Nitric oxide-dependent long-term potentiation revealed by real-time imaging of nitric oxide production and neuronal excitation in the dorsal horn of rat spinal cord slices.

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.

Depression of presynaptic excitation by the activation of vanilloid receptor 1 in the rat spinal dorsal horn revealed by optical imaging.

In this study, we show that capsaicin (CAP) depresses primary afferent fiber terminal excitability by acting on vanilloid receptor 1 (TRPV1 channels) of primary afferent fibers in adenosine 5'-triphosphate (ATP)- and temperature-dependent manner using two optical imaging methods. First, transverse slices of spinal cord were stained with a voltage-sensitive dye and the net excitation in the spinal dorsal horn was recorded. Prolonged treatment (>20 min) with the TRPV1 channel agonist, CAP, resulted in a long-lasting inhibition of the net excitation evoked by single-pulse stimulation of C fiber-activating strength. A shorter application of CAP inhibited the excitation in a concentration-dependent manner and the inhibition was reversed within several minutes. This inhibition was Ca(++)-dependent, was antagonized by the TRPV1 channel antagonist, capsazepine (CPZ), and the P(2)X and P(2)Y antagonist, suramin, and was facilitated by the P(2)Y agonist, uridine 5'-triphosphate (UTP). The inhibition of excitation was unaffected by bicuculline and strychnine, antagonists of GABA(A) and glycine receptors, respectively. Raising the perfusate temperature to 39 degrees C from 27 degrees C inhibited the excitation (-3%/ degrees C). This depressant effect was antagonized by CPZ and suramin, but not by the P(2)X antagonist, 2', 3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP). Second, in order to record the presynaptic excitation exclusively, we stained the primary afferent fibers anterogradely from the dorsal root. CAP application and a temperature increase from 27 degrees C to 33 degrees C depressed the presynaptic excitation, and CPZ antagonized these effects. Thus, this study showed that presynaptic excitability is modulated by CAP, temperature, and ATP under physiological conditions, and explains the reported central actions of CAP. These results may have clinical importance, especially for the control of pain.

Effects of corticotropin-releasing factor on plasticity of optically recorded neuronal activity in the substantia gelatinosa of rat spinal cord slices.

We examined the effects of corticotropin-releasing factor (CRF) on plasticity of optically recorded neuronal activity in the substantia gelatinosa (lamina II) of 12-18-day-old rat spinal cord slices stained with a voltage-sensitive dye. Single-pulse test stimulation to the dorsal root that activated A and C fibres evoked prolonged (>100 ms) light-absorption change in the lamina II. This response represents the gross membrane potential change of all elements along the slice depth. After conditioning high-frequency stimulation of A-fibre-activating strength, test stimulus elicited less neuronal activity [-27+/-1% (7), (average+/-SE (n)), P<0.01 (*) at 45-60 min after conditioning]. When CRF (1 microM, 10 min) was applied during conditioning, the neuronal activity was facilitated rather than suppressed [+20+/-3% (5), P<0.05]. CRF alone exhibited insignificant effect [-5+/-1% (4), P=0.2]. In the presence of the inhibitory amino acid antagonists bicuculline (1 microM) and strychnine (0.3 microM) in the perfusate, in contrast, the conditioning facilitated it [+27+/-1% (12)*], and CRF treatment during conditioning inhibited the facilitation dose-dependently [0.1 microM: +18+/-2% (5)*, 1 microM: +13+/-1% (7)*]. Although interneuronal actions might contribute, these results suggest that CRF may have dual effects on excitatory synaptic transmission within the lamina II depending upon cellular conditions: a conversion from the induction of long-term depression to long-term potentiation (LTP), and inhibition of LTP induction. Since the LTP is thought to be responsible at least in part for the persistent pain, CRF could regulate the induction.

Inhibitory effect of caffeine on C-fibre-evoked excitation in the rat spinal dorsal horn recorded under Ca2+-free condition: an interaction with halothane.

Effect of caffeine on C-fibre-evoked excitation in the superficial dorsal horn of rat spinal cord slices was investigated in Ca(2+)-free medium using the optical imaging technique. Perfusing slices with Ca(2+)-free solution reversibly suppressed the late phase of dorsal-root stimulus-induced excitation, leaving short-lasting optical responses that primarily represent the excitation of presynaptic elements. Under the Ca(2+)-free condition, 0.1-10 mM caffeine depressed the peak optical amplitude in a concentration-dependent and reversible manner (IC(50) 4.5 mM, I(max) 84%). The volatile anaesthetic halothane at a clinical concentration (0.6 mM) also depressed the peak optical amplitude. Pretreatment with caffeine augmented the inhibitory effect of halothane. These results suggest that caffeine and halothane may interact presynaptically to cause synergistic inhibition of C-fibre-induced excitation in the dorsal horn.

Intrinsic optical signals in the dorsal horn of rat spinal cord slices elicited by brief repetitive stimulation.

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.

Effect of halothane on neuronal excitation in the superficial dorsal horn of rat spinal cord slices: evidence for a presynaptic action.

The action of the volatile anaesthetic halothane on optically recorded neuronal excitation in juvenile rat spinal cord slices was investigated. Prolonged neuronal excitation lasting approximately 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 (IC(50) 0.21 mm, I(max) 28%). In Ca(2+)-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 (IC(50) 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 Ba(2+) 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 [Mg(2+)](o) reduced the excitation similar to that of halothane treatment, and the degree of halothane inhibition became larger with lower [Mg(2+)](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 in the hyperpolarizing direction.