG exerts two opposing actions, suppression of the synaptic transmission and facilitation of postsynaptic responses. However, the presynaptic action dominates, since the net effect of monosynaptic activation is a reduction of response.5. Perfusion of DHPG reduced three calcium-dependent responses in CA3 pyramidal neurons, which are presynaptic to CA1 neurons. These are calcium spike width and amplitude, after-hyperpolarization (AHP), and spike frequency adaptation (SFA).6. These results suggest that the DHPG-LTD results from modulation of the presynaptic calcium currents by group l mGluRs.     

Autoinactivation of the Stargazin–AMPA Receptor Complex: Subunit-Dependency and Independence from Physical Dissociation

Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This “autoinactivation” has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1–A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.     

Contactin-associated protein 1 (Caspr1) regulates the traffic and synaptic content of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors

Glutamate receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type mediate fast excitatory synaptic transmission in the CNS. Synaptic strength is modulated by AMPA receptor binding partners, which regulate receptor synaptic targeting and functional properties. We identify Contactin-associated protein 1 (Caspr1) as an AMPA receptor interactor. Caspr1 is present in synapses and interacts with AMPA receptors in brain synaptic fractions. Coexpression of Caspr1 with GluA1 increases the amplitude of glutamate-evoked currents. Caspr1 overexpression in hippocampal neurons increases the number and size of synaptic GluA1 clusters, whereas knockdown of Caspr1 decreases the intensity of synaptic GluA1 clusters. Hence, Caspr1 is a regulator of the trafficking of AMPA receptors to synapses.     

A novel form of synaptic plasticity in field CA3 of hippocampus requires GPER1 activation and BDNF release

Estrogen is an important modulator of hippocampal synaptic plasticity and memory consolidation through its rapid action on membrane-associated receptors. Here, we found that both estradiol and the G-protein–coupled estrogen receptor 1 (GPER1) specific agonist G1 rapidly induce brain-derived neurotrophic factor (BDNF) release, leading to transient stimulation of activity-regulated cytoskeleton-associated (Arc) protein translation and GluA1-containing AMPA receptor internalization in field CA3 of hippocampus. We also show that type-I metabotropic glutamate receptor (mGluR) activation does not induce Arc translation nor long-term depression (LTD) at the mossy fiber pathway, as opposed to its effects in CA1, and it only triggers LTD after GPER1 stimulation. Furthermore, this form of mGluR-dependent LTD is associated with ubiquitination and proteasome-mediated degradation of GluA1, and is prevented by proteasome inhibition. Overall, our study identifies a novel mechanism by which estrogen and BDNF regulate hippocampal synaptic plasticity in the adult brain.     

NMDA receptor, PKC and ERK prevent fos expression induced by the activation of group I metabotropic glutamate receptors in the spinal trigeminal subnucleus oralis

Fos, a protein product of immediate early gene c-fos, has been used as a marker for activation of nociceptive neurons in central nervous system including spinal trigeminal nucleus (Vsp). By noxious stimulation applied to orofacial area, the expression of Fos occurred in the Vsp pars oralis (Vo), the subnucleus receiving inputs from trigeminal primary afferents that predominantly innervate intraoral receptive fields. The present study demonstrates that the in vitro activation of group I metabotropic glutamate receptors (mGluRs; mGluR1 and 5) by bath-application of their well-known agonist (S)-3,5-dihydroxyphenylglycine (DHPG) increased the number of Fos-expressing neurons in the Vo area. In addition, bath application of DHPG caused inward currents, a parameter of neuronal excitation, in the Vo neurons held at −70 mV in voltage-clamp mode of whole-cell recordings. In further experiments characterizing two phenomena, the increased Fos expression in the Vo was mediated by an additive activation of both mGluR1 and mGluR5, which required the activation of N-methyl-D-aspartate (NMDA) receptors, protein kinase C (PKC) and extracellular signal-regulated kinase (ERK). In contrast, the inward currents were mediated only by mGluR1, but not by others. The data resulting from this in vitro study indicate that the DHPG-induced membrane depolarisation or neuronal excitation may be upstream to, or skip, the NMDA receptor, PKC and ERK pathways for the DHPG-induced Fos expression.     

The Structure of (?)-Kaitocephalin Bound to the Ligand Binding Domain of the (S)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA)/Glutamate Receptor,GluA2

Glutamate receptors mediate the majority of excitatory synaptic transmission in the central nervous system, and excessive stimulation of these receptors is involved in a variety of neurological disorders and neuronal damage from stroke. The development of new subtype-specific antagonists would be of considerable therapeutic interest. Natural products can provide important new lead compounds for drug discovery. The only natural product known to inhibit glutamate receptors competitively is (−)-kaitocephalin, which was isolated from the fungus Eupenicillium shearii and found to protect CNS neurons from excitotoxicity. Previous work has shown that it is a potent antagonist of some subtypes of glutamate receptors (AMPA and NMDA, but not kainate). The structure of kaitocephalin bound to the ligand binding domain of the AMPA receptor subtype, GluA2, is reported here. The structure suggests how kaitocephalin can be used as a scaffold to develop more selective and high affinity antagonists for glutamate receptors.     

Distinct Subunit-specific α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Trafficking Mechanisms in Cultured Cortical and Hippocampal Neurons in Response to Oxygen and Glucose Deprivation

Brain ischemia occurs when the blood supply to the brain is interrupted, leading to oxygen and glucose deprivation (OGD). This triggers a cascade of events causing a synaptic accumulation of glutamate. Excessive activation of glutamate receptors results in excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts. The mechanisms that underlie this difference are unclear. Cultured hippocampal neurons respond to OGD with a rapid internalization of AMPA receptor (AMPAR) subunit GluA2, resulting in a switch from GluA2-containing Ca²⁺-impermeable receptors to GluA2-lacking Ca²⁺-permeable subtypes (CP-AMPARs). GluA2 internalization is a critical component of OGD-induced cell death in hippocampal neurons. It is unknown how AMPAR trafficking is affected in cortical neurons following OGD. Here, we show that cultured cortical neurons are resistant to an OGD insult that causes cell death in hippocampal neurons. GluA1 is inserted at the plasma membrane in both cortical and hippocampal neurons in response to OGD. In contrast, OGD causes a rapid endocytosis of GluA2 in hippocampal neurons, which is absent in cortical neurons. These data demonstrate that populations of neurons with different vulnerabilities to OGD recruit distinct cell biological mechanisms in response to insult, and that a crucial aspect of the mechanism leading to OGD-induced cell death is absent in cortical neurons. This strongly suggests that the absence of OGD-induced GluA2 trafficking contributes to the relatively low vulnerability of cortical neurons to ischemia.     

PTEN regulates AMPA receptor-mediated cell viability in iPS-derived motor neurons

Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast–iPS–motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.     

Sustained activation of metabotropic glutamate receptor 5 and protein tyrosine phosphatases mediate the expression of (S)-3,5-dihydroxyphenylglycine-induced long-term depression in the hippocampal CA1 region

Previous studies have shown that brief application of group I metabotropic glutamate receptor (mGluR) agonist (S)-3, 5-dihydroxyphenylglycine (DHPG) to hippocampal slices can induce a chemical form of long-term depression (DHPG-LTD) in the hippocampal CA1 region; however, the expression mechanisms of this LTD remain unclear. We show here that the expression of DHPG-LTD can be specifically reversed by application of the broad-spectrum mGluR antagonists, (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) and LY341495, and mGluR5 antagonist, 2-methyl-6-(phenylethyl)pyridine, but not by NMDA receptor antagonist, D-2-amino-5-phosphonopentanoic acid, mGluR1 antagonist, LY367385, group II mGluR antagonist, (2S)-alpha-ethylglutamic acid, or group III mGluR antagonist, (S)-2-amino-2-methyl-4-phosphonobutanic acid (MAP4). In addition, the ability of MCPG to reverse DHPG-LTD was mimicked by the protein tyrosine phosphatase inhibitors, phenylarsine oxide and orthovanadate, but not phospholipase C inhibitor, U73122, protein kinase C inhibitor, bisindolylmaleimide 1, p38 mitogen-activated protein kinase inhibitor, SB203580, or protein phosphatases 1/2 A inhibitor, okadaic acid. Moreover, MCPG reversed the DHPG-LTD without affecting the paired-pulse facilitation. The expression of DHPG-LTD was associated with the reduction of both tyrosine phosphorylation and surface expression of AMPA receptor GluR2 subunits. Together, these results suggest that sustained activation of mGluR5 and in turn triggering a protein tyrosine phosphatase-dependent regulation of postsynaptic expression of AMPA receptors may contribute to the expression of DHPG-LTD.     

Syntaxin13 Expression Is Regulated by Mammalian Target of Rapamycin (mTOR) in Injured Neurons to Promote Axon Regeneration

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.     

AMPA and NMDA glutamate receptors are found in both peptidergic and non-peptidergic primary afferent neurons in the rat

Two distinct classes of nociceptive primary afferents, peptidergic and non-peptidergic, respond similarly to acute noxious stimulation; however the peptidergic afferents are more likely to play a role in inflammatory pain, while the non-peptidergic afferents may be more characteristically involved in neuropathic pain. Using multiple immunofluorescence, we determined the proportions of neurons in the rat L4 dorsal root ganglion (DRG) that co-express AMPA or NMDA glutamate receptors and markers for the peptidergic and non-peptidergic classes of primary afferents, substance P and P2X(3), respectively. The fraction of DRG neurons immunostained for the NR1 subunit of the NMDA receptor (40%) was significantly higher than that of DRG neurons immunostained for the GluR2/3 (27%) or the GluR4 (34%) subunits of the AMPA receptor. Of all DRG neurons double-immunostained for glutamate receptor subunits and either marker for peptidergic and non-peptidergic afferents, a significantly larger proportion expressed GluR4 than GluR2/3 or NR1 and in a significantly larger proportion of P2X(3)- than SP-positive DRG neurons. These observations support the idea that nociceptors, involved primarily in the mediation of neuropathic pain, may be presynaptically modulated by GluR4-containing AMPA receptors.