Convergent neural representations of experimentally-induced acute pain in healthy volunteers: A large-scale fMRI meta-analysis.

Characterizing a reliable, pain-related neural signature is critical for translational applications. Many prior fMRI studies have examined acute nociceptive pain-related brain activation in healthy participants. However, synthesizing these data to identify convergent patterns of activation can be challenging due to the heterogeneity of experimental designs and samples. To address this challenge, we conducted a comprehensive meta-analysis of fMRI studies of stimulus-induced pain in healthy participants. Following pre-registration, two independent reviewers evaluated 4,927 abstracts returned from a search of 8 databases, with 222 fMRI experiments meeting inclusion criteria. We analyzed these experiments using Activation Likelihood Estimation with rigorous type I error control (voxel height p < 0.001, cluster p < 0.05 FWE-corrected) and found a convergent, largely bilateral pattern of pain-related activation in the secondary somatosensory cortex, insula, midcingulate cortex, and thalamus. Notably, these regions were consistently recruited regardless of stimulation technique, location of induction, and participant sex. These findings suggest a highly-conserved core set of pain-related brain areas, encouraging applications as a biomarker for novel therapeutics targeting acute nociceptive pain.

Primary Afferent-Derived BDNF Contributes Minimally to the Processing of Pain and Itch.

BDNF is a critical contributor to neuronal growth, development, learning, and memory. Although extensively studied in the brain, BDNF is also expressed by primary afferent sensory neurons in the peripheral nervous system. Unfortunately, anatomical and functional studies of primary afferent-derived BDNF have been limited by the availability of appropriate molecular tools. Here, we used targeted, inducible molecular approaches to characterize the expression pattern of primary afferent BDNF and the extent to which it contributes to a variety of pain and itch behaviors. Using a BDNF-LacZ reporter mouse, we found that BDNF is expressed primarily by myelinated primary afferents and has limited overlap with the major peptidergic and non-peptidergic subclasses of nociceptors and pruritoceptors. We also observed extensive neuronal, but not glial, expression in the spinal cord dorsal horn. In addition, because BDNF null mice are not viable and even Cre-mediated deletion of BDNF from sensory neurons could have developmental consequences, here we deleted BDNF selectively from sensory neurons, in the adult, using an advillin-Cre-ER line crossed to floxed BDNF mice. We found that BDNF deletion in the adult altered few itch or acute and chronic pain behaviors, beyond sexually dimorphic phenotypes in the tail immersion, histamine, and formalin tests. Based on the anatomical distribution of sensory neuron-derived BDNF and its limited contribution to pain and itch processing, we suggest that future studies of primary afferent-derived BDNF should examine behaviors evoked by activation of myelinated primary afferents.

Neuronal aromatase expression in pain processing regions of the medullary and spinal cord dorsal horn.

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion-derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.

Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells.

The heat and capsaicin receptor, TRPV1, is required for the detection of painful heat by primary afferent pain fibers (nociceptors), but the extent to which functional TRPV1 channels are expressed in the CNS is debated. Because previous evidence is based primarily on indirect physiological responses to capsaicin, here we genetically modified the Trpv1 locus to reveal, with excellent sensitivity and specificity, the distribution of TRPV1 in all neuronal and non-neuronal tissues. In contrast to reports of widespread and robust expression in the CNS, we find that neuronal TRPV1 is primarily restricted to nociceptors in primary sensory ganglia, with minimal expression in a few discrete brain regions, most notably in a contiguous band of cells within and adjacent to the caudal hypothalamus. We confirm hypothalamic expression in the mouse using several complementary approaches, including in situ hybridization, calcium imaging, and electrophysiological recordings. Additional in situ hybridization experiments in rat, monkey, and human brain demonstrate that the restricted expression of TRPV1 in the CNS is conserved across species. Outside of the CNS, we find TRPV1 expression in a subset of arteriolar smooth muscle cells within thermoregulatory tissues. Here, capsaicin increases calcium uptake and induces vasoconstriction, an effect that likely counteracts the vasodilation produced by activation of neuronal TRPV1.

Pain behavior in the formalin test persists after ablation of the great majority of C-fiber nociceptors.

Although the formalin test is a widely used model of persistent pain, the primary afferent fiber types that underlie the cellular and behavioral responses to formalin injection are largely unknown. Here we used a combined genetic and pharmacological approach to investigate the effect of ablating subsets of primary afferent nociceptors on formalin-induced nocifensive behaviors and spinal cord Fos protein expression. Intrathecal capsaicin-induced ablation of the central terminals of TRPV1+neurons greatly reduced the behavioral responses and Fos elicited by low-dose (0.5%) formalin. In contrast, genetic ablation of the MrgprD-expressing subset of non-peptidergic unmyelinated afferents, which constitute a largely non-overlapping population, altered neither the behavior nor the Fos induced by low-dose formalin. Remarkably, nocifensive behavior following high-dose (2%) formalin was unchanged in mice lacking either afferent population, or even in mice lacking both populations, which together make up the great majority of C-fiber nociceptors. Thus, at high doses, which are routinely used in the formalin test, formalin-induced "pain" behavior persists in the absence of the vast majority of C-fiber nociceptors, which points to a contribution of a large spectrum of afferents secondary to non-specific formalin-induced tissue and nerve damage.

Differential ATF3 expression in dorsal root ganglion neurons reveals the profile of primary afferents engaged by diverse noxious chemical stimuli.

Although transgenic and knockout mice have helped delineate the mechanisms of action of diverse noxious compounds, it is still difficult to determine unequivocally the subpopulations of primary afferent nociceptor that these molecules engage. As most noxious stimuli lead to tissue and/or nerve injury, here we used induction of activating transcription factor 3 (ATF3), a reliable marker of nerve injury, to assess the populations of primary afferent fibers that are activated after peripheral administration of noxious chemical stimuli. In wild-type mice, hindpaw injections of capsaicin, formalin, mustard oil or menthol induce expression of ATF3 in distinct subpopulations of sensory neurons. Interestingly, even though these noxious chemicals are thought to act through subtypes of transient receptor potential (TRP) channels, all compounds also induced ATF3 in neurons that appear not to express the expected TRP channel subtypes. On the other hand, capsaicin failed to induce ATF3 in mice lacking TRPV1, indicating that TRPV1 is required for both the direct and indirect induction of ATF3 in sensory neurons. By contrast, only low doses of formalin or mustard oil failed to induce ATF3 in TRPA1 null mice, indicating that injections of high doses (>0.5%) of formalin or mustard oil recruit both TRPA1- and non-TRPA1 expressing primary afferent fibers. Finally, peripheral injection of menthol, a TRPM8 receptor agonist, induced ATF3 in a wide variety of sensory neurons, but in a TRPM8-independent manner. We conclude that purportedly selective agonists can activate a heterogeneous population of sensory neurons, which ultimately could contribute to the behavioral responses evoked.

Profound reduction of somatic and visceral pain in mice by intrathecal administration of the anti-migraine drug, sumatriptan.

Sumatriptan and the other triptan drugs target the serotonin receptor subtypes1B, 1D, and 1F (5-HT(1B/D/F)), and are prescribed widely in the treatment of migraine. An anti-migraine action of triptans has been postulated at multiple targets, within the brain and at both the central and peripheral terminals of trigeminal "pain-sensory" fibers. However, as triptan receptors are also located on "pain-sensory" afferents throughout the body, it is surprising that triptans only reduce migraine pain in humans, and experimental cranial pain in animals. Here we tested the hypothesis that sumatriptan can indeed reduce non-cranial, somatic and visceral pain in behavioral models in mice. Because sumatriptan must cross the blood brain barrier to reach somatic afferent terminals in the spinal cord, we compared systemic to direct spinal (intrathecal) sumatriptan. Acute nociceptive thresholds were not altered by sumatriptan pre-treatment, regardless of route. However, in behavioral models of persistent inflammatory pain, we found a profound anti-hyperalgesic action of intrathecal, but not systemic, sumatriptan. By contrast, sumatriptan was completely ineffective in an experimental model of neuropathic pain. The pronounced activity of intrathecal sumatriptan against inflammatory pain in mice raises the possibility that there is a wider spectrum of therapeutic indications for triptans beyond headache.

TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents.

TRPA1 is an excitatory ion channel targeted by pungent irritants from mustard and garlic. TRPA1 has been proposed to function in diverse sensory processes, including thermal (cold) nociception, hearing, and inflammatory pain. Using TRPA1-deficient mice, we now show that this channel is the sole target through which mustard oil and garlic activate primary afferent nociceptors to produce inflammatory pain. TRPA1 is also targeted by environmental irritants, such as acrolein, that account for toxic and inflammatory actions of tear gas, vehicle exhaust, and metabolic byproducts of chemotherapeutic agents. TRPA1-deficient mice display normal cold sensitivity and unimpaired auditory function, suggesting that this channel is not required for the initial detection of noxious cold or sound. However, TRPA1-deficient mice exhibit pronounced deficits in bradykinin-evoked nociceptor excitation and pain hypersensitivity. Thus, TRPA1 is an important component of the transduction machinery through which environmental irritants and endogenous proalgesic agents depolarize nociceptors to elicit inflammatory pain.

Characterization of wide dynamic range neurons in the deep dorsal horn of the spinal cord in preprotachykinin-a null mice in vivo.

We previously reported that mice with a deletion of the preprotachykinin-A (pptA) gene, from which substance P (SP) and neurokinin A (NKA) are derived, exhibit reduced behavioral responses to intense stimuli, but that behavioral hypersensitivity after injury is unaltered. To understand the contribution of SP and NKA to nociceptive transmission in the spinal cord, we recorded single-unit activity from wide dynamic range neurons in the lamina V region of the lumbar dorsal horn of urethane-anesthetized wild-type and ppt-A null mutant (-/-) mice. We found that intensity coding to thermal stimuli was largely preserved in the ppt-A -/- mice. Neither the peak stimulus-evoked firing nor the neuronal activity during the initial phase (0-4 s) of the 41-49 degrees C thermal stimuli differed between the genotypes. However, electrophysiological responses during the late phase of the stimulus (5-10 s) and poststimulus (11-25 s) were significantly reduced in ppt-A -/- mice. To activate C-fibers and to sensitize the dorsal horn neurons we applied mustard oil (MO) topically to the hindpaw. We found that neither total MO-evoked activity nor sensitization to subsequent stimuli differed between the wild-type and ppt-A -/- mice. However, the time course of the sensitization and the magnitude of the poststimulus discharges were reduced in ppt-A -/- mice. We conclude that SP and/or NKA are not required for intensity coding or sensitization of nociresponsive neurons in the spinal cord, but that these peptides prolong thermal stimulus-evoked responses. Thus whereas behavioral hypersensitivity after injury is preserved in ppt-A -/- mice, our results suggest that the magnitude and duration of these behavioral responses would be reduced in the absence of SP and/or NKA.

Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T.

Subunit non-selective N-methyl-D-aspartate (NMDA) receptor antagonists reduce injury-induced pain behavior, but generally produce unacceptable side effects. In this study, we examined the antinociceptive and motor effects of cone snail venom-derived peptides, conantokins G and T (conG and conT), which are selective inhibitors of the NR2B or NR2A and NR2B subtypes of the NMDA receptor, respectively. We tested the effects of conG and conT in models of tissue (formalin test), nerve injury (partial sciatic nerve ligation) and inflammation-induced (intraplantar Complete Freund's Adjuvant; CFA) pain in mice. In the formalin test, intrathecal (i.t.) conG or conT suppressed the ongoing pain behavior (ED(50) and 95% confidence intervals (CI), 11 (7-19) and 19 (11-33), respectively) at doses that were 17-27 times lower than those required to impair motor function (accelerating rotarod treadmill test: ED(50) and 95% CI, 300 (120-730) and 320 (190-540) pmol, respectively). By comparison, SNX-111, an N-type voltage-sensitive calcium channel antagonist that is also derived from cone snail venom, produced significant motor impairment at a dose (3.0 pmol, i.t.) that was only partially efficacious in the formalin test. Furthermore, conG reversed the allodynia produced by nerve injury, with greater potency on thermal (ED50 and 95% CI, 24 (10-55) pmol) than on mechanical allodynia (59 (33-105) pmol). Finally, a single dose of conG (100 pmol, i.t.) also reduced CFA-evoked thermal and mechanical allodynia. Taken together, these results demonstrate that conantokins exhibit potent antinociceptive effects in several models of injury-induced pain. The study supports the notion that drugs directed against subtypes of the NMDA receptor, by virtue of their reduced side-effect profile, hold promise as novel therapeutic agents for the control of pain.