TRESK K+ Channel Activity Regulates Trigeminal Nociception and Headache.

Although TWIK-related spinal cord K+ (TRESK) channel is expressed in all primary afferent neurons in trigeminal ganglia (TG) and dorsal root ganglia (DRG), whether TRESK activity regulates trigeminal pain processing is still not established. Dominant-negative TRESK mutations are associated with migraine but not with other types of pain in humans, suggesting that genetic TRESK dysfunction preferentially affects the generation of trigeminal pain, especially headache. Using TRESK global knock-out mice as a model system, we found that loss of TRESK in all TG neurons selectively increased the intrinsic excitability of small-diameter nociceptors, especially those that do not bind to isolectin B4 (IB4-). Similarly, loss of TRESK resulted in hyper-excitation of the small IB4- dural afferent neurons but not those that bind to IB4 (IB4+). Compared with wild-type littermates, both male and female TRESK knock-out mice exhibited more robust trigeminal nociceptive behaviors, including headache-related behaviors, whereas their body and visceral pain responses were normal. Interestingly, neither the total persistent outward current nor the intrinsic excitability was altered in adult TRESK knock-out DRG neurons, which may explain why genetic TRESK dysfunction is not associated with body and/or visceral pain in humans. We reveal for the first time that, among all primary afferent neurons, TG nociceptors are the most vulnerable to the genetic loss of TRESK. Our findings indicate that endogenous TRESK activity regulates trigeminal nociception, likely through controlling the intrinsic excitability of TG nociceptors. Importantly, we provide evidence that genetic loss of TRESK significantly increases the likelihood of developing headache.

Characterization of a mouse model of headache.

Migraine and other primary headache disorders affect a large population and cause debilitating pain. Establishing animal models that display behavioral correlates of long-lasting and ongoing headache, the most common and disabling symptom of migraine, is vital for the elucidation of disease mechanisms and identification of drug targets. We have developed a mouse model of headache, using dural application of capsaicin along with a mixture of inflammatory mediators (IScap) to simulate the induction of a headache episode. This elicited intermittent head-directed wiping and scratching as well as the phosphorylation of c-Jun N-terminal kinase in trigeminal ganglion neurons. Interestingly, dural application of IScap preferentially induced FOS protein expression in the excitatory but not inhibitory cervical/medullary dorsal horn neurons. The duration of IScap-induced behavior and the number of FOS-positive neurons correlated positively in individual mice; both were reduced to the control level by the pretreatment of antimigraine drug sumatriptan. Dural application of CGRP(8-37), the calcitonin gene-related peptide (CGRP) receptor antagonist, also effectively blocked IScap-induced behavior, which suggests that the release of endogenous CGRP in the dura is necessary for IScap-induced nociception. These data suggest that dural IScap-induced nocifensive behavior in mice may be mechanistically related to the ongoing headache in humans. In addition, dural application of IScap increased resting time in female mice. Taken together, we present the first detailed study using dural application of IScap in mice. This headache model can be applied to genetically modified mice to facilitate research on the mechanisms and therapeutic targets for migraine headache.

Metabotropic glutamate receptor 5 (mGluR5) regulates bladder nociception.

BACKGROUND: Interstitial cystitis/painful bladder syndrome (IC/PBS), is a severely debilitating chronic condition that is frequently unresponsive to conventional pain medications. The etiology is unknown, however evidence suggests that nervous system sensitization contributes to enhanced pain in IC/PBS. In particular, central nervous system plasticity of glutamatergic signaling involving NMDA and metabotropic glutamate receptors (mGluRs) has been implicated in a variety of chronic pain conditions. Here, we test the hypothesis that mGluR5 mediates both non-inflammatory and inflammatory bladder pain or nociception in a mouse model by monitoring the visceromotor response (VMR) during graded bladder distention. RESULTS: Using a combination of genetic and pharmacologic approaches, we provide evidence indicating that mGluR5 is necessary for the full expression of VMR in response to bladder distention in the absence of inflammation. Furthermore, we observed that mice infected with a uropathogenic strain of Escherichia coli (UPEC) develop inflammatory hyperalgesia to bladder distention, and that the selective mGluR5 antagonist fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl) urea], reduces the VMR to bladder distention in UPEC-infected mice. CONCLUSIONS: Taken together, these data suggest that mGluR5 modulates both inflammatory and non-inflammatory bladder nociception, and highlight the therapeutic potential for mGluR5 antagonists in the alleviation of bladder pain.

Activation of spinal extracellular signal-regulated kinases (ERK) 1/2 is associated with the development of visceral hyperalgesia of the bladder.

Activation of extracellular signal-regulated kinases (ERK) 1/2 in dorsal horn neurons is important for the development of somatic hypersensitivity and spinal central sensitization after peripheral inflammation. However, data regarding the roles of spinal ERK1/2 in the development of visceral hyperalgesia are sparse. Here we studied the activation of ERK1/2 in the lumbosacral spinal cord after innocuous and noxious distention of the inflamed (cyclophosphamide-treated) and noninflamed urinary bladder in mice. We also correlated the spinal ERK1/2 activation to distention-evoked bladder nociception as quantified by the abdominal visceromotor response (VMR). Cyclophosphamide treatment (bladder inflammation) evoked increased bladder hyperalgesia and allodynia to bladder distention, as evident from an upward and leftward shift of the VMR stimulus-response curve compared with that of noninflamed mice. Development of bladder hyperalgesia was associated with robust enhancement of ERK1/2 activation in the dorsal horn and deeper laminae bilaterally in the L6-S1 spinal cord. Functional blockade of spinal ERK1/2 activity via intrathecal administration of the upstream MEK inhibitor U0126 attenuated distention-evoked bladder nociception and caused a significant downward shift of the VMR stimulus-response curve. In summary, we have provided functional and immunohistochemical evidence that activation of lumbosacral spinal ERK1/2 is associated with the development of primary visceral (bladder) hyperalgesia. Our results suggest that aberrant processing of visceral nociceptive information at the level of the lumbosacral spinal cord via activation of ERK1/2 signaling may contribute to chronic bladder pain in the context of inflammation.

Antinociceptive effects of MSVIII-19, a functional antagonist of the GluK1 kainate receptor.

The ionotropic glutamate receptor subunit, GluK1 (GluR5), is expressed in many regions of the nervous system related to sensory transmission. Recently, a selective ligand for the GluK1 receptor, MSVIII-19 (8,9-dideoxy-neodysiherbaine), was synthesized as a derivative of dysiherbaine, a toxin isolated from the marine sponge Lendenfeldia chondrodes. MSVIII-19 potently desensitizes GluK1 receptors without channel activation, rendering it useful as a functional antagonist. Given the high selectivity for GluK1 and the proposed role for this glutamate receptor in nociception, we sought to test the analgesic potential of MSVIII-19 in a series of models of inflammatory, neuropathic, and visceral pain in mice. MSVIII-19 delivered intrathecally dose-dependently reduced formalin-induced spontaneous behaviors and reduced thermal hypersensitivity 3 hours after formalin injection and 24 hours after complete Freund's adjuvant-induced inflammation, but had no effect on mechanical sensitivity in the same models. Intrathecal MSVIII-19 significantly reduced both thermal hyperalgesia and mechanical hypersensitivity in the chronic constriction injury model of neuropathic pain, but had no effect in the acetic acid model of visceral pain. Peripheral administration of MSVIII-19 had no analgesic efficacy in any of these models. Finally, intrathecal MSVIII-19 did not alter responses in Tail-flick tests or performance on the accelerating RotaRod. These data suggest that spinal administration of MSVIII-19 reverses hypersensitivity in several models of pain in mice, supporting the clinical potential of GluK1 antagonists for the management of pain.

Transcription factor Egr-1 is required for long-term fear memory and anxiety.

The zinc finger transcription factor Egr-1 is critical for coupling extracellular signals to changes in cellular gene expression. In the hippocampus and amygdala, two major central regions for memory formation and storage, Egr-1 is up-regulated by long-term potentiation (LTP) and learning paradigms. Using Egr-1 knockout mice, we showed that Egr-1 was selectively required for late auditory fear memory while short term, trace and contextual memory were not affected. Additionally, synaptic potentiation induced by theta burst stimulation in the amygdala and auditory cortex was significantly reduced or blocked in Egr-1 knockout mice. Our study suggests that the transcription factor Egr-1 plays a selective role in late auditory fear memory.

Pavlovian fear memory induced by activation in the anterior cingulate cortex.

Identifying higher brain central region(s) that are responsible for the unpleasantness of pain is the focus of many recent studies. Here we show that direct stimulation of the anterior cingulate cortex (ACC) in mice produced fear-like freezing responses and induced long-term fear memory, including contextual and auditory fear memory. Auditory fear memory required the activation of N-methyl-D-aspartate (NMDA) receptors in the amygdala. To test the hypothesis that neuronal activity in the ACC contributes to unpleasantness, we injected a GABAA receptor agonist, muscimol bilaterally into the ACC. Both contextual and auditory memories induced by foot shock were blocked. Furthermore, activation of metabotropic glutamate receptors in the ACC enhanced behavioral escape responses in a noxious hot-plate as well as spinal nociceptive tail-flick reflex. Our results provide strong evidence that the excitatory activity in the ACC contribute to pain-related fear memory as well as descending facilitatory modulation of spinal nociception.

Altered behavioral responses to noxious stimuli and fear in glutamate receptor 5 (GluR5)- or GluR6-deficient mice.

Different kainate receptor (KAR) subtypes contribute to the regulation of both excitatory and inhibitory transmission. However, no study has reported a role for KAR subtypes in behavioral responses to persistent pain and fear memory. Here we show that responses to capsaicin or inflammatory pain were significantly reduced in mice lacking glutamate receptor 5 (GluR5) but not GluR6 subunits. In classic fear-memory tests, mice lacking GluR6 but not GluR5 showed a significant reduction in fear memory when measured 3, 7, or 14 d after training. Additionally, synaptic potentiation was significantly reduced in the lateral amygdala of GluR6 but not GluR5 knock-out mice. Our findings provide evidence that distinct KAR subtypes contribute to chemical/inflammatory pain and fear memory. Selectively targeting different KAR subtypes may provide a useful strategy for treating persistent pain and fear-related mental disorders.

Calmodulin regulates synaptic plasticity in the anterior cingulate cortex and behavioral responses: a microelectroporation study in adult rodents.

We developed a microelectroporation method for the transfer of genes into neurons in the cerebral cortex of adult rodents, both rats and mice. We selectively expressed either green-fluorescent protein (GFP) or a Ca2+-binding deficient calmodulin (CaM) mutant in the anterior cingulate cortex (ACC). In mice that expressed GFP, positive neuronal cell bodies were found specifically at the injection site in the ACC. Mice that expressed CaM12, a mutant CaM with two impaired Ca2+ binding sites in the N-terminal lobe, exhibited significant changes in vocalization, locomotion, and sensory functions. Long-term potentiation and long-term depression, two major forms of central plasticity, were completely abolished by expression of CaM12. Mice that expressed CaM34, a mutant CaM with two impaired Ca2+ binding sites in the C-terminal lobe, did not show any significant behavioral or electrophysiological alterations. These findings provide strong evidence that CaM is critical for bidirectional synaptic plasticity. This new method will be useful for investigating gene function in specific brain regions of freely moving animals. Furthermore, this approach also may facilitate gene therapy in adult human brains.

Genetic elimination of behavioral sensitization in mice lacking calmodulin-stimulated adenylyl cyclases.

Adenylyl cyclase types 1 (AC1) and 8 (AC8), the two major calmodulin-stimulated adenylyl cyclases in the brain, couple NMDA receptor activation to cAMP signaling pathways. Cyclic AMP signaling pathways are important for many brain functions, such as learning and memory, drug addiction, and development. Here we show that wild-type, AC1, AC8, or AC1&8 double knockout (DKO) mice were indistinguishable in tests of acute pain, whereas behavioral responses to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant, were reduced or abolished in AC1&8 DKO mice. AC1 and AC8 are highly expressed in the anterior cingulate cortex (ACC), and contribute to inflammation-induced activation of CREB. Intra-ACC administration of forskolin rescued behavioral allodynia defective in the AC1&8 DKO mice. Our studies suggest that AC1 and AC8 in the ACC selectively contribute to behavioral allodynia.

Calcium calmodulin-dependent protein kinase IV is required for fear memory.

The ability to remember potential dangers in an environment is necessary to the survival of animals and humans. The cyclic AMP responsive element binding protein (CREB) is a key transcription factor in synaptic plasticity and memory consolidation. We have found that in CaMKIV(-/-) mice--which are deficient in a component of the calcium calmodulin-dependent protein kinase (CaMK) pathway, a major pathway of CREB activation--fear memory, but not persistent pain, was significantly reduced. CREB activation by fear conditioning and synaptic potentiation in the amygdala and cortical areas was reduced or blocked. We propose that cognitive memory related to a noxious shock can be disassociated from behavioral responses to tissue injury and inflammation.

Short-term endothelin receptor blockade with tezosentan has both immediate and long-term beneficial effects in rats with myocardial infarction.

OBJECTIVES: We investigated the effects of short-term tezosentan treatment on cardiac function, pulmonary edema and long-term evolution of heart failure (HF) in a rat model of myocardial infarction (MI). BACKGROUND: Endothelin (ET) may play a major role in the progression from MI to HF. Tezosentan is a new dual ET(A)/ET(B) receptor antagonist. METHODS: Rats were subjected to coronary artery ligation and were treated with either vehicle or tezosentan (10 mg/kg IV bolus) at 1 h and 24 h after MI. Cardiac hemodynamics and lung weight were measured at 48 h after MI. Survival was assessed over a five-month period. RESULTS: At 48 h after ligation, vehicle-treated rats developed HF, as evidenced by a marked increase in left ventricular end-diastolic pressure (LVEDP), reduction in dP/dt(max) and mean arterial pressure (MAP), and development of pulmonary edema. Tezosentan treatment attenuated the increase in LVEDP and in lung weight and slightly reduced MAP without affecting dP/dt(max). Infarct size was not modified by tezosentan. Despite the fact that treatment with tezosentan was stopped after 24 h, the initial tezosentan administration significantly reduced cardiac hypertrophy (22%) and decreased mortality by 51% at five months (50% survival vs. 19% survival in vehicle-treated rats, p < 0.001). CONCLUSIONS: Tezosentan administered during the first day after MI in rats, in addition to improving acutely hemodynamic conditions, markedly increases long-term survival. This increase is associated with a decrease of pulmonary edema and prevention of cardiac hypertrophy. Tezosentan could be a safe and useful therapeutic agent in the prevention and treatment of ischemic HF.