Involvement of myosin Vb in glutamate receptor trafficking

Myosin V motors mediate cargo transport; however, the identity of neuronal molecules transported by these proteins remains unknown. Here we show that myosin Vb is expressed in several neuronal populations and associates with the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor subunit GluR1. In developing hippocampal neurons, expression of the tail domain of myosin Vb, but not myosin Va, enhanced GluR1 accumulation in the soma and reduced its surface expression. These changes were accompanied by reduced GluR1 clustering and diminished frequency of excitatory but not inhibitory synaptic currents. Similar effects were observed upon expression of full-length myosin Vb lacking a C-terminal region required for binding to the small GTPase Rab11. In contrast, mutant myosin Vb did not change the localization of several other neurotransmitter receptors, including the glutamate receptor subunit NR1. These results reveal a novel mechanism for the transport of a specific glutamate receptor subunit in neurons mediated by a member of the myosin V family.     

Synaptic transmission and plasticity in the absence of AMPA glutamate receptor GluR2 and GluR3

The AMPA glutamate receptor (AMPAR) subunits GluR2 and GluR3 are thought to be important for synaptic targeting/stabilization of AMPARs and the expression of hippocampal long-term depression (LTD). In order to address this hypothesis genetically, we generated and analyzed knockout mice deficient in the expression of both GluR2 and GluR3. We show here that the double knockout mice are severely impaired in basal synaptic transmission, demonstrating that GluR2/3 are essential to maintain adequate synaptic transmission in vivo. However, these mutant mice are competent in establishing several forms of long-lasting synaptic changes in the CA1 region of the hippocampus, including LTD, long-term potentiation (LTP), depotentiation, and dedepression, indicating the presence of GluR2/3-independent mechanisms of LTD expression and suggesting that AMPA receptor GluR1 alone is capable of various forms of synaptic plasticity.     

谷氨酸下调培养海马神经元AMPA受体G1uR2亚单位的表达

目的 研究在癫痫发病过程中,谷氨酸对AMPA受体G1uR2亚单位表达变化的影响。方法 用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体G1uR2亚单位mRNA和蛋白的表达变化。结果 在谷氨酸刺激后2h,8h,12h,培养海马神经元G1uR2 mRNA和蛋白表达明显下降,与对照组相比,差异有显著性（P〈0．05）,而非NMDA受体拮抗剂CNQX能阻断此变化。结论 在癫痫等疾病中,谷氨酸能通过激活AMAP／KA受体下调AMPA受体G1uR2亚单位的表达,参与发病过程。     

谷氨酸下调培养海马神经元AMPA 受体GluR2亚单位的表达

目的研究在癫痫发病过程中,谷氨酸对AMPA受体Glu R2亚单位表达变化的影响。方法用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体Glu R2亚单位mRNA和蛋白的表达变化。结果在谷氨酸刺激后2h,8h,12h,培养海马神经元Glu R2mRNA和蛋白表达明显下降,与对照组相比,差异有显著性(P<0.05),而非NMDA受体拮抗剂CNQX能阻断此变化。结论在癫痫等疾病中,谷氨酸能通过激活AMAP/KA受体下调AMPA受体GluR2亚单位的表达,参与发病过程。     

Brain-derived neurotrophic factor regulates surface expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors by enhancing the N-ethylmaleimide-sensitive factor/GluR2 interaction in developing neocortical neurons

In hippocampal neurons, the exocytotic process of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors is known to depend on activation of N-methyl-d-aspartate channels and its resultant Ca(2+) influx from extracellular spaces. Here we found that brain-derived neurotrophic factor (BDNF) induced a rapid surface translocation of AMPA receptors in an activity-independent manner in developing neocortical neurons. The receptor translocation became evident within hours as monitored by [(3)H]AMPA binding and was resistant against ionotropic glutamate receptor antagonists as evidenced with surface biotinylation assay. This process required intracellular Ca(2+) and was inhibited by the blockers of conventional exocytosis, brefeldin A, botulinum toxin B, and N-ethylmaleimide. To explore the translocation mechanism of individual AMPA receptor subunits, we utilized the human embryonic kidney (HEK) 293 cells carrying the BDNF receptor TrkB. After the single transfection of GluR2 cDNA or GluR1 cDNA into HEK/TrkB cells, BDNF triggered the translocation of GluR2 but not that of GluR1. Subsequent mutation analysis of GluR2 carboxyl-terminal region indicated that the translocation of GluR2 subunit in HEK293 cells involved its N-ethylmaleimide-sensitive factor-binding domain but not its PDZ-interacting site. Following co-transfection of GluR1 and GluR2 cDNAs, solid phase cell sorting revealed that GluR1 subunits were also able to translocate to the cell surface in response to BDNF. An immunoprecipitation assay confirmed that BDNF stimulation can enhance the interaction of GluR2 with N-ethylmaleimide-sensitive factor. These results reveal a novel role of BDNF in regulating the surface expression of AMPA receptors through a GluR2-NSF interaction.     

Regulation of Kainate Receptor Subunit mRNA by Stress and Corticosteroids in the Rat Hippocampus

Kainate receptors are a class of ionotropic glutamate receptors that have a role in the modulation of glutamate release and synaptic plasticity in the hippocampal formation. Previous studies have implicated corticosteroids in the regulation of these receptors and recent clinical work has shown that polymorphisms in kainate receptor subunit genes are associated with susceptibility to major depression and response to anti-depressant treatment. In the present study we sought to examine the effects of chronic stress and corticosteroid treatments upon the expression of the mRNA of kainate receptor subunits GluR5-7 and KA1-2. Our results show that, after 7 days, adrenalectomy results in increased expression of hippocampal KA1, GluR6 and GluR7 mRNAs, an effect which is reversed by treatment with corticosterone in the case of KA1 and GluR7 and by aldosterone treatment in the case of GluR6. 21 days of chronic restraint stress (CRS) elevated the expression of the KA1 subunit, but had no effect on the expression of the other subunits. Similarly, 21 days of treatment with a moderate dose of corticosterone also increased KA1 mRNA in the dentate gyrus, whereas a high corticosterone dose has no effect. Our results suggest an interaction between hippocampal kainate receptor composition and the hypothalamic-pituitary-adrenal (HPA) axis and show a selective chronic stress induced modulation of the KA1 subunit in the dentate gyrus and CA3 that has implications for stress-induced adaptive structural plasticity.     

Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death

Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-d-aspartate (300 microm, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nm) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the N-methyl-d-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca(2+)-activated neutral protease calpain I was readily detectable after the exposure to N-methyl-d-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-d-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.     

Subunit rules governing the sorting of internalized AMPA receptors in hippocampal neurons

Removal of synaptic AMPA receptors is important for synaptic depression. Here, we characterize the roles of individual subunits in the inducible redistribution of AMPA receptors from the cell surface to intracellular compartments in cultured hippocampal neurons. The intracellular accumulation of GluR2 and GluR3 but not GluR1 is enhanced by AMPA, NMDA, or synaptic activity. After AMPA-induced internalization, homomeric GluR2 enters the recycling pathway, but following NMDA, GluR2 is diverted to late endosomes/lysosomes. In contrast, GluR1 remains in the recycling pathway, and GluR3 is targeted to lysosomes regardless of NMDA receptor activation. Interaction with NSF plays a role in regulated lysosomal targeting of GluR2. GluR1/GluR2 heteromeric receptors behave like GluR2 homomers, and endogenous AMPA receptors show differential activity-dependent sorting similar to homomeric GluR2. Thus, GluR2 is a key subunit that controls recycling and degradation of AMPA receptors after internalization.     

NPC-16, a novel naphthalimide–polyamine conjugate,induced apoptosis and autophagy in human hepatoma HepG2 cells and Bel-7402 cells

The antitumor effects and molecular mechanism of NPC-16, a novel naphthalimide–polyamine conjugate, were evaluated in HepG2 cells and Bel-7402 cells. Apoptosis and necrosis were evaluated by Annexin V-FITC detection kit, and autophagy by acridine orange and Lyso-Tracker Red staining. The change of mitochondrial transmembrane potential was measured using rhodamine 123 staining. The protein expression of Beclin 1, LC3 II and mTOR, p70S6 K, 14-3-3, caspase, and Bcl-2 family members was detected by immunofluorescence assays and Western Blot. Here, we elucidated the nature of cellular response of HepG2 cells and Bel-7402 cells to NPC-16 at IC₅₀. NPC-16 induced caspase-dependent apoptosis via the mitochondrial pathway and death receptor pathway in Bel-7402 cells. Differently, NPC-16 triggered HepG2 cells both apoptosis and autophagy, further autophagy facilitated cellular apoptosis. Furthermore, mTOR signal pathway was involved in NPC-16-mediated autophagy in HepG2 cells. Thus, NPC-16 may be useful as a potential template for investigation the molecular mechanism of naphthalimide–polyamine conjugate against hepatocellular carcinoma.     

Learning-induced glutamate receptor phosphorylation resembles that induced by long term potentiation

Long term potentiation and long term depression of synaptic responses in the hippocampus are thought to be critical for certain forms of learning and memory, although until recently it has been difficult to demonstrate that long term potentiation or long term depression occurs during hippocampus-dependent learning. Induction of long term potentiation or long term depression in hippocampal slices in vitro modulates phosphorylation of the alpha-amino-3-hydrozy-5-methylisoxazole-4-propionic acid subtype of glutamate receptor subunit GluR1 at distinct phosphorylation sites. In long term potentiation, GluR1 phosphorylation is increased at the Ca2+/calmodulin-dependent protein kinase and protein kinase C site serine 831, whereas in long term depression, phosphorylation of the protein kinase A site serine 845 is decreased. Indeed, phosphorylation of one or both of these sites is required for long term synaptic plasticity and for certain forms of learning and memory. Here we demonstrate that training in a hippocampus-dependent learning task, contextual fear conditioning is associated with increased phosphorylation of GluR1 at serine 831 in the hippocampal formation. This increased phosphorylation is specific to learning, has a similar time course to that in long term potentiation, and like memory and long term potentiation, is dependent on N-methyl-D-aspartate receptor activation during training. Furthermore, the learning-induced increase in serine 831 phosphorylation is present at synapses and is in heteromeric complexes with the glutamate receptor subunit GluR2. These data indicate that a biochemical correlate of long term potentiation occurs at synapses in receptor complexes in a final, downstream, postsynaptic effector of long term potentiation during learning in vivo, further strengthening the link between long term potentiation and memory.     

Tyrosine phosphorylation of GluR2 is required for insulin-stimulated AMPA receptor endocytosis and LTD

The alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors is subject to functionally distinct constitutive and regulated clathrin-dependent endocytosis, contributing to various forms of synaptic plasticity. In HEK293 cells transiently expressing GluR1 or GluR2 mutants containing domain deletions or point mutations in their intracellular carboxyl termini (CT), we found that deletion of the first 10 amino acids (834-843) selectively reduced the rate of constitutive AMPA receptor endocytosis, whereas truncation of the last 15 amino acids of the GluR2 CT, or point mutation of the tyrosine residues in this region, only eliminated the regulated (insulin-stimulated) endocytosis. Moreover, in hippocampal slices, both insulin treatment and low-frequency stimulation (LFS) specifically stimulated tyrosine phosphorylation of the GluR2 subunits of native AMPA receptors, and the enhanced phosphorylation appears necessary for both insulin- and LFS-induced long-term depression of AMPA receptor-mediated excitatory postsynaptic currents. Thus, our results demonstrate that constitutive and regulated AMPA receptor endocytosis requires different sequences within GluR CTs and tyrosine phosphorylation of GluR2 CT is required for the regulated AMPA receptor endocytosis and hence the expression of certain forms of synaptic plasticity.     

Human T cells express a functional ionotropic glutamate receptor GluR3, and glutamate by itself triggers integrin-mediated adhesion to laminin and fibronectin and chemotactic migration

T cells may encounter glutamate, the major excitatory neurotransmitter in the nervous system, when patrolling the brain and in glutamate-rich peripheral organs. Moreover, glutamate levels increase in the CNS in many pathological conditions in which T cells exert either beneficial or detrimental effects. We discovered that normal human T cells, human T leukemia cells, and mouse anti-myelin basic protein T cells express high levels of glutamate ion channel receptor (ionotropic) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype 3 (GluR3). The evidence for GluR3 on T cells includes GluR3-specific RT-PCR, Western blot, immunocytochemical staining and flow cytometry. Sequencing showed that the T cell-expressed GluR3 is identical with the brain GluR3. Glutamate (10 nM), in the absence of any additional molecule, triggered T cell function: integrin-mediated T cell adhesion to laminin and fibronectin, a function normally performed by activated T cells only. The effect of glutamate was mimicked by AMPA receptor-agonists and blocked specifically by the selective receptor-antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulfamoylbenzo[f]quinoxalin-2,3-dione (NBQX), and by relevant anti-integrin mAbs. Glutamate also increased the CXCR4-mediated T cell chemotactic migration toward the key chemokine CXCL12/stromal cell-derived factor-1. GluR3 expression on normal, cancer and autoimmune-associated T cells and the ability of glutamate to directly activate T cell function could be of substantial scientific and clinical importance to normal neuroimmune dialogues and to CNS diseases and injury, and especially to: 1) T cell transmigration to the CNS and patrolling in the brain, 2) T cell-mediated multiple sclerosis, and 3) autoimmune epilepsy, as neurotoxic anti-GluR3 Abs are found and suspected to cause/potentiate seizures and neuropathology in several types of human epilepsies. Thus far, GluR3 was found only on neurons and glia cells; our results reveal a novel peripheral source of this antigenic receptor.     

Fimbria-Fornix Transections Selectively Down-Regulate Subtypes of Glutamate Transporter and Glutamate Receptor Proteins in Septum and Hippocampus

Abstract: The effects of CNS axotomy on glutamate transporter and glutamate receptor expression were evaluated in adult rats following unilateral fimbria-fornix transections. The septum and hippocampus were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted by using antibodies directed against glutamate transporters (GLT-1, GLAST, and EAAC1) and glutamate receptors (GluR1, GluR2/3, GluR6/7, and NMDAR1), and they were assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 was decreased at 7 and 14 days postlesion within the ipsilateral septum and at 7 days postlesion in the hippocampus. GLAST was decreased within the ipsilateral septum and hippocampus at 7 and 14 days postlesion. No postlesion alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 7, 14, and 30 days postlesion within the ipsilateral septum and 14 days postlesion in the hippocampus. GluR2/3 expression was down-regulated at 30 days postlesion within the ipsilateral septum, whereas GluR1, GluR6/7, and NMDAR1 immunoreactivity was unchanged. In addition, no alterations in glutamate receptor expression were detected within hippocampal homogenates. This study demonstrates a selective down-regulation of primarily glial, and not neuronal, glutamate transporters and a delayed, subtype-specific down-regulation of septal GluR2/3 receptor expression after regional deafferentation within the CNS.     

Cannabidiol activates PINK1-Parkin-dependent mitophagy and mitochondrial-derived vesicles

The PINK1/Parkin pathway plays an important role in maintaining a healthy pool of mitochondria. Activation of this pathway can lead to apoptosis, mitophagy, or mitochondrial-derived vesicle formation, depending on the nature of mitochondrial damage. The signaling by which PINK/Parkin activation leads to these different mitochondrial outcomes remains understudied. Here we present evidence that cannabidiol (CBD) activates the PINK1-Parkin pathway in a unique manner. CBD stimulates PINK1-dependent Parkin mitochondrial recruitment similarly to other well-studied Parkin activators but with a distinctive shift in the temporal dynamics and mitochondrial fates. The mitochondrial permeability transition pore inhibitor cyclosporine A exclusively diminished the CBD-induced PINK1/Parkin activation and its associated mitochondrial effects. Unexpectedly, CBD treatment also induced elevated production of mitochondrial-derived vesicles (MDV), a potential quality control mechanism that may help repair partial damaged mitochondria. Our results suggest that CBD may engage the PINK1-Parkin pathway to produce MDV and repair mitochondrial lesions via mitochondrial permeability transition pore opening. This work uncovered a novel link between CBD and PINK1/Parkin-dependent MDV production in mitochondrial health regulation.     

Autoantibodies to neuronal glutamate receptors in patients with paraneoplastic neurodegenerative syndrome enhance receptor activation.

BACKGROUND: Paraneoplastic syndromes are "remote" complications of cancer characterized clinically by neurological disease. The sera and cerebrospinal fluid (CSF) from patients with paraneoplastic neurological syndromes (PNS) frequently contain autoantibodies to ill-defined neuronal antigens. We report here that neuronal glutamate receptors are targets for autoantibodies found in the serum from some patients with well-characterized PNS. MATERIALS AND METHODS: We have analyzed the serum from seven patients with well-characterized PNS for the presence of autoreactive antibodies to non-NMDA glutamate receptor subunits. Autoantibodies were assessed using Western blot, immunohistochemistry, and immunocytochemistry. Whole-cell electrophysiological recordings were used to examine the effect of antibodies on glutamate receptors expressed by cortical neurons in culture. RESULTS: Six of seven patients' serum contained autoantibodies to the non-NMDA glutamate receptor (GluR) subunits GluR1, GluR4, and/or GluR5/6. No patient had autoantibodies to GluR2, and only one patient exhibited weak immunoreactivity to GluR3. Electrophysiological analysis demonstrated that the serum from four of the six GluR-antibody-positive patients enhanced glutamate-elicited currents on cultured cortical neurons but had no effect on receptor function alone. Enhancement of glutamate-elicited currents was also produced by affinity-purified antibody to GluR5. CONCLUSIONS: The occurrence of autoantibodies to specific neuronal neurotransmitter subunits in the sera of patients with PNS and the ability of these autoantibodies to modulate glutaminergic receptor function suggest that some paraneoplastic neurological injury could result from glutamate-mediated excitotoxicity.     

Impact of High-Fat Diet and Early Stress on Depressive-Like Behavior and Hippocampal Plasticity in Adult Male Rats

During development, the brain goes through fundamental processes, including organization of neural networks and plasticity. Environmental interventions may change initial brain programming, leading to long-lasting effects and altering the susceptibility to psychopathologies, including depression disorder. It is known that depression is a psychiatric disorder with a high prevalence worldwide, including high rates among adolescents. In this study, we evaluated whether social isolation in the prepubertal period and chronic use of high-fat diet (HFD) may induce depressive-like behavior in male adult rats. We also investigated hippocampal plasticity markers and neurotransmitter systems. We found both social isolation and HFD induced a depressive-like behavior in the forced swimming task. Moreover, chronic HFD reduced synaptic markers in hippocampus, demonstrated by reductions in βIII-tubulin (neuronal marker), PSD-95, SNAP-25, and neurotrophin-3. The HFD group also presented decreased glutamatergic and GABAergic receptors subunits. On the other hand, stress affected hippocampal brain-derived neurotrophic factor (BDNF) signaling pathways, and increased expression of subunit of the NMDA receptor (NR2A). Both factors (stress and diet) decreased GR in the hippocampus without affecting plasma corticosterone at basal levels. Interactions between early stress and HFD access were observed only in the BNDF receptor (tropomyosin receptor kinase B; TrkB) and synaptophysin. In summary, these findings showed that a brief social isolation and chronic HFD, during a sensitive developmental period, cause depressive-like behavior in adulthood. The mechanisms underlying these behavioral effects may involve changes in the levels of synaptic proteins in hippocampus: HFD consumption appears to affect synaptic markers, while social isolation affected BDNF signaling more significantly.     

Developmental Hypothyroxinemia Caused by Mild Iodine Deficiency Leads to HFS-Induced LTD in Rat Hippocampal CA1 Region: Involvement of AMPA Receptor

Hypothyroidism induced by severe iodine deficiency (ID) during developmental period seriously damages the central nervous system function. In addition to developmental hypothyroidism induced by severe ID, developmental hypothyroxinemia induced by mild ID is potentially damaging for neurodevelopment and learning and memory in children. Wistar rats were treated with iodine-deficient diet or methimazole (MMZ) during pregnancy and lactation to induce developmental hypothyroxinemia or hypothyroidism in the present study. Pups were weaned on postnatal day (PN) 21 and used for electrophysiological recordings on PN80. It is generally accepted that long-term depression (LTD) is induced at low-frequency stimulation (LFS) in hippocampal CA1 region. Surprisingly, we observed developmental hypothyroxinemia as well as developmental hypothyroidism led to high-frequency stimulation (HFS)-induced LTD in hippocampal CA1 region. The abnormal HFS-induced LTD suggests not only developmental hypothyroidism but also developmental hypothyroxinemia impairs learning and memory. To explore the mechanisms responsible for the HFS-induced LTD, the phosphorylation status of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) was investigated. The results showed that developmental hypothyroxinemia as well as developmental hypothyroidism decreased the phosphorylation of AMPAR subunit glutamate receptor 1 (GluR1) at serine 831 and serine 845 in hippocampal CA1 region. Neither developmental hypothyroxinemia nor developmental hypothyroidism altered the phosphorylation of AMPAR subunit glutamate receptor 2 (GluR2) at serine 880. Increased levels of protein phosphatase-1 (PP1) were also observed in hippocampal CA1 regions of pups subjected to developmental hypothyroxinemia or hypothyroidism. Taken together, our results suggest that the increased levels of PP1 caused by developmental hypothyroxinemia or hypothyroidism may account for the dephosphorylation of GluR1 at serine 831 and serine 845, which may contribute to HFS-induced LTD in hippocampal CA1 region.     

Nicotine preconditioning antagonizes activity-dependent caspase proteolysis of a glutamate receptor.

Neuronal excitation is required for normal brain function including processes of learning and memory, yet if this process becomes dysregulated there is reduced neurotransmission and possibly death through excitotoxicity. Nicotine, through interaction with neuronal nicotinic acetylcholine receptors, possesses the ability to modulate neurotransmitter systems through numerous mechanisms that define this critical balance. We examined the modulatory role of nicotine in primary mixed cortical neuronal-glial cultures on activity-dependent caspase cleavage of a glutamate receptor, GluR1. We find that GluR1, but not GluR2 or GluR3, is a substrate for agonist (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-initiated rapid proteolytic cleavage at aspartic acid 865 through the activation of caspase 8-like activity that is independent of membrane fusion and is not coincident with apoptosis. Dose-dependent nicotine preconditioning for 24 h antagonizes agonist-initiated caspase cleavage of GluR1 through a mechanism that is coincident with desensitization of both nAChRalpha4beta2 and nAChRalpha7 receptors and the delayed activation of a caspase 8-like activity. The modulation of GluR1 agonist-initiated caspase-mediated cleavage by nicotine preconditioning offers a novel insight into how this agent can impart its numerous effects on the nervous system.     

Immunocytochemical localization of the AMPA receptor subunits in the mesencephalic trigeminal nucleus of the rat

The mesencephalic trigeminal nucleus is composed of large (35-50 microns) pseudo-unipolar neurons. Closely associated with them are small (< 20 microns) multipolar neurons. An unique peculiarity of the pseudo-unipolar perikarya is that they receive synaptic input from various sources, which sets them apart from the dorsal root and cranial nerves sensory ganglia neurons. Whereas glutamate is the best neurotransmitter candidate in pseudo-unipolar neurons, glutamatergic input into them has not yet been reported. AMPA glutamate receptors are implicated in fast excitatory glutamatergic synaptic transmission. They have been localized ultrastructurally at postsynaptic sites. This study demonstrates that the pseudo-unipolar neurons of the mesencephalic trigeminal nucleus express AMPA glutamate receptor subunits, which indicates that these neurons receive glutamatergic input. Serial sections from the rostral pons and midbrain of Sprague-Dawley rats were immunostained with antibodies against C-terminus of AMPA receptor subunits: GluR1, GluR2/3, and GluR4. The immunoreaction was visualized with avidin-biotin-peroxidase/DAB for light and electron microscopy. With GluR1 antibody only the smallest multipolar neurons were recognized as immunopositive within the mesencephalic trigeminal nucleus. GluR2/3 stained the pseudo-unipolar neurons intensely within the entire rostro-caudal extent of the nucleus. In addition the former antibody stained small multipolar neurons within the mesencephalic trigeminal nucleus, though with somewhat larger dimensions than those immunoreactive for GluR1. Whereas the overall staining with GluR4 antibody was scant, those pseudo-unipolar neurons that were stained, were strongly stained. Furthermore, a considerable number of microglial cells within and surrounding the mesencephalic trigeminal nucleus displayed very intense immunoreactivity for GluR4. These results are discussed in the light of the glutamate receptor subunit composition.     

Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors

Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca²⁺, resulting from Ca²⁺ influxes through calcium-permeable AMPA receptors, voltage-gated Ca²⁺ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca²⁺ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca²⁺ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain.