6-cyano-7-nitroquinoxaline-2-3-dione and rhod-2

The trigeminal subnucleus caudalis (Vc) plays a critical role in transmission and modulation of nociceptive afferent inputs, and exhibits a similar layer construction to the spinal dorsal horn. However, afferent inputs enter the brainstem and project to a separately located nucleus. It has previously been difficult to record responses of the Vc to afferent fiber activation in a brainstem slice preparation. The aim of the present study was to establish a novel brainstem slice preparation method to study trigeminal nociceptive transmission mechanisms.. Thirty adult 6-7-week-old C57/BL6J male mice were included in the study. Obliquely sliced brainstem sections at a thickness of 600μm, which included the Vc and the root entry zone to the brainstem, were prepared. The Vc response to electrical stimulation of afferent fibers was observed as a change in intracellular calcium concentration by fluorescence intensity response.. Electrical stimulation of afferent inputs to the trigeminal nerve increased fluorescent intensity in the Vc, which was completely diminished by tetrodotoxin and significantly suppressed by the AMPA/kainate antagonist CNQX (paired t-test, P<0.001), although the non-competitive NMDA antagonist (+)-MK801 maleate resulted in no changes. These results suggested a glutamate receptor-mediated response.. This brainstem slice preparation will be useful for investigating nociceptive transmission mechanisms of the trigeminal nerve.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Afferent Pathways; Animals; Calcium; Dizocilpine Maleate; Electric Stimulation; Excitatory Amino Acid Antagonists; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Nociception; Nociceptors; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin; Time Factors; Trigeminal Nucleus, Spinal

Mitochondrial uptake of Ca(2+) has recently been found to play an important role in glutamate-induced neurotoxicity (GNT) as well as in the activation of Ca(2+)-dependent molecules, such as calmodulin and neuronal nitric oxide synthase (nNOS), in the cytoplasm. Prolonged exposure to glutamate injures motor neurons predominantly through the activation of Ca(2+)/calmodulin-nNOS, as previously reported, and is, in part, associated with the pathogenesis of amyotrophic lateral sclerosis (ALS). In the present study, we investigated how mitochondrial uptake of Ca(2+) is involved in GNT in spinal motor neurons. Acute excitotoxicity induced by exposure to 0.5 mM glutamate for 5 min was found in both motor and nonmotor neurons in cultured spinal cords from rat embryos and was dependent on extracellular Ca(2+) and on N-methyl-D-aspartate (NMDA) receptor activation. Mitochondrial uncouplers markedly blocked acute excitotoxicity, and membrane-permeable superoxide dismutase mimics attenuated acute excitotoxicity induced by glutamate and NMDA but not by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainate. Fluorimetric analysis showed that mitochondrial Ca(2+) was elevated promptly with subsequent accumulation of reactive oxygen species (ROS) in the mitochondria. An NMDA receptor antagonist and a mitochondrial uncoupler eliminated the increase in fluorescence of mitochondrial Ca(2+) and ROS indicators. These data indicate that acute excitotoxicity in spinal neurons is mediated by mitochondrial Ca(2+) overload and ROS generation through the activation of NMDA receptors. This mechanism is different from that of chronic GNT.

Topics: 2,4-Dinitrophenol; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cyclosporine; Dibucaine; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluoresceins; Fluorescent Dyes; Glutamic Acid; Heterocyclic Compounds, 3-Ring; Imidazoles; Kainic Acid; Mitochondria; Motor Neuron Disease; Motor Neurons; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Neurotoxins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Rhodamines; Spinal Cord; Superoxide Dismutase; Uncoupling Agents

Mechanisms underlying the Ca2+ increase during tetanic synaptic inputs in layer II/III of visual cortical slices of young rats were investigated with microfluorometry using a Ca2+ indicator, rhod-2, and simultaneous recordings of field potentials evoked by white matter stimulation. Application of an antagonist for N-methyl-D-aspartate (NMDA) receptors, 2-amino-5-phosphonovalerate, did not significantly affect field potentials but reduced the tetanus-induced fluorescence rise to 56%, on average, of the control values. Application of a broad-spectrum antagonist for both NMDA and non-NMDA receptors, kynurenate, completely abolished the synaptically evoked component of field potentials and decreased the tetanus-induced fluorescence rise to 42%. Application of a non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, in the Mg(2+)-free medium diminished the field potentials but did not decrease the tetanus-induced fluorescence increase. Nifedipine and diltiazem, L-type Ca2+ channel blockers, and Ni2+, a relatively selective blocker for T-type Ca2+ channels, did not affect the tetanus-induced fluorescence rise. These results indicate that NMDA receptors play a significant role in the increase of intracellular Ca2+ during tetanic synaptic inputs in the visual cortex of young rats.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Calcium Channel Blockers; Electric Stimulation; Evoked Potentials; Female; Fluorescent Dyes; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Kynurenic Acid; Long-Term Potentiation; Male; Microscopy, Fluorescence; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spectrometry, Fluorescence; Synapses; Visual Cortex

The expression of a receptor subtype for one type of excitatory amino acid agonist, t-ACPD, was examined in developing Purkinje cells of cerebellar slices. The t-ACPD-induced responses were compared with those induced by QA in current response, single cell Ca2+ imaging and changes in the miniature currents in the same preparation. It was found that t-ACPD induced a single component of inward current, and an increase in the frequency of miniature currents associated with the presence of external Ca2+, but was ineffective at mobilizing intracellular Ca2+ even in the presence of external Ca2+. The present study suggests the expression of at least two types of metabotropic receptors in the Purkinje cell region, one of which, expressed in the Purkinje cell dendrites, is highly sensitive to QA, but relatively insensitive to t-ACPD, and the other of which is a t-ACPD-sensitive receptor expressed on the presynaptic terminals of the neurons making synapses onto Purkinje cells.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Cerebellum; Cycloleucine; Dendrites; Evoked Potentials; Fluorescent Dyes; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Membrane Potentials; Neurotoxins; Purkinje Cells; Quinoxalines; Quisqualic Acid; Rats; Receptors, Metabotropic Glutamate; Receptors, Neurotransmitter; Synapses