Vasopressin casts light on the suprachiasmatic nucleus.

KEY POINTS: A subpopulation of retinal ganglion cells expresses the neuropeptide vasopressin. These retinal ganglion cells project predominately to our biological clock, the suprachiasmatic nucleus (SCN). Light-induced vasopressin release enhances the responses of SCN neurons to light. It also enhances expression of genes involved in photo-entrainment of biological rhythms. ABSTRACT: In all animals, the transition between night and day engages a host of physiological and behavioural rhythms. These rhythms depend not on the rods and cones of the retina, but on retinal ganglion cells (RGCs) that detect the ambient light level in the environment. These project to the suprachiasmatic nucleus (SCN) of the hypothalamus to entrain circadian rhythms that are generated within the SCN. The neuropeptide vasopressin has an important role in this entrainment. Many SCN neurons express vasopressin, and it has been assumed that the role of vasopressin in the SCN reflects the activity of these cells. Here we show that vasopressin is also expressed in many retinal cells that project to the SCN. Light-evoked vasopressin release contributes to the responses of SCN neurons to light, and enhances expression of the immediate early gene c-fos in the SCN, which is involved in photic entrainment of circadian rhythms.

The SH3 domain of postsynaptic density 95 mediates inflammatory pain through phosphatidylinositol-3-kinase recruitment.

Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino-acid substitution in the polyproline-binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol-3-kinase-C2alpha to the SH3-binding site of PSD95. In wild-type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.

TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction.

TRPA1, a member of the transient receptor potential (TRP) family of ion channels, is expressed by dorsal root ganglion neurons and by cells of the inner ear, where it has proposed roles in sensing sound, painful cold, and irritating chemicals. To test the in vivo roles of TRPA1, we generated a mouse in which the essential exons required for proper function of the Trpa1 gene were deleted. Knockout mice display behavioral deficits in response to mustard oil, to cold ( approximately 0 degrees C), and to punctate mechanical stimuli. These mice have a normal startle reflex to loud noise, a normal sense of balance, a normal auditory brainstem response, and normal transduction currents in vestibular hair cells. TRPA1 is apparently not essential for hair-cell transduction but contributes to the transduction of mechanical, cold, and chemical stimuli in nociceptor sensory neurons.

Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.

Microarray expression profiles reveal substantial changes in gene expression in the ipsilateral dorsal horn of the spinal cord in response to three peripheral nerve injury models of neuropathic pain. However, only 54 of the 612 regulated genes are commonly expressed across all the neuropathic pain models. Many of the commonly regulated transcripts are immune related and include the complement components C1q, C3, and C4, which we find are expressed only by microglia. C1q and C4 are, moreover, the most strongly regulated of all 612 regulated genes. In addition, we find that the terminal complement component C5 and the C5a receptor (C5aR) are upregulated in spinal microglia after peripheral nerve injury. Mice null for C5 had reduced neuropathic pain sensitivity, excluding C3a as a pain effector. C6-deficient rats, which cannot form the membrane attack complex, have a normal neuropathic pain phenotype. However, C5a applied intrathecally produces a dose-dependent, slow-onset cold pain in naive animals. Furthermore, a C5aR peptide antagonist reduces cold allodynia in neuropathic pain models. We conclude that induction of the complement cascade in spinal cord microglia after peripheral nerve injury contributes to neuropathic pain through the release and action of the C5a anaphylatoxin peptide.

The voltage-gated sodium channel Na(v)1.9 is an effector of peripheral inflammatory pain hypersensitivity.

We used a mouse with deletion of exons 4, 5, and 6 of the SCN11A (sodium channel, voltage-gated, type XI, alpha) gene that encodes the voltage-gated sodium channel Na(v)1.9 to assess its contribution to pain. Na(v)1.9 is present in nociceptor sensory neurons that express TRPV1, bradykinin B2, and purinergic P2X3 receptors. In Na(v)1.9-/- mice, the non-inactivating persistent tetrodotoxin-resistant sodium TTXr-Per current is absent, whereas TTXr-Slow is unchanged. TTXs currents are unaffected by the mutation of Na(v)1.9. Pain hypersensitivity elicited by intraplantar administration of prostaglandin E2, bradykinin, interleukin-1beta, capsaicin, and P2X3 and P2Y receptor agonists, but not NGF, is either reduced or absent in Na(v)1.9-/- mice, whereas basal thermal and mechanical pain sensitivity is unchanged. Thermal, but not mechanical, hypersensitivity produced by peripheral inflammation (intraplanatar complete Freund's adjuvant) is substantially diminished in the null allele mutant mice, whereas hypersensitivity in two neuropathic pain models is unchanged in the Na(v)1.9-/- mice. Na(v)1.9 is, we conclude, an effector of the hypersensitivity produced by multiple inflammatory mediators on nociceptor peripheral terminals and therefore plays a key role in mediating peripheral sensitization.

Delayed sympathetic dependence in the spared nerve injury (SNI) model of neuropathic pain.

BACKGROUND: Clinical and experimental studies of neuropathic pain support the hypothesis that a functional coupling between postganglionic sympathetic efferent and sensory afferent fibers contributes to the pain. We investigated whether neuropathic pain-related behavior in the spared nerve injury (SNI) rat model is dependent on the sympathetic nervous system. RESULTS: Permanent chemical sympathectomy was achieved by daily injection of guanethidine (50 mg/kg s.c.) from age P8 to P21. SNI was performed at adulthood followed by 11 weeks of mechanical and thermal hypersensitivity testing. A significant but limited effect of the sympathectomy on SNI-induced pain sensitivity was observed. The effect was delayed and restricted to cold allodynia-like behavior: SNI-related cold scores were lower in the sympathectomized group compared to the control group at 8 and 11 weeks after the nerve injury but not before. Mechanical hypersensitivity tests (pinprick and von Frey hair threshold tests) showed no difference between groups during the study period. Concomitantly, pericellular tyrosine-hydroxylase immunoreactive basket structures were observed around dorsal root ganglia (DRG) neurons 8 weeks after SNI, but were absent at earlier time points after SNI and in sham operated controls. CONCLUSION: These results suggest that the early establishment of neuropathic pain-related behavior after distal nerve injury such as in the SNI model is mechanistically independent of the sympathetic system, whereas the system contributes to the maintenance, albeit after a delay of many weeks, of response to cold-related stimuli.

RNAi blocks DYT1 mutant torsinA inclusions in neurons.

Early onset generalized dystonia is a dominantly inherited movement disorder caused by neuronal dysfunction without an apparent loss of neurons. The same single mutation (GAG deletion) causes most cases and results in loss of a glutamic acid (E) in the carboxy terminal region of torsinA (Delta302/303). To model the neuronal involvement, adult rat primary sensory dorsal root ganglion neurons in culture were infected with lentivirus vectors expressing human wild-type or mutant torsinA. Expression of the mutant protein resulted in formation of torsinA-positive perinuclear inclusions. When the cells were co-infected with lentivirus vectors expressing the mutant torsinA message and a shRNA selectively targeting this message, inclusion formation was blocked. Vector-delivered siRNAs have the potential to decrease the adverse effects of this mutant protein in neurons without affecting wild-type protein.

Detection of cold pain, cold allodynia and cold hyperalgesia in freely behaving rats.

BACKGROUND: Pain is elicited by cold, and a major feature of many neuropathic pain states is that normally innocuous cool stimuli begin to produce pain (cold allodynia). To expand our understanding of cold induced pain states we have studied cold pain behaviors over a range of temperatures in several animal models of chronic pain. RESULTS: We demonstrate that a Peltier-cooled cold plate with +/- 1 degrees C sensitivity enables quantitative measurement of a detection withdrawal response to cold stimuli in unrestrained rats. In naïve rats the threshold for eliciting cold pain behavior is 5 degrees C. The withdrawal threshold for cold allodynia is 15 degrees C in both the spared nerve injury and spinal nerve ligation models of neuropathic pain. Cold hyperalgesia is present in the spared nerve injury model animals, manifesting as a reduced latency of withdrawal response threshold at temperatures that elicit cold pain in naïve rats. We also show that following the peripheral inflammation produced by intraplantar injection of complete Freund's adjuvant, a hypersensitivity to cold occurs. CONCLUSION: The peltier-cooled provides an effective means of assaying cold sensitivity in unrestrained rats. Behavioral testing of cold allodynia, hyperalgesia and pain will greatly facilitate the study of the neurobiological mechanisms involved in cold/cool sensations and enable measurement of the efficacy of pharmacological treatments to reduce these symptoms.

Peripheral axonal injury results in reduced mu opioid receptor pre- and post-synaptic action in the spinal cord.

In both the spared nerve injury (SNI) and spinal nerve ligation (SNL) rat peripheral neuropathic pain models the presynaptic inhibitory effect of the mu opioid receptor (MOR) agonist (DAMGO) on primary afferent-evoked excitatory postsynaptic currents (EPSCs) and miniature EPSCs in superficial dorsal horn neurons is substantially reduced, but only in those spinal cord segments innervated by injured primary afferents. The two nerve injury models also reduce the postsynaptic potassium channel opening action of DAMGO on lamina II spinal cord neurons, but again only in segments receiving injured afferent input. The inhibitory action of DAMGO on ERK (extracellular signal-regulated kinase) activation in dorsal horn neurons is also reduced in affected segments following nerve injury. MOR expression decreases substantially in injured dorsal root ganglion neurons (DRG), while intact neighboring DRGs are unaffected. Decreased activation of MOR on injured primary afferent central terminals and the second order neurons they innervate may minimize any reduction by opioids of the spontaneous pain mediated by ectopic input from axotomized small diameter afferents. Retention of MOR expression and activity in nearby non-injured afferents will enable, however, an opioid-mediated reduction of stimulus-evoked and spontaneous pain carried by intact nociceptor afferents and we find that intrathecal DAMGO (1000 ng) reduces mechanical hypersensitivity in rats with SNL. Axotomy-induced changes in MOR may contribute to opioid- insensitive components of neuropathic pain while the absence of these changes in intact afferents may contribute to the opioid sensitive components.

Morphine, the NMDA receptor antagonist MK801 and the tachykinin NK1 receptor antagonist RP67580 attenuate the development of inflammation-induced progressive tactile hypersensitivity.

Normally-innocuous low-intensity tactile stimuli applied to inflamed tissue induce a progressive decrease in the mechanical flexion withdrawal threshold, the phenomenon of progressive tactile hypersensitivity (PTH). The effects of the mu opioid receptor agonist morphine, the non-competitive NMDA receptor antagonist MK801 and the tachykinin NK1 receptor antagonist RP67580 on the development and maintenance of PTH has now been investigated behaviourally in rats inflamed 48 h earlier by intraplantar complete Freund's adjuvant injection. A standard protocol of eight light tactile stimuli applied to the dorsum of the inflamed paw every 4 s at 5 min intervals resulted, over 60 min, in a 70% fall in mechanical threshold from the pre-conditioning baseline value. Morphine administered before the tactile stimuli at 0.05 mg/kg i.p. had no effect on either baseline thresholds or PTH. At 0.5 mg/kg, morphine prevented the establishment of PTH without changing baseline thresholds. At 5 mg/kg morphine produced analgesia, increasing thresholds above the baseline. MK801 pre-treatment at 0.01 and 0.001 mg/kg i.p. significantly attenuated the development of progressive tactile hyperalgesia without an effect on basal thresholds. RP67580 pre-treatment at 0.1 mg/kg i.p. had no effect, but at both I and 10 mg/kg, attenuated progressive tactile hypersensitivity without changing baseline values. To test the effect of the drugs on established PTH, they were administered 90 min after the commencement of intermittent tactile stimulation to the inflamed hindpaw, when thresholds had reached a plateau. Morphine (0.5 mg/kg) and MK801 (0.01 mg/kg) produced only a small reduction in sensitivity and RP67580 (1 mg/kg) had no effect. These results suggest that the induction of inflammatory progressive tactile hypersensitivity is sensitive to morphine, and to a lesser extent NMDA and NKI receptor antagonists, but these compounds at a dose that do not alter baseline values, do not normalise established tactile hypersensitivity.

A novel model of combined neuropathic and inflammatory pain displaying long-lasting allodynia and spontaneous pain-like behaviour.

Many clinical cases of chronic pain exhibit both neuropathic and inflammatory components. In contrast, most animal models of chronic pain focus on one type of injury alone. Here we present a novel combined model of both neuropathic and inflammatory pain and characterise its distinctive properties. This combined model of chronic constriction injury (CCI) and intraplantar Complete Freund's Adjuvant (CFA) injection results in enhanced mechanical allodynia, thermal hyperalgesia, a static weight bearing deficit, and notably pronounced spontaneous foot lifting (SFL) behaviour (which under our conditions was not seen in either individual model and may reflect ongoing/spontaneous pain). Dorsal root ganglion (DRG) expression of Activating Transcription Factor-3 (ATF-3), a marker of axonal injury, was no greater in the combined model than CCI alone. Initial pharmacological characterisation of the new model showed that the SFL was reversed by gabapentin or diclofenac, typical analgesics for neuropathic or inflammatory pain respectively, but not by mexiletine, a Na(+) channel blocker effective in both neuropathic and inflammatory pain models. Static weight bearing deficit was moderately reduced by gabapentin, whereas only diclofenac reversed mechanical allodynia. This novel animal model of chronic pain may prove a useful test-bed for further analysing the pharmacological susceptibility of complicated clinical pain states.

GDNF selectively promotes regeneration of injury-primed sensory neurons in the lesioned spinal cord.

Axonal regeneration within the CNS fails due to the growth inhibitory environment and the limited intrinsic growth capacity of injured neurons. Injury to DRG peripheral axons induces expression of growth associated genes including members of the glial-derived neurotrophic factor (GDNF) signaling pathway and "preconditions" the injured cells into an active growth state, enhancing growth of their centrally projecting axons. Here, we show that preconditioning DRG neurons prior to culturing increased neurite outgrowth, which was further enhanced by GDNF in a bell-shaped growth response curve. In vivo, GDNF delivered directly to DRG cell bodies facilitated the preconditioning effect, further enhancing axonal regeneration beyond spinal cord lesions. Consistent with the in vitro results, the in vivo effect was seen only at low GDNF concentrations. We conclude that peripheral nerve injury upregulates GDNF signaling pathway components and that exogenous GDNF treatment selectively promotes axonal growth of injury-primed sensory neurons in a concentration-dependent fashion.

The transcription factor ATF-3 promotes neurite outgrowth.

Dorsal root ganglion (DRG) neurons regenerate after a peripheral nerve injury but not after injury to their axons in the spinal cord. A key question is which transcription factors drive the changes in gene expression that increase the intrinsic growth state of peripherally injured sensory neurons? A prime candidate is activating transcription factor-3 (ATF-3), a transcription factor that we find is induced in all DRG neurons after peripheral, but not central axonal injury. Moreover, we show in adult DRG neurons that a preconditioning peripheral, but not central axonal injury, increases their growth, correlating closely with the pattern of ATF-3 induction. Using viral vectors, we delivered ATF-3 to cultured adult DRG neurons and find that ATF-3 enhances neurite outgrowth. Furthermore, ATF-3 promotes long sparsely branched neurites. ATF-3 overexpression did not increase c-Jun expression. ATF-3 may contribute, therefore, to neurite outgrowth by orchestrating the gene expression responses in injured neurons.