Melissa E. Martenson

A possible neural basis for stress-induced hyperalgesia

ABSTRACT Intense stress and fear have long been known to give rise to a suppression of pain termed “stress‐induced analgesia”, mediated by brainstem pain‐modulating circuitry, including pain‐inhibiting neurons of the rostral ventromedial medulla. However, stress does not invariably suppress pain, and indeed, may exacerbate it. Although there is a growing support for the idea of “stress‐induced hyperalgesia”, the neurobiological basis for this effect remains almost entirely unknown. Using simultaneous single‐cell recording and functional analysis, we show here that stimulation of the dorsomedial nucleus of the hypothalamus, known to be a critical component of central mechanisms mediating neuroendocrine, cardiovascular and thermogenic responses to mild or “emotional” stressors such as air puff, also triggers thermal hyperalgesia by recruiting pain‐facilitating neurons, “ON‐cells”, in the rostral ventromedial medulla. Activity of identified RVM ON‐cells, OFF‐cells and NEUTRAL cells, nociceptive withdrawal thresholds, rectal temperature, and heart rate were recorded in lightly anesthetized rats. In addition to the expected increases in body temperature and heart rate, disinhibition of the DMH induced a robust activation of ON‐cells, suppression of OFF‐cell firing and behavioral hyperalgesia. Blocking ON‐cell activation prevented hyperalgesia, but did not interfere with DMH‐induced thermogenesis or tachycardia, pointing to differentiation of neural substrates for autonomic and nociceptive modulation within the RVM. These data demonstrate a top‐down activation of brainstem pain‐facilitating neurons, and suggest a possible neural circuit for stress‐induced hyperalgesia.

Sensitization of Pain-Modulating Neurons in the Rostral Ventromedial Medulla after Peripheral Nerve Injury

Nerve injury can lead to mechanical hypersensitivity in both humans and animal models, such that innocuous touch produces pain. Recent functional studies have demonstrated a critical role for descending pain-facilitating influences from the rostral ventromedial medulla (RVM) in neuropathic pain, but the underlying mechanisms and properties of the relevant neurons within the RVM are essentially unknown. We therefore characterized mechanical responsiveness of physiologically characterized neurons in the RVM after spinal nerve ligation, a model of neuropathic pain that produces robust mechanical hyperalgesia and allodynia. RVM neurons were studied 7–14 d after spinal nerve ligation, and classified as “on-cells,” “off-cells,” or “neutral cells” using standard criteria of changes in firing associated with heat-evoked reflexes. On-cells are known to promote nociception, and off-cells to suppress nociception, whereas the role of neutral cells in pain modulation remains an open question. Neuronal and behavioral responses to innocuous and noxious mechanical stimulation were tested using calibrated von Frey filaments (4–100 g) applied to the hindpaws ipsilateral and contralateral to the injury, and in sham-operated and unoperated control animals. On- and off-cells recorded in nerve-injured animals exhibited novel responses to innocuous mechanical stimulation, and enhanced responses to noxious mechanical stimulation. Neuronal hypersensitivity in the RVM was correlated with behavioral hypersensitivity. Neutral cells remained unresponsive to cutaneous stimulation after nerve injury. These data demonstrate that both on- and off-cells in the RVM are sensitized to innocuous and noxious mechanical stimuli after nerve injury. This sensitization likely contributes to allodynia and hyperalgesia of neuropathic pain states.

Responses of adult human dorsal root ganglion neurons in culture to capsaicin and low pH

&NA; This study examined the responses of cultured adult human dorsal root ganglion (hDRG) neurons to protons and capsaicin, two substances known to produce pain and hyperalgesia in humans. Both substances were applied to each neuron and responses were examined under both voltage‐ and current‐clamp recording conditions. Sensitivity to protons was tested with rapid acidification of the extracellular fluid from pH 7.35 to 6.0. In neurons nominally clamped near −60 mV, low pH evoked a transient inward current which, in all 40 hDRG neurons tested, was followed by a more sustained inward current. The sustained current was associated with an increase in membrane conductance in 10 neurons, a decrease in 27 neurons, and no overt change in conductance (<10%) in 3 neurons. Current‐clamp recordings in the same neurons showed that the proton‐induced sustained net inward current caused a prolonged depolarization of the membrane potential in all 40 hDRG neurons. The prolonged depolarization was associated with action potential discharge in 5 neurons. Unlike low pH, capsaicin evoked a sustained net inward current in only a subset of neurons tested (10 nM: Symbol, 30 nM: Symbol, 100 nM: Symbol, and 10 &mgr;M: Symbol neurons tested). The capsaicin‐evoked currents were accompanied by an increase in membrane conductance in 15 neurons, a decrease in 2, and no overt change in conductance in 9 neurons. Capsaicin currents, like proton‐induced currents, resulted in prolonged depolarizations (10 nM: Symbol, 30 nM: Symbol, 100 nM: Symbol,d and 10 &mgr;M: Symbol neurons tested). The depolarization resulted in the discharge of action potentials in 14 neurons. It is concluded that, while both protons and capsaicin exert excitatory effects on human sensory neurons, multiple membrane mechanisms lead to the depolarization of cultured hDRG neurons by low pH. Inhibition of resting membrane conductances contributes to the responses to low pH in some hDRG neurons. Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available Symbol. No caption available

Background potassium channel block and TRPV1 activation contribute to proton depolarization of sensory neurons from humans with neuropathic pain

Protons cause a sustained depolarization of human dorsal root ganglion (DRG) neurons [Baumann et al. (1996) Pain, 65, 31–38]. In the present study we sought to determine which ion channels are expressed in human DRG neurons that could mediate the sustained responses observed in the patch‐clamp recordings. RT‐PCR of material from the DRG tissue revealed the presence of mRNAs for a nonselective cation channel that is activated by protons (TRPV1) and background potassium channels that are blocked by protons (TASK‐1, TASK‐3 and Kir2.3). Highly acidic solution (pH 5.4) applied to cultured DRG neurons evoked prolonged currents that were associated with a net increase in membrane conductance. Consistent with the involvement of TRPV1, these proton‐evoked currents were blocked by capsazepine and were only found in neurons that responded to capsaicin with an increase in membrane conductance. Less acidic extracellular solution (pH 6.0) evoked such currents only rarely, but was able to strongly enhance the currents evoked by capsaicin. Capsazepine (1 µm) blocked the currents evoked by capsaicin at pH 7.35, as well as the potentiated responses to capsaicin at pH 6.0. In neurons that were not excited by capsaicin, moderate extracellular acidification (pH 6.0) caused a sustained decrease in resting membrane conductance. The decrease in membrane conductance by protons was associated with inhibition of background potassium channels. This excitatory effect of protons was not blocked by capsazepine. We conclude that in most neurons the sustained depolarization in response to moderately acidic solutions is the result of blocked background potassium channels. In a subset of neurons, TRPV1 also contributes.

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 μm). All non-responders had a soma diameter > 33 μm. Capsazepine (1 μM) 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.

Prostaglandin E2 in the midbrain periaqueductal gray produces hyperalgesia and activates pain-modulating circuitry in the rostral ventromedial medulla

&NA; Recent years have seen significant advances in our understanding of the peripheral and spinal mechanisms through which prostaglandins contribute to nociceptive sensitization. By contrast, the possibility of a supraspinal contribution of these compounds to facilitated pain states has received relatively little attention. One possible mechanism through which prostaglandins could act supraspinally to facilitate nociception would be by recruitment of descending facilitation from brainstem pain‐modulating systems. The rostral ventromedial medulla (RVM) is now known to contribute to enhanced responding in a variety of inflammatory and nerve injury models. Its major supraspinal input, the midbrain periaqueductal gray (PAG), expresses prostanoid receptors and synthetic enzymes. The aim of the present study was to determine whether direct application of prostaglandin E2 (PGE2) within the ventrolateral PAG is sufficient to produce hyperalgesia, and whether any hyperalgesia could be mediated by recruiting nociceptive modulating neurons in the RVM. We determined the effects of focal application of PGE2 in the PAG on paw withdrawal latency and activity of identified nociceptive modulating neurons in the RVM of lightly anesthetized rats. Microinjection of PGE2 (50 fg in 200 nl) into the PAG produced a significant decrease in paw withdrawal latency. 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. These data demonstrate a prostaglandin‐sensitive descending facilitation from the PAG, and suggest that this is mediated by on‐ and off‐cells in the RVM.

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.

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.

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.

A possible neural mechanism for photosensitivity in chronic pain.

Abstract Patients with functional pain disorders often complain of generalized sensory hypersensitivity, finding sounds, smells, or even everyday light aversive. The neural basis for this aversion is unknown, but it cannot be attributed to a general increase in cortical sensory processing. Here, we quantified the threshold for aversion to light in patients with fibromyalgia, a pain disorder thought to reflect dysregulation of pain-modulating systems in the brain. These individuals expressed discomfort at light levels substantially lower than that of healthy control subjects. Complementary studies in lightly anesthetized rat demonstrated that a subset of identified pain-modulating neurons in the rostral ventromedial medulla unexpectedly responds to light. Approximately half of the pain-facilitating “ON-cells” and pain-inhibiting “OFF-cells” sampled exhibited a change in firing with light exposure, shifting the system to a pronociceptive state with the activation of ON-cells and suppression of OFF-cell firing. The change in neuronal firing did not require a trigeminal or posterior thalamic relay, but it was blocked by the inactivation of the olivary pretectal nucleus. Light exposure also resulted in a measurable but modest decrease in the threshold for heat-evoked paw withdrawal, as would be expected with engagement of this pain-modulating circuitry. These data demonstrate integration of information about light intensity with somatic input at the level of single pain-modulating neurons in the brain stem of the rat under basal conditions. Taken together, our findings in rodents and humans provide a novel mechanism for abnormal photosensitivity and suggest that light has the potential to engage pain-modulating systems such that normally innocuous inputs are perceived as aversive or even painful.