Identification of peripheral oxytocin-expressing cells using systemically applied cell-type specific adeno-associated viral vector.

Oxytocin is primarily synthesised in the brain and is widely known for its role in lactation and parturition after being released into the blood from the posterior pituitary gland. Nevertheless, peripheral tissues have also been reported to express oxytocin. Using systemic injection of a recombinant adeno-associated virus vector, we investigated the expression of the green fluorescent protein Venus under the control of the oxytocin promoter in the gastrointestinal tract, pancreas and testes of adult rats. Here, we confirm that the vector infects oxytocin neurones of the enteric nervous system in ganglia of the myenteric and submucosal plexuses. Venus was detected in 25%-60% of the ganglia in the myenteric and submucosal plexuses identified by co-staining with the neuronal marker PGP9.5. Oxytocin expression was also detected in the islets of Langerhans in the pancreas and the Leydig cells of the testes. Our data illustrate that peripheral administration of the viral vector represents a powerful method for selectively labelling oxytocin-producing cells outside the brain.

Effects of optogenetic stimulation of vasopressinergic retinal afferents on suprachiasmatic neurones.

Physiological circadian rhythms are orchestrated by the hypothalamic suprachiasmatic nucleus (SCN). The activity of SCN cells is synchronised by environmental signals, including light information from retinal ganglion cells (RGCs). We recently described a population of vasopressin-expressing RGCs (VP-RGC) that send axonal projections to the SCN. To determine how these VP-RGCs influence the activity of cells in the SCN, we used optogenetic tools to specifically activate their axon terminals within the SCN. Rats were intravitreally injected with a recombinant adeno-associated virus to express the channelrhodopsin-2 and the red fluorescent protein mCherry under the vasopressin promoter (VP-ChR2mCherry). In vitro recordings in acute brain slices showed that approximately 30% of ventrolateral SCN cells responded to optogenetic stimulation with an increase in firing rate that progressively increased during the first 200 seconds of stimulation and which persisted after the end of stimulation. Finally, application of a vasopressin V1A receptor antagonist dampened the response to optogenetic stimulation. Our data suggest that optogenetic stimulation of VP-RGC axons within the SCN influences the activity of SCN cells in a vasopressin-dependent manner.

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 TRPM8 channel forms a complex with the 5-HT(1B) receptor and phospholipase D that amplifies its reversal of pain hypersensitivity.

Effective relief from chronic hypersensitive pain states remains an unmet need. Here we report the discovery that the TRPM8 ion channel, co-operating with the 5-HT(1B) receptor (5-HT(1B)R) in a subset of sensory afferents, exerts an influence at the spinal cord level to suppress central hypersensitivity in pain processing throughout the central nervous system. Using cell line models, ex vivo rat neural tissue and in vivo pain models, we assessed functional Ca(2+) fluorometric responses, protein:protein interactions, immuno-localisation and reflex pain behaviours, with pharmacological and molecular interventions. We report 5-HT(1B)R expression in many TRPM8-containing afferents and direct interaction of these proteins in a novel multi-protein signalling complex, which includes phospholipase D1 (PLD1). We provide evidence that the 5-HT(1B)R activates PLD1 to subsequently activate PIP 5-kinase and generate PIP2, an allosteric enhancer of TRPM8, achieving a several-fold increase in potency of TRPM8 activation. The enhanced activation responses of synaptoneurosomes prepared from spinal cord and cortical regions of animals with a chronic inflammatory pain state are inhibited by TRPM8 activators that were applied in vivo topically to the skin, an effect potentiated by co-administered 5-HT(1B)R agonists and attenuated by 5-HT(1B)R antagonists, while 5-HT(1B)R agents alone had no detectable effect. Corresponding results are seen when assessing reflex behaviours in inflammatory and neuropathic pain models. Control experiments with alternative receptor/TRP channel combinations reveal no such synergy. Identification of this novel receptor/effector/channel complex and its impact on nociceptive processing give new insights into possible strategies for enhanced analgesia in chronic pain.

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.

Multiple chronic pain states are associated with a common amino acid-changing allele in KCNS1.

Not all patients with nerve injury develop neuropathic pain. The extent of nerve damage and age at the time of injury are two of the few risk factors identified to date. In addition, preclinical studies show that neuropathic pain variance is heritable. To define such factors further, we performed a large-scale gene profiling experiment which plotted global expression changes in the rat dorsal root ganglion in three peripheral neuropathic pain models. This resulted in the discovery that the potassium channel alpha subunit KCNS1, involved in neuronal excitability, is constitutively expressed in sensory neurons and markedly downregulated following nerve injury. KCNS1 was then characterized by an unbiased network analysis as a putative pain gene, a result confirmed by single nucleotide polymorphism association studies in humans. A common amino acid changing allele, the 'valine risk allele', was significantly associated with higher pain scores in five of six independent patient cohorts assayed (total of 1359 subjects). Risk allele prevalence is high, with 18-22% of the population homozygous, and an additional 50% heterozygous. At lower levels of nerve damage (lumbar back pain with disc herniation) association with greater pain outcome in homozygote patients is P = 0.003, increasing to P = 0.0001 for higher levels of nerve injury (limb amputation). The combined P-value for pain association in all six cohorts tested is 1.14 E-08. The risk profile of this marker is additive: two copies confer the most, one intermediate and none the least risk. Relative degrees of enhanced risk vary between cohorts, but for patients with lumbar back pain, they range between 2- and 3-fold. Although work still remains to define the potential role of this protein in the pathogenic process, here we present the KCNS1 allele rs734784 as one of the first prognostic indicators of chronic pain risk. Screening for this allele could help define those individuals prone to a transition to persistent pain, and thus requiring therapeutic strategies or lifestyle changes that minimize nerve injury.

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.

Neuropathic changes in equine laminitis pain.

Laminitis is a common debilitating disease in horses that involves painful disruption of the lamellar dermo-epidermal junction within the hoof. This condition is often refractory to conventional anti-inflammatory analgesia and results in unremitting pain, which in severe cases requires euthanasia. The mechanisms underlying pain in laminitis were investigated using quantification of behavioural pain indicators in conjunction with histological studies of peripheral nerves innervating the hoof. Laminitic horses displayed consistently altered or abnormal behaviours such as increased forelimb lifting and an increased proportion of time spent at the back of the box compared to normal horses. Electron micrographic analysis of the digital nerve of laminitic horses showed peripheral nerve morphology to be abnormal, as well as having reduced numbers of unmyelinated (43.2%) and myelinated fibers (34.6%) compared to normal horses. Sensory nerve cell bodies innervating the hoof, in cervical, C8 dorsal root ganglia (DRG), showed an upregulated expression of the neuronal injury marker, activating transcription factor-3 (ATF3) in both large NF-200-immunopositive neurons and small neurons that were either peripherin- or IB4-positive. A significantly increased expression of neuropeptide Y (NPY) was also observed in myelinated afferent neurons. These changes are similar to those reported in other neuropathic pain states and were not observed in the C4 DRG of laminitic horses, which is not associated with innervation of the forelimb. This study provides novel evidence for a neuropathic component to the chronic pain state associated with equine laminitis, indicating that anti-neuropathic analgesic treatment may well have a role in the management of this condition.

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.

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.

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.

GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence.

We report that GTP cyclohydrolase (GCH1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, is a key modulator of peripheral neuropathic and inflammatory pain. BH4 is an essential cofactor for catecholamine, serotonin and nitric oxide production. After axonal injury, concentrations of BH4 rose in primary sensory neurons, owing to upregulation of GCH1. After peripheral inflammation, BH4 also increased in dorsal root ganglia (DRGs), owing to enhanced GCH1 enzyme activity. Inhibiting this de novo BH4 synthesis in rats attenuated neuropathic and inflammatory pain and prevented nerve injury-evoked excess nitric oxide production in the DRG, whereas administering BH4 intrathecally exacerbated pain. In humans, a haplotype of the GCH1 gene (population frequency 15.4%) was significantly associated with less pain following diskectomy for persistent radicular low back pain. Healthy individuals homozygous for this haplotype exhibited reduced experimental pain sensitivity, and forskolin-stimulated immortalized leukocytes from haplotype carriers upregulated GCH1 less than did controls. BH4 is therefore an intrinsic regulator of pain sensitivity and chronicity, and the GTP cyclohydrolase haplotype is a marker for these traits.

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.

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.

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.

Mutant SPTLC1 dominantly inhibits serine palmitoyltransferase activity in vivo and confers an age-dependent neuropathy.

Mutations in enzymes involved in sphingolipid metabolism and trafficking cause a variety of neurological disorders, but details of the molecular pathophysiology remain obscure. SPTLC1 encodes one subunit of serine palmitoyltransferase (SPT), the rate-limiting enzyme in sphingolipid synthesis. Mutations in SPTLC1 cause hereditary sensory and autonomic neuropathy (type I) (HSAN1), an adult onset, autosomal dominant neuropathy. HSAN1 patients have reduced SPT activity. Expression of mutant SPTLC1 in yeast and mammalian cell cultures dominantly inhibits SPT activity. We created transgenic mouse lines that ubiquitously overexpress either wild-type (SPTLC1(WT)) or mutant SPTLC1 (SPTLC1(C133W)). We report here that SPTLC1(C133W) mice develop age-dependent weight loss and mild sensory and motor impairments. Aged SPTLC1(C133W) mice lose large myelinated axons in the ventral root of the spinal cord and demonstrate myelin thinning. There is also a loss of large myelinated axons in the dorsal roots, although the unmyelinated fibers are preserved. In the dorsal root ganglia, IB4 staining is diminished, whereas expression of the injury-induced transcription factor ATF3 is increased. These mice represent a novel mouse model of peripheral neuropathy and confirm the link between mutant SPT and neuronal dysfunction.

Bradykinin produces pain hypersensitivity by potentiating spinal cord glutamatergic synaptic transmission.

Bradykinin, an inflammatory mediator, sensitizes nociceptor peripheral terminals reducing pain threshold. We now show that the B2 kinin receptor is expressed in rat dorsal horn neurons and that bradykinin, a B2-specific agonist, augments AMPA- and NMDA-induced, and primary afferent-evoked EPSCs, and increases the frequency and amplitude of miniature EPSCs in superficial dorsal horn neurons in vitro. Administration of bradykinin to the spinal cord in vivo produces, moreover, an NMDA-dependent hyperalgesia. We also demonstrate that nociceptive inputs result in the production of bradykinin in the spinal cord and that an intrathecal B2-selective antagonist suppresses behavioral manifestations of central sensitization, an activity-dependent increase in glutamatergic synaptic efficacy. Primary afferent-evoked central sensitization is, in addition, reduced in B2 receptor knock-out mice. We conclude that bradykinin is released in the spinal cord in response to nociceptor inputs and acts as a synaptic neuromodulator, potentiating glutamatergic synaptic transmission to produce pain hypersensitivity.

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.