Neural basis for improgan antinociception.

Improgan, the prototype compound of a novel class of non-opioid analgesic drugs derived from histamine antagonists, attenuates thermal and mechanical nociception in rodents following intracerebroventricular (i.c.v.) administration. Improgan does not bind to known opioid, histamine or cannabinoid receptors, and its molecular target has not been identified. It is known however, that improgan acts directly in the periaqueductal gray and the rostral ventromedial medulla to produce its antinociceptive effects, and that inactivation of the rostral ventromedial medulla prevents the antinociceptive effect of improgan given i.c.v. Here we used in vivo single-cell recording in lightly anesthetized rats to show that improgan engages pain-modulating neurons in the medulla to produce antinociception. Following improgan administration, OFF-cells, which inhibit nociception, became continuously active and no longer paused during noxious stimulation. The increase in OFF-cell firing does not represent a non-specific neuroexcitant effect of this drug, since ON-cell discharge, associated with net nociceptive facilitation, was depressed. NEUTRAL-cell firing was unaffected by improgan. The net response of rostral ventromedial medulla (RVM) neurons to improgan is thus comparable to that evoked by mu-opioids and cannabinoids, well known RVM-active analgesic drugs. This common basis for improgan, opioid, and cannabinoid antinociception in the RVM supports the idea that improgan functions as a specific analgesic agent.

Prostaglandin E2 in the medial preoptic area produces hyperalgesia and activates pain-modulating circuitry in the rostral ventromedial medulla.

Prostaglandin E2 (PGE2) produced in the medial preoptic region (MPO) in response to immune signals is generally accepted to play a major role in triggering the illness response, a complex of physiological and behavioral changes induced by infection or injury. Hyperalgesia is now thought to be an important component of the illness response, yet the specific mechanisms through which the MPO acts to facilitate nociception have not been established. However, the MPO does project to the rostral ventromedial medulla (RVM), a region with a well-documented role in pain modulation, both directly and indirectly via the periaqueductal gray. To test whether PGE2 in the MPO produces thermal hyperalgesia by recruiting nociceptive modulating neurons in the RVM, we recorded the effects of focal application of PGE2 in the MPO on paw withdrawal latency and activity of identified nociceptive modulating neurons in the RVM of lightly anesthetized rats. Microinjection of a sub-pyrogenic dose of PGE2 (50 fg in 200 nl) into the MPO produced thermal hyperalgesia, as measured by a significant decrease in paw withdrawal latency. In animals displaying behavioral hyperalgesia, the PGE2 microinjection activated on-cells, RVM neurons thought to facilitate nociception, and suppressed the firing of off-cells, RVM neurons believed to have an inhibitory effect on nociception. A large body of evidence has implicated prostaglandins in the MPO in generation of the illness response, especially fever. The present study indicates that the MPO also contributes to the hyperalgesic component of the illness response, most likely by recruiting the nociceptive modulating circuitry of the RVM.

Vanilloid receptor expression and capsaicin excitation of rat dental primary afferent neurons.

Little is known about the molecular mechanisms that cause excitation of neurons which innervate the teeth. We investigated whether rat dental sensory neurons express the vanilloid (capsaicin) receptor (VR1). Dental sensory neurons were identified by retrograde transport of the fluorescent dye DiIC18 placed in maxillary molars. Patch-clamp recordings in culture showed that 65% of DiIC18-labeled rat trigeminal ganglion neurons are excited by capsaicin. Responders covered the entire range of cell sizes examined (soma diameter, 24 to 48 microm). All non-responders had a soma diameter > 33 microm. Capsazepine (1 microM) reduced the capsaicin-evoked membrane current (6/6) and depolarization (7/7 responders). RT-PCR amplified a 375-bp product from DiIC18-labeled neurons which was identical to that expected for VR1. Thus, many rat dental primary afferent neurons are excited by capsaicin, and the response appears to be mediated by VR1. These results suggest that pharmacological blockers of VR1 may provide significant relief of dental pain.

Extracellular protons both increase the activity and reduce the conductance of capsaicin- gated channels.

Capsaicin evokes a membrane current in trigeminal ganglion neurons that is increased substantially in a moderately acidic extracellular environment. Using excised outside-out membrane patches, we studied the mechanism by which protons enhance the sustained response to capsaicin. In the absence of capsaicin, extracellular exposure to a moderately acidic physiological solution (pH 6.6) did not result in sustained channel openings in any capsaicin-sensitive outside-out patches. When co-applied with capsaicin, the acidic extracellular solution greatly increased the probability of capsaicin-gated channels being in the open state. In addition, acidic extracellular solution appeared to increase the number of channels available to be opened by capsaicin. The amplitude of the unitary currents was reduced by the acidic extracellular solution. These results show that the proton enhancement of the capsaicin-evoked whole-cell excitatory current is attributable to proton-receptive site(s) causing a marked increase in the activity of capsaicin-gated channels.

Preferential inhibition of Ih in rat trigeminal ganglion neurons by an organic blocker.

The potency and specificity of a novel organic Ih current blocker DK-AH 268 (DK, Boehringer) was studied in cultured rat trigeminal ganglion neurons using whole-cell patch-clamp recording techniques. In neurons current-clamped at the resting potential, the application of 10 microM DK caused a slight hyperpolarization of the membrane potential and a small increase in the threshold for action potential discharge without any major change in the shape of the action potential. In voltage-clamped neurons, DK caused a reduction of a hyperpolarization-activated current. Current subtraction protocols revealed that the time-dependent, hyperpolarization-activated currents blocked by 10 microM DK or external Cs+ (3 mM) had virtually identical activation properties, suggesting that DK and Cs+ caused blockade of the same current, namely Ih. The block of Ih by DK was dose-dependent. At the intermediate and higher concentrations of DK (10 and 100 microM) a decrease in specificity was observed so that time-independent, inwardly rectifying and noninactivating, voltage-gated outward potassium currents were also reduced by DK but to a much lesser extent than the time-dependent, hyperpolarization-activated currents. Blockade of the time-dependent, hyperpolarization-activated currents by DK appeared to be use-dependent since it required hyperpolarization for the effect to take place. Relief of DK block was also aided by membrane hyperpolarization. Since both the time-dependent current blocked by DK and the Cs+-sensitive time-dependent current behaved as Ih, we conclude that 10 microM DK can preferentially reduce Ih without a major effect on other potassium currents. Thus, DK may be a useful agent in the investigation of the function of Ih in neurons.

Enhancement of rat trigeminal ganglion neuron responses to piperine in a low-pH environment and block by capsazepine.

Both trigeminal and spinal ganglion neurons show a strong potentiation of responses to the irritant capsaicin in an acidic environment. The present study revealed that there is also a strong interaction between protons and piperine, another vanilloid irritant. We studied the mechanism of the interaction between protons and piperine. Whole-cell patch clamp recordings were performed on cultured adult rat trigeminal ganglion (TG) neurons voltage-clamped near their resting membrane potential (-60 mV). Piperine (10 microM) caused a sustained net inward current associated with either an increase or decrease in membrane conductance. When protons and piperine were co-applied, the membrane currents evoked in piperine-sensitive TG neurons far exceeded the algebraic sum of the responses to the two stimuli applied in isolation. Capsazepine blocked the response of TG neurons to piperine at both physiological and acidic pH. In the presence of capsazepine, responses to the mixture of piperine and protons resembled the response to the low pH stimulus applied alone. Capsazepine had no effect on the sustained proton-induced current. These findings suggest that protons enhance the piperine current by altering the vanilloid receptor/channel complex or increasing the length constant of the space clamp.

Potentiation of rabbit trigeminal responses to capsaicin in a low pH environment.

The sensitivity of 35 adult rabbit trigeminal ganglion neurons to low pH (pH 6.0), 10 microM capsaicin (CAP) and 10 microM capsaicin at low pH (CAP@pH6.0) was studied using voltage-clamp whole-cell recording techniques. Neurons responded to pH 6.0 with a transient inward current, followed by a more slowly activating (sustained) net inward current. Responses to capsaicin showed only a sustained current. Capsaicin caused an increase in membrane conductance, whereas responses to low pH were associated with either a net increase or decrease in conductance. A subset of neurons (n = 14) responded to CAP@pH6.0 with a sustained current which exceeded the sum of the peak sustained currents evoked by CAP and pH 6.0 applied singularly by approximately a factor of 4. The current was associated with a substantial increase in membrane conductance. The present results indicate that, in addition to a direct conductance activating effect, protons have the ability to enhance the current evoked by capsaicin.

Responses of cultured adult monkey trigeminal ganglion neurons to capsaicin.

The sensitivity of adult primate (Macaca mulatta) trigeminal ganglion neurons to capsaicin was studied using whole-cell recording techniques. Neurons responding to capsaicin (9 out of 14) generated inward currents of up to 3.0 nA (median = 0.23 nA; interquartile range = 1.19 nA) upon drug application measured at -60 mV. Capsaicin-sensitive neurons had longer action potential (AP) durations than capsaicin-insensitive neurons.