tetrodotoxin 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

Enteric neurons controlling various gut functions are prone to oxidative insults that might damage mitochondria (e.g., intestinal inflammation). To resume local energy supply, mitochondria need to be transported. We used MitoTracker dyes and confocal microscopy to investigate basic characteristics of mitochondrial transport in guinea pig myenteric neurites. During a 10-s observation of 1 mm nerve fiber, on average, three mitochondria were transported at an average speed of 0.41 +/- 0.02 microm/s. Movement patterns were clearly erratic, and velocities were independent of mitochondrial size. The velocity oscillated periodically ( approximately 6 s) but was not consistently affected by structures such as en route boutons, bifurcations, or stationary mitochondria. Also, mitochondria transported in opposite directions did not necessarily affect each others' mobility. Transport was blocked by microtubule disruption (100 microM colchicine), and destabilization (1 microM cytochalasin-D) or stabilization (10 microM phalloidin) of actin filaments, respectively, decreased (0.22 +/- 0.02 microm/s, P < 0.05) or increased (0.53 +/- 0.02 microm/s, P < 0.05) transport speed. Transport was inhibited by TTX (1 microM), and removal of extracellular Ca(2+) (plus 2 mM EGTA) had no effect. However, depletion of intracellular stores (thapsigargin) reduced (to 33%) and slowed the transport significantly (0.18 +/- 0.02 microm/s, P < 0.05), suggesting an important role for stored Ca(2+) in mitochondrial transport. Transport was also reduced (to 21%) by the mitochondrial uncoupler FCCP (1 microM) in a time-dependent fashion and slowed by oligomycin (10 microM). We conclude that mitochondrial transport is remarkably independent of structural nerve fiber properties. We also show that mitochondrial transport is TTX sensitive and speeds up by stabilizing actin and that functional Ca(2+) stores are required for efficient transport.

Topics: Actins; Action Potentials; Algorithms; Anesthetics, Local; Animals; Biological Transport, Active; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Enteric Nervous System; Fluorescent Dyes; Guinea Pigs; Heterocyclic Compounds, 3-Ring; Image Processing, Computer-Assisted; Immunohistochemistry; In Vitro Techniques; Microscopy, Confocal; Microtubules; Mitochondria; Myenteric Plexus; Nerve Fibers; Oligomycins; Tetrodotoxin

Changes in intracellular Ca2+ evoked by electrical stimulation of the white matter were observed by means of microfluorometry with a Ca2+ indicator, rhod-2, in slice preparations of the visual cortex obtained from young rats. Tetanic stimulation at 5 Hz for 1 min induced a marked fluorescence increase, while single-shock stimulation did not induce a sizable increase in normal perfusate. The tetanus-induced increase took place in a column-like manner from layer VI near the stimulation site to layer II/III of the cortex, although it spread horizontally in layer II/III. The magnitude of fluorescence rise was largest in layer II/III of the cortex. Since N-methyl-D-aspartate (NMDA) receptors are known to exist only on neurons, the following results are taken to indicate that the fluorescent signal is derived mostly from postsynaptic neurons: Application of NMDA in the presence of tetrodotoxin induced a marked fluorescence increase with the same laminar bias as tetanic stimulation did, and the fluorescence increase by single-shock stimulation in Mg(2+)-free medium was almost completely blocked by an antagonist for NMDA receptors. These results support the hypothesis that input-associated entry of Ca2+ into postsynaptic neurons triggers processes for induction of long-term potentiation of synaptic efficacy.

Topics: Animals; Brain Chemistry; Calcium; Electric Stimulation; Evoked Potentials; Female; Fluorescent Dyes; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Long-Term Potentiation; Magnesium; Male; Neuroglia; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spectrometry, Fluorescence; Tetrodotoxin; Visual Cortex