EDITOR'S NOTE: Shan Lou is a graduate student in the lab of Quifu Ma, Dana Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, where she studies the development of mechanical sensory neurons in the dorsal root ganglia (DRG). She agreed to serve as a roving reporter for the PRF at the recent Society for Neuroscience meeting in Washington, DC. Below are Lou’s picks of the most interesting posters and talks of the meeting.
Technology Progress: Automation Facilitates Pain Research
In pain research, conventional behavioral assays usually involve video recording and manual quantification of specific animal behaviors. To increase efficiency and reduce bias, several techniques have been invented. Jasenka Borzan, Johns Hopkins University, Baltimore, Maryland, presented data on the use of rodent ultrasonic vocalizations as a measure of spontaneous pain. The researchers found that rats injected with formalin produced more ultrasonic signals (short whistles) than saline-injected controls. Meanwhile, injection of the itch-producing 5-hydroxytryptamine induced a specific non-vocal ultrasonic signal called "swipe," which is indicative of scratching. By combining waves and frequencies of the ultrasonic signals, an easier andmoreprecise measurement of animal behavior could be achieved. This research is not the first to report the indicative role of ultrasonic signals of rodent behaviors. It has been reported that rats can "laugh" at 50 kHz while tickled, while 22kHz indicates negative emotions (Panskepp and Burgdorf, 2003; Panskepp and Burgdorf 2000). Currently, the signature ultrasonic signals that could correlate to specific behaviors are still limited, and further screening is needed to establish a more complete profile.
Skin as a neurotrophic organ
It is widely agreed that skin plays an important role in sensory neuron development, innervation, and specification, by producing a variety of neurotrophic factors, including nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), artemin, neurturin, and others. Yet the molecular mechanisms of the target-derived signal at the skin level and neuronal specification in the DRG are not understood. In a talk, Kathryn Albers of the University of Pittsburgh, Pennsylvania, described her work using a skin-specific Cre mouse line (K14-Cre) to overexpress various neurotrophic factors in keratinocytes in vivo, and evaluate the effects on sensory afferent neurons. Albers reported that artemin overexpression led to increased expression of the mRNA for the growth factor receptor GFRa3+, and the TrpA and TrpV1 channels in DRG neurons. A lower threshold and higher firing rate were observed in the heat-sensitive C fibers in the skin nerve preparation, corresponding to an increase in noxious heat and cold sensitivity in the artemin-overexpressing mice. Overexpressing GDNF led to an increase in IB4-positive, P2X3-expressing neurons as well as mechanical and heat hypersensitivity. When neurturin was overexpressed, an increase of neurons bearing the TrpM8 channel was observed, as well as increased TrpM8/IB4 colocalization. It remains to be seen whether the correlations between neurotrophins and neuron specifications observed in neurotrophin overexpressing mice can be confirmedin other animal models. One interesting observation is that nerve injury induced artemin expression in the skin. Based on the overexpression experiment, artemin might be expected to increase heat sensitivity of neurons within the mechanically sensitive population, which could explain the heat hypersensitization that occurs in nerve injury conditions.
To further dissect the different roles of neurotrophins, Leslie Ponce and colleagues, Mannheim, Germany, used a compartmented chamber to look into the correlation between neurite outgrowth and neurotrophins. In their poster, they reported that when NGF was applied to DRG soma in the central compartment, there was little neurite growth in the side compartment, while co-culturing keratinocytes dissociated from the skin with GDNF or artemin in the side compartments caused neurite outgrowth. When NGF or GDNF was applied to the side (neurite-containing) compartments, a higher percentage of capsaicin-sensitive neuritis grew compared to the controls. This in vitro model suggested that different neurotrophins have various targets: While some function to sustain neuron survival by targeting the soma, some function to promote neurite growth targeting the axons. The interaction of target-derived signals and the corresponding neurons could be the underlying mechanism of sensory modality specification. Ponce also showed data that neuronal activity (stimulated by KCl application to neurites) caused calcium mobilization in the co-cultured keratinocytes, suggesting a dynamic relationship between the neurons and target tissues.
Autism and tactile touch
C-tactile neurons are recently discovered, low threshold, unmyelinated neurons. Their functions are unknown, but they have been suggested to play a role in the affective sensory system. Testing the affective role of C-tactile neurons has been quite challenging, due to lack of established tactile behavioral assays, as well as the lack of loss-of-function models. Francis McGlone, Liverpool John Moores University, suggested that a well-known disease, autism, could be used as a model to study C-tactile fibers and their functions in brain. It was well known that the autism patients show significant aversion to social interactions with other human beings. McGlone suggested that this lack of affective tactile stimuli since birth would lead to underdevelopment of the C-tactile pathway. Indeed, in fMRI scans, autism patients showed much less activity in the insular cortex area of the brain than in a control group in response to strokes witha soft brush on the arm. This observation indicated that people with autism have impaired development of the tactile pathway. More experiments need to be done to test if the impairment is caused by a lack of C-tactile fiber stimulation or other pathological factors of autism.
TrpA1 as a potential pain drug target
A painkiller that is specific to the chronic pain but not affecting acute and normal pain sensation is always the goal of pain researchers. To dissect the chronic pain and normal pain pathways during inflammation, Cheryl Stucky, Medical College of Wisconsin, Milwaukee, used an ex vivo skin nerve preparation system to record unmyelinated nerve activities in response to mechanical, cold, and heat stimulation in preparations from animals that had been treated with complete Freund’s adjuvant (CFA). In her talk, Stucky described the response of multiple varieties of C fibers based on their sensitivities to different stimuli. Only the cold and mechanical sensitive C fibers are mechanically sensitized by CFA-induced inflammation. It was also found that application of TrpA1 antagonist could block the hypersensitization caused by inflammation. To achieve long-term blockage of the TrpA1 channel, a combination of TrpA1 agonist cinnamaldehyde and the ion channel blocker QX314 were applied to the inflamed skin. This application was successful in reversing the mechanical hypersensitivity while having little effect on baseline mechanical sensitivity.
Explanation for two phases of the formalin test
Injection of formalin into the paw of a rodent generates a characteristic two-phase sensory hypersensitivity, with the first phase explained by a peripheral mechanism, and the second phase due to spinal cord sensitization. But the quiescent interphase between the two has not been studied in detail. Michael Fischer, University of Cambridge, U.K., and colleagues revisited the mechanisms of formalin-induced sensitization. Using whole cell patch clamp recordings of DRG neurons from pigs, the group found that, after a transient depolarization caused by formalin infusion, a hyperpolarization occurred that last five to 10 minutes, followed by the second depolarization. The timeline of the dosage-dependent hyperpolarization correlatedwell with the interphase of the animal inflammation model. But it remains to be seen what is the sensor of formalin-induced hyperpolarization and which channels participate in this process.
Does protein translation play a significant role in regulating pain sensitivity?
Some say yes, but only in some pain pathways. Robert Sorge of McGill University, Montreal, Canada, used a knock-in mouse to disrupt normal protein translation and found that only thermal pain sensitivities were changed. The mouse line carries one allele of mutated eukaryotic initiation factor2 α(eIF2α), a subunit of eukaryotic initiation factor, which mediates the binding of Met-tRNA to the ribosome in a GTP-dependent manner. Activity of eIF2α can be regulated by environmental factors, such as stress, via the receptor GCN2, or by endoplasmic reticulum stress, which is signaled by the PERK receptor. Activation of GCN2 or PERK results in phosphorylation of eIF2, attenuation of overall translation, and increased ATF4 expression for stress responses. The phosphorylation of eIF2α can be reversed by the growth arrest- and DNA damage-inducible protein 34 (GADD34). The point mutation affects the phosphorylation site of eIF2α, and abolishes the decrease in translation. The knock-in mice showed increased thermal sensitivity and greater inflammation-induced thermal hypersensitivity in the hot-plate, tail withdrawal, and radiant heat paw withdrawal tests compared to the controls.Meanwhile, there was no significant change in mechanical sensitivity or hypersensitivity. Consistent with previous observations, ATF4+/- and GADD34 knockout mice also showed defects in thermal sensitivities. However, no thermal sensitivity differences were shown in GCN2-null mice. This study suggested that the thermal sensitivity is translation dependent, and could have different mechanisms compared to mechanical sensitivity. It will be important to find out if specific translational products or overall translation regulates thermal sensitivities, and to identify upstream regulators of eIF2α phosphorylation.
References:
Borzan J, Turnquist BP, Carteret FA, Hartke TV, Raja SN. Rodent ultrasonic signals are a sensitive measure of itch and persistent pain. Program No. 583.04. 2011 Neuroscience Meeting Planner. Washington, DC: Society for Neuroscience, 2011.
Ponce L, Klusch A, Ringkamp M, Schäfer I, Holloschi A, Schmelz M, Hafner M, Petersen M. An in vitro model to study phenotype of trophic factor specific sensory neuron terminals and interaction with keratinocytes. Program No. 380.01. 2011 Neuroscience Meeting Planner. Washington, DC: Society for Neuroscience, 2011.
McGlone FP, Ackerly R, Curran A, Hassan E, Kaiser M. Pleasant touch in autism: A role for c-tactile afferents? Program No. 385.19. 2011 Neuroscience Meeting Planner. Washington, DC: Society for Neuroscience, 2011.
Fischer MJ, Hoffmann T, Leffler A, McNaughton PA, Reeh P. The interphase of the formalin test. Program No. 380.02. 2011 Neuroscience Meeting Planner. Washington, DC: Society for Neuroscience, 2011.
Sorge RE, Khoutorsky A, Sonenberg N, Mogil J. Alterations in translational regulation play a key role in thermal pain sensitivity in mice. Program No. 380.06. 2011 Neuroscience Meeting Planner. Washington, DC: Society for Neuroscience, 2011.
