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The neural processing of pain and itch took center stage at the Neurobiology of Pain and Itch meeting held April 10-11, 2014, at the University of California, San Francisco (UCSF).
Chaired by Allan Basbaum and David Julius, both from UCSF, and organized by the life sciences reagent company Abcam, the conference featured talks on the circuitry of two related sensations whose molecular and cellular mechanisms are under keen investigation. The following report summarizes selected presentations from the meeting.
Executive Editor at Pain Research Forum
“The field is becoming revolutionized,” said Patrick Mantyh, University of Arizona, Tucson, US, at the beginning of a talk on recent advances in understanding skeletal pain, as he expressed his optimism about a new potential avenue of treatment involving sclerostin, a natural inhibitor of bone formation.
Rodent studies of bone pain have revealed three essential themes, Mantyh said (for review, see Mantyh, 2014). First, compared to the skin, the skeleton is innervated by a more limited repertoire of sensory nerve fibers, 80 percent of which express TrkA, the receptor for nerve growth factor (NGF).
This finding provides a rationale for anti-NGF therapy to relieve bone pain, an approach that has proven effective in rodent models of malignant and non-malignant bone pain, and in people with osteoarthritis pain.
Second, bone that is subjected to consistent mechanical stress, through physical activity, for instance, is always poorly innervated. Third, when fractured bone heals poorly, sprouting of sensory nerves is observed.
This basic neurobiology helps to explain the increased prevalence of skeletal pain in the elderly. People lose bone mass, bone strength, and bone mineral density as they age, a process abetted by disease (both malignant and non-malignant) and physical inactivity.
These factors elevate the risk of fracture, and together with delayed bone healing in the elderly, create a favorable environment for the sensory (and sympathetic) nerve sprouting in bone that is thought to contribute to skeletal pain. Considering these dynamics, a therapeutic approach that builds new bone and speeds fracture healing should prove beneficial for the treatment of bone pain.
One approach that Mantyh described targets the protein sclerostin. Released by osteocytes, bone’s most plentiful cell type, sclerostin is a natural inhibitor of bone formation.
Preclinical studies in rats and monkeys showed that sclerostin-neutralizing antibodies can build bone in osteoporosis models and enhance fracture healing (e.g., see Li et al., 2010).
Anti-sclerostin antibodies are now in Phase 3 testing in patients with osteoporosis (ClinicalTrials.gov).
Could anti-sclerostin antibodies relieve skeletal pain in the elderly by building new bone, improving fracture healing, and, in combination with anti-NGF therapy, denying the permissive environment for nerve sprouting that slow healing favors? Mantyh is currently investigating this exciting hypothesis by using rodent fracture models.
Rohini Kuner, University of Heidelberg, Germany, discussed her work elucidating the molecular signaling events leading to persistent inflammatory pain.
In work published last year, Kuner and colleagues reported that nuclear calcium signaling in spinal dorsal horn neurons is a key player in those events, regulating transcription of a number of genes, including the gene encoding C1q, which is increasingly recognized for its role in synaptic remodeling (see PRF related news story).
Kuner and co-workers discovered that C1q was downregulated in spinal neurons from mice after injection of complete Freund’s adjuvant (CFA), resulting in increased density of dendritic spines, which could underlie the transition from acute to chronic pain in that animal model.
In unpublished research presented at the meeting, Kuner showed data implicating kalirin-7 as another key player driving nociceptive activity-induced synaptic remodeling in inflammatory pain.
Kalirin-7 is a Rho-guanine nucleotide exchange factor (Rho-GEF) that regulates dendritic spines and synapse formation (for review, see Mandela and Ma, 2012). While kalirin-7 is known to be important in many neuropsychiatric and neurological diseases, only recently has it been linked to pain (Peng et al., 2013).
Adding to that work, Kuner reported that kalirin-7 is expressed in spinal dorsal horn neurons, and that knockdown of kalirin-7 by using an adeno-associated virus (AAV)/Cre recombinase method to selectively delete the protein in spinal neurons attenuated inflammatory and mechanical hypersensitivity in mouse models of inflammatory pain.
Her studies also showed that loss of kalirin-7 decreased the density of mature spines and inhibited nociceptive activity-induced structural remodeling.
Furthermore, downregulation of Rac1, a Rho family GTPase, reproduced the phenotype of kalirin-7 knockout animals. The research linked these structural changes to functional alterations, as disruption of the synaptic interactions of kalirin-7 eliminated activity-induced long-term potentiation (LTP) at synapses of neurons projecting to the periaqueductal gray.
Together, said Kuner, the new findings indicate that kalirin-7 acts as a coordinator of structural and functional plasticity at spinal nociceptive synapses. She is planning to extend the current work into neuropathic pain models to further elucidate kalirin-7 actions.
How is itch coded by the nervous system at the spinal cord level? That question was addressed by Sarah Ross, University of Pittsburgh, Pennsylvania, US, in one of several talks on itch.
In previous work, Ross and colleagues found that mice missing the transcription factor Bhlhb5 developed self-inflicted skin lesions and showed elevated scratching in response to itch-producing agents (Ross et al., 2010). Further experiments showed that Bhlhb5 was required for the survival of a subset of inhibitory interneurons (B5-I neurons) in the dorsal horn spinal cord that negatively regulate itch.
At the meeting, Ross described recently published follow-up work performed in collaboration with Andrew Todd, University of Glasgow, UK, that aimed to characterize B5-I neurons (Kardon et al., 2014). They found that these cells express a receptor for somatostatin (SST), an inhibitory neuropeptide, and demonstrated that intrathecal injection of an SST analog, octreotide, caused spontaneous scratching in wild-type mice.
Ross reasoned that if this effect was due specifically to inhibition of B5-I neurons, then the effect should be lessened in knockout mice missing those cells, and, as expected, octreotide had little impact in the Bhlhb5 knockout animals.
Further experiments characterized the B5-I neurons neurochemically into two mostly non-overlapping subpopulations: galanin-expressing and neuronal nitric oxide synthase (nNOS)-expressing cells; knockout mice missing Bhlhb5 exhibited significant losses of both subpopulations.
How did the B5-I neurons function to inhibit itch? Ross and colleagues found that the cells released dynorphin, a κ-opioid receptor agonist. Previous work has suggested that κ-opioids can block itch, and the researchers hypothesized that decreased κ-opioid signaling resulting from loss of the dynorphin-producing B5-I neurons was responsible for the increased scratching in the knockout mice.
After confirming that κ-opioid agonists inhibited scratching behavior in response to pruritogens in wild-type mice, the group tested κ-agonists in the knockout mice. In support of the hypothesis, κ-agonists blocked chloroquine-induced scratching in the knockouts.
In further experiments using chloroquine or capsaicin injection into the cheek of wild-type mice, the researchers showed that the κ-agonists affected only itch, and not pain, behaviors.
Results from a calf model of itch suggested that the κ-agonists functioned at the spinal cord level to regulate itch; the investigators found that intrathecal injection of κ-agonists inhibited itch, while intrathecal administration of κ-antagonists resulted in exaggerated itch responses.
Finally, electrophysiological work revealed that the B5-I neurons mediated the inhibition of itch by counter-stimuli such as noxious chemicals or menthol. These experiments demonstrated that the B5-I neurons received input from sensory neurons that respond to menthol, capsaicin, and mustard oil.
Further experiments using a mouse model of inhibition of itch by menthol showed that B5-I neurons were required for menthol’s anti-itch effects.
Ross’ work suggests that κ-opioids could be effective agents for treating pathological itch. Ross said that the κ-opioid agonist nalfurafine, the only clinically approved drug for itch (marketed in Japan for treatment of itch associated with chronic kidney failure), is now being tested in the US in patients with end-stage renal disease who are receiving hemodialysis (ClinicalTrials.gov).
And in the setting of morphine-induced itch, administering μ- and κ-opioid agonists together, or using agonists that target both receptors, could be a way to ease pain without producing itch.
Qiufu Ma, Dana-Farber Cancer Institute and Harvard Medical School, Boston, US, described his research on the ontogeny of distinct populations of excitatory neurons in the spinal cord, with the goal of better understanding the spinal microcircuitry of mechanical pain processing.
Ma had previously found that conditional knockout mice missing the homeobox gene Tlx3 specifically in two subsets of glutamatergic excitatory neurons in the dorsal horn (dI5 and dILB cells) exhibited impaired behavioral responses to mechanical stimulation, heat, and capsaicin, as well as decreased scratching in response to pruritic agents (Xu et al., 2013).
At the meeting, Ma presented unpublished follow-up work characterizing Tlx3-dependent neurons. In collaboration with the Martyn Goulding lab at the Salk Institute, La Jolla, US, Ma and colleagues have been using an intersectional genetic manipulation strategy to mark and ablate specific sub-populations of dorsal horn neurons.
Preliminary behavioral studies have revealed a specific population of spinal excitatory neurons that are required to process information associated with mechanical pain and itch, but are dispensable for thermal information processing, thereby suggesting the existence of distinct circuits processing different sensory modalities in the spinal cord..
The role of the insular cortex (IC) in painful and pleasurable sensation was the focus of a talk by M. Catherine Bushnell of the National Center for Complementary and Alternative Medicine (NCCAM), US National Institutes of Health, Bethesda, Maryland, US.
The insula is a critical cortical region in pain processing and modulation in both healthy subjects and in chronic pain patients, and also plays a crucial role in emotional aspects of pain (Apkarian et al., 2005).
The picture that emerges from the recent studies described by Bushnell is of the IC as a key brain region involved in many facets of somatosensory experience, that is capable of adapting through plastic changes to the unique sensory input characteristic of both healthy and disease states.
It is well established that the IC is involved in processing pain intensity information (e.g., see Coghill et al., 1999), and Bushnell’s work has shown that heightened activation of the IC is associated with tactile allodynia in painful conditions such as vulvodynia (Pukall et al., 2005) and neuropathic pain (Hofbauer et al., 2006).
But research has also revealed the participation of the IC in the processing of pleasant touch. Bushnell described her work with two patients lacking Aβ afferents, the large myelinated fibers that respond to pleasant touch sensation.
Studying these patients let Bushnell and colleagues study input from a less explored type of fiber, C tactile (CT) fibers (unmyelinated afferents in hairy skin), which also signal pleasant touch.
The first patient, seen by Bushnell in 2002, said she could not sense touch (Olausson et al., 2002). But psychophysical testing indicated the patient could faintly detect a type of pleasant tactile stimulus (a brush stroking her skin), a stimulus designed to activate CT fibers.
Functional magnetic resonance imaging (fMRI) subsequently revealed activation of the IC, but deactivation in somatosensory cortices during CT stimulation, a pattern also observed in a second patient (Olausson et al., 2008).
In addition to underscoring the role of the IC in pleasant touch, that finding also suggested that the CT system primarily mediates emotional aspects of touch, but plays a small role in discriminative aspects (which are mediated by somatosensory cortices).
A more recent study using a heat and capsaicin model of tactile allodynia further supports a multifaceted role of the IC in sensory experience.
Here, Bushnell and colleagues identified, both in healthy subjects and in one of the Aβ-de-afferented patients, different fMRI activation patterns in the IC in response to tactile stimulation (stroking) in the allodynic skin zone versus stroking in a control zone (Liljencrantz et al., 2013).
Another study in one of the patients revealed widespread cortical thinning compared to healthy controls, but increased cortical thickness in the insula and increased functional connectivity both within the insula and between the insula and visual cortex (Čeko et al. 2013).
These alterations could reflect the brain’s capacity to compensate for the lack of input from the missing Aβ fibers, thus revealing the plasticity of the IC.
Plastic changes in the IC are also observed in response to yoga in healthy people. For instance, Bushnell’s studies have revealed increased pain tolerance in yoga practitioners compared to non-yogis that is correlated with increased gray matter in the IC (Villemure et al., 2013).
Yogis also displayed increased anatomical connectivity within the insula, which could account for their increased tolerance to pain.
Finally, the IC also plays a part in itch. For example, an fMRI study of healthy volunteers found that, in comparison to passive (investigator-controlled) scratching, active scratching (performed by subjects themselves) was associated with higher pleasurability and a greater deactivation of the IC (Papoiu et al., 2013).
In sum, Bushnell’s work has further delineated the role of the IC in painful and pleasurable sensation. It has also provided a convincing physiological basis for the ability of complementary and alternative therapies, such as yoga, to alter pain pathways
Visitors leave their hearts in San Francisco, but what do they take home? Attendees at the Neurobiology of Pain and Itch meeting could answer: a cutting-edge knowledge of the latest science on pain and itch circuitry, and a strong sense that many more exciting findings are soon to come.