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By imaging neuronal excitation in rat spinal cord slices with a voltage-sensitive dye, we examined the role of glial cells in the P2X receptor agonist αβ-methylene ATP (αβmeATP)-triggered long-term potentiation (LTP) in the dorsal horn. Bath application of αβmeATP potentiated neuronal excitation in the superficial dorsal horn. The potentiation was inhibited in the presence of the P2X receptor antagonists TNP-ATP, PPADS and A-317491, and was not induced in slices taken from rats neonatally treated with capsaicin. These results suggest that αβmeATP acts on P2X receptors, possibly P2X3 and/or P2X2/3, in capsaicin-sensitive primary afferent terminals. Furthermore, the potentiation was inhibited by treatment with the glial metabolism inhibitor monofluoroacetic acid. Results obtained with the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, tumour necrosis factor-α (TNF-α) and interleukin (IL)-6, and antibodies to TNF-α and IL-6, as well as by double immunolabelling of activated p38 MAPK with markers of astrocytes and microglia, demonstrated that αβmeATP activated p38 MAPK in astrocytes, and that the presence of proinflammatory cytokines and p38 MAPK activation were necessary for the induction of αβmeATP-triggered LTP. These findings indicate that glial cells contribute to the αβmeATP-induced LTP, which might be part of a cellular mechanism for the induction of persistent pain. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is thought to be involved in the central mechanism of hyperalgesia and allodynia at the spinal level. Recently, we reported that NO played an important role in the induction of long-term potentiation (LTP) of synaptic strength in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. In this study, to elucidate the relationship of NO to LTP in spinal dorsal horn, we measured the spatiotemporal distribution of NO signal with the NO-sensitive dye, DAR-4M, and neuronal excitation with the voltage-sensitive dye, RH482, in rat spinal cord slices, elicited by dorsal root stimulation. In superficial dorsal horn, neuronal excitation evoked by C fiber-activating dorsal root stimulation was potentiated for more than 2 h after low-frequency conditioning stimulation (LFS, 240 pulses at 2 Hz for 2 min). In the same slices that exhibited LTP, NO was produced and distributed in the superficial dorsal horn during the delivery of LFS, and the amplitude of LTP and amount of NO production showed close correlation from slice to slice. LTP and production of NO were inhibited in the presence of the NO synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (CO). These results suggest that production and distribution of NO is necessary for the induction of LTP in spinal dorsal horn, and that CO contributes to the LTP induction and NO production by LFS. [ABSTRACT FROM AUTHOR]

Abstract With repetitive electrical stimulation of the dorsal root (20 Hz for 1 s at C-fibre strength), intrinsic optical signals (IOSs), measured as changes in light transmittance, were recorded in the superficial dorsal horn of rat spinal cord slices using a photodiode array imaging device. The mechanism underlying the induction of IOSs was investigated. IOSs elicited by brief repetitive stimulation persisted for 1–2 min and were decreased by reducing external Cl– concentration or by cation–chloride cotransport inhibitors. Furosemide was most effective whilst bumetanide was least effective among the inhibitors tested. A 1-min elevation of external K+ concentration evoked IOSs in the dorsal horn in the absence of stimulation, and K+ -induced IOSs were inhibited by furosemide. These results suggest that the uptake of excess K+ via the furosemide-sensitive, cation–chloride cotransporters underlies the induction of the IOSs. One-minute exposure to hypotonic solutions, which would cause cell swelling, induced IOSs in the superficial dorsal horn. Whilst osmotic-induced IOSs were not affected by furosemide, they were inhibited by HgCl2 in a 2-mercaptoethanol-sensitive manner. The stimulation-induced IOSs were similarly depressed by HgCl2 . In contrast, voltage-sensitive dye signals and field potentials, evoked by single electrical stimuli, were significantly less affected by HgCl2 . These results suggest that there is a specialized water transport pathway in the superficial dorsal horn, and that IOSs elicited by brief repetitive activation of C-fibres are attributable to cell swelling caused by water influx through this pathway, as an osmotic gradient is established by the uptake of K+ via the furosemide-sensitive cotransporters. [ABSTRACT FROM AUTHOR]

Abstract The action of the volatile anaesthetic halothane on optically recorded neuronal excitation in juvenile rat spinal cord slices was investigated. Prolonged neuronal excitation lasting ≈ 100 ms was evoked in the superficial dorsal horn after single-pulse dorsal root stimulation that activated both A- and C-fibres. Halothane depressed the neuronal excitation in a concentration-dependent manner (IC50 0.21 mm, I max 28%). In Ca2+ -free solution, dorsal root stimulation induced excitation with a short duration of several tens of milliseconds, in which the excitation of the postsynaptic component was largely eliminated. Under these conditions, halothane also depressed the excitation concentration-dependently (IC50 0.46 mm, I max 60%). Most of the suppression occurred within 5 min of halothane application, and the effect of halothane was fully reversible upon washout of the anaesthetic. Application of bicuculline and strychnine or picrotoxin, or reduction of extracellular Cl– concentration ([Cl– ]o ), had no effect on halothane inhibition. Applications of K+ channel blockers tetraethyl ammonium, 4-aminopyridine, Cs+ or Ba2+ either had no effect or augmented the inhibitory effect of halothane. On the other hand, the degree of inhibition by halothane was found to be dependent on [K+ ]o ; the higher [K+ ]o , the larger the depression. In addition, decreases in [Na+ ]o and [Mg2+ ]o reduced the excitation similar to that of halothane treatment, and the degree of halothane inhibition became larger with lower [Mg2+ ]o . These results lead to a hypothesis that halothane suppresses the excitation of presynaptic elements by inhibiting presynaptic Na+ channels by shifting the steady-state inactivation curve... [ABSTRACT FROM AUTHOR]

Abstract The neuronal circuitry through which prolonged excitation is generated in the spinal dorsal horn was investigated using optical imaging of neuronal excitation in transverse slices of rat spinal cords. It is known that tetanic stimulation (20 Hz for 1 s) of the dorsal root that activates both A and C primary afferent fibres elicits slow intrinsic optical signals (IOS) in the dorsal horn, seen most intensely in the substantia gelatinosa (SG), lamina II, and that IOS expresses in part the slow synaptic response recorded intracellularly in dorsal horn neurons. We here report that the slow IOS within the SG were completely abolished after an incision was made at the border between the SG and the deeper laminae, but not after an incision within the deeper dorsal horn of the laminae III–V. The result demonstrates directly that, in order to generate prolonged excitation in the SG, the neuronal elements in the deeper dorsal horn must be intact. Thus, the afferent information might be received first by the deeper elements and then transmitted to the SG region, and/or collaboration between the SG and deeper elements is necessary to maintain prolonged excitation in the SG. [ABSTRACT FROM AUTHOR]