Carmen De Felipe

Altered nociception, analgesia and aggression in mice lacking the receptor for substance P

The peptide neurotransmitter substance P modulates sensitivity to pain by activating the neurokinin-1 (NK-1) receptor, which is expressed by discrete populations of neurons throughout the central nervous system. Substance P is synthesized by small-diameter sensory ‘pain’ fibres, and release of the peptide into the dorsal horn of the spinal cord following intense peripheral stimulation promotes central hyperexcitability and increased sensitivity to pain. However, despite the availability of specific NK-1 antagonists, the function of substance P in the perception of pain remains unclear. Here we investigate the effect of disrupting the gene encoding the NK-1 receptor in mice. We found that the mutant mice were healthy and fertile, but the characteristic amplification (‘wind up’) and intensity coding of nociceptive reflexes was absent. Although substance P did not mediate the signalling of acute pain or hyperalgesia, it was essential for the full development of stress-induced analgesia and for an aggressive response to territorial challenge, demonstrating that the peptide plays an unexpected role in the adaptive response to stress.

Attenuation of thermal nociception and hyperalgesia by VR1 blockers

Vanilloid receptor subunit 1 (VR1) appears to play a critical role in the transduction of noxious chemical and thermal stimuli by sensory nerve endings in peripheral tissues. Thus, VR1 antagonists are useful compounds to unravel the contribution of this receptor to pain perception, as well as to induce analgesia. We have used a combinatorial approach to identify new, nonpeptidic channel blockers of VR1. Screening of a library of trimers of N-alkylglycines resulted in the identification of two molecules referred to as DD161515 {N-[2-(2-(N-methylpyrrolidinyl)ethyl]glycyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl)glycinamide} and DD191515 {[N-[3-(N,N-diethylamino)propyl]glycyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl)glycinamide} that selectively block VR1 channel activity with micromolar efficacy, rivaling that characteristic of vanilloid-related inhibitors. These compounds appear to be noncompetitive VR1 antagonists that recognize a receptor site distinct from that of capsaicin. Intraperitoneal administration of both trialkylglycines into mice significantly attenuated thermal nociception as measured in the hot plate test. It is noteworthy that these compounds eliminated pain and neurogenic inflammation evoked by intradermal injection of capsaicin into the animal hindpaw, as well as the thermal hyperalgesia induced by tissue irritation with nitrogen mustard. In contrast, responses to mechanical stimuli were not modified by either compound. Modulation of sensory nerve fibers excitability appears to underlie the peptoid analgesic activity. Collectively, these results indicate that blockade of VR1 activity attenuates chemical and thermal nociception and hyperalgesia, supporting the tenet that this ionotropic receptor contributes to chemical and thermal sensitivity and pain perception in vivo. These trialkylglycine-based, noncompetitive VR1 antagonists may likely be developed into analgesics to treat inflammatory pain.

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3‐directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence‐activated cell sorting from these transgenic mouse embryos. In culture, Pitx3–GFP is coexpressed in a proportion of ES‐derived DA neurons. Furthermore, ES cell‐derived Pitx3–GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA‐specific neurotoxin N‐4‐methyl‐1, 2, 3, 6‐tetrahydropyridine. We anticipate that the Pitx3–GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock‐in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain‐specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo‐ or adult‐derived neural stem cells.

Vanilloid receptor TRPV1, sensory C-fibers, and vascular autoregulation: a novel mechanism involved in myogenic constriction.

Myogenic constriction describes the innate ability of resistance arteries to constrict in response to elevations in intraluminal pressure and is a fundamental determinant of peripheral resistance and, hence, organ perfusion and systemic blood pressure. However, the receptor/cell-type that senses changes in pressure on the blood vessel wall and the pathway that couples this to constriction of vascular smooth muscle remain unclear. In this study, we show that elevation of intraluminal transmural pressure of mesenteric small arteries in vitro results in a myogenic response that is profoundly suppressed following ablation of sensory C-fiber activity (using in vitro capsaicin desensitization resulted in 72.8±10.3% inhibition, n=8; P<0.05). Activation of C-fiber nerve endings by pressure was attributable to stimulation of neuronal vanilloid receptor, TRPV1, because blockers of this channel, capsazepine (71.9±11.1% inhibition, n=9; P<0.001) and ruthenium red (46.1±11.7% inhibition, n=4; P<0.05), suppressed the myogenic constriction. In addition, this C-fiber dependency is likely related to neuropeptide substance P release and activity because blockade of tachykinin NK1 receptors (66.3±13.7% inhibition, n=6; P<0.001), and not NK2 receptors (n=4, NS), almost abolished the myogenic response. Previous studies support a role for 20-hydroxyeicosatetraenoic acid (20-HETE) in myogenic constriction responses; herein, we show that 20-HETE–induced constriction of mesenteric resistance arteries is blocked by capsazepine. Together, these results suggest that elevation of intraluminal pressure is associated with generation of 20-HETE that, in turn, activates TRPV1 on C-fiber nerve endings resulting in depolarization of nerves and consequent vasoactive neuropeptide release. These findings identify a novel mechanism contributing to Bayliss’ myogenic constriction and highlights an alternative pathway that may be targeted in the therapeutics of vascular disease, such as hypertension, where enhanced myogenic constriction plays a role in the pathogenesis.

Lack of self-administration and behavioural sensitisation to morphine, but not cocaine, in mice lacking NK1 receptors

Mice lacking the NK1 receptor, the preferred receptor for substance P, demonstrate normal analgesic responses to morphine on the hot plate assay, but have been predicted, on the basis of conditioned place preference studies, to be insensitive to the rewarding properties of opiates. In this study, self-administration and the development and maintenance of locomotor sensitisation of both morphine and cocaine were investigated in mice that lacked the NK1 gene (NK1 knockout mice, NK1(-/-)). Both wildtype and NK1(-/-) mice learned an operant lever-press response to obtain food. When intravenous infusions of morphine (0.2 mg/kg/infusion) were substituted for the food reward, the wildtype mice initially reduced rates of lever pressing, but then increased them on the rewarded lever to obtain approx. 10 infusions per 90 min session; in contrast, NK1(-/-) mice continued to operate both the rewarded, and non-rewarded levers at low rates. Additionally, NK1(-/-) mice failed, following repeated administration, to sensitise to the locomotor stimulant effects of morphine (15 mg/kg, i.p.). These deficits were specific to opiates, since NK1(-/-) mice responding for food or cocaine self-administration (0.65 mg/kg/infusion) did not differ from wildtypes, and they showed normal behavioural sensitisation to repeated cocaine administration (10 mg/kg, i.p.). These results demonstrate that NK1 receptors are critical for the reinforcing properties of morphine, and for adaptive responses elicited by repeated opiate administration. It is postulated that substance P and the NK1 receptor may be necessary for the development of opiate, but not cocaine addiction.

Increased neurogenesis and brain-derived neurotrophic factor in neurokinin-1 receptor gene knockout mice

It has previously been shown that chronic treatment with antidepressant drugs increases neurogenesis and levels of brain‐derived neurotrophic factor in the hippocampus. These changes have been correlated with changes in learning and long‐term potentiation and may contribute to the therapeutic efficacy of antidepressant drug treatment. Recently, antagonists at the neurokinin‐1 receptor, the preferred receptor for the neuropeptide substance P, have been shown to have antidepressant activity. Mice with disruption of the neurokinin‐1 receptor gene are remarkably similar both behaviourally and neurochemically to mice maintained chronically on antidepressant drugs. We demonstrate here that there is a significant elevation of neurogenesis but not cell survival in the hippocampus of neurokinin‐1 receptor knockout mice. Neurogenesis can be increased in wild‐type but not neurokinin‐1 receptor knockout mice by chronic treatment with antidepressant drugs which preferentially target noradrenergic and serotonergic pathways. Hippocampal levels of brain‐derived neurotrophic factor are also two‐fold higher in neurokinin‐1 receptor knockout mice, whereas cortical levels are similar. Finally, we examined hippocampus‐dependent learning and memory but found no clear enhancement in neurokinin‐1 receptor knockout mice. These data argue against a simple correlation between increased levels of neurogenesis or brain‐derived neurotrophic factor and mnemonic processes in the absence of increased cell survival. They support the hypothesis that increased neurogenesis, perhaps accompanied by higher levels of brain‐derived neurotrophic factor, may contribute to the efficacy of antidepressant drug therapy.

Bone cancer pain: The effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis

Abstract Patients with metastatic breast, lung or prostate cancer frequently have significant bone cancer pain. In the present report we address, in a single in vivo mouse model, the effects the bisphosphonate alendronate has on bone cancer pain, bone remodeling and tumor growth and necrosis. Following injection and confinement of green fluorescent protein‐transfected murine osteolytic tumor cells into the marrow space of the femur of male C3H/HeJ mice, alendronate was administered chronically from the time the tumor was established until the bone cancer pain became severe. Alendronate therapy reduced ongoing and movement‐evoked bone cancer pain, bone destruction and the destruction of sensory nerve fibers that innervate the bone. Whereas, alendronate treatment did not change viable tumor burden, both tumor growth and tumor necrosis increased. These data emphasize that it is essential to utilize a model where pain, skeletal remodeling and tumor growth can be simultaneously assessed, as each of these can significantly impact patient quality of life and survival.

The NK1 receptor is essential for the full expression of noxious inhibitory controls in the mouse.

Behavioral analysis of the NK1 receptor gene knock-out (NK1−/−) mouse indicated that substance P was closely involved in orchestrating the physiological and behavioral response of the animal to major environmental stressors. In particular, endogenous pain control mechanisms, such as stress-induced analgesia were substantially impaired in mutant mice, suggesting a reduction in descending inhibitory controls to the spinal cord from the brainstem. To directly test the integrity of descending controls in NK1−/− mice, we have analyzed c-Fos expression in laminae I-II of the lumbar and cervical cord and in the rostral ventromedial medulla in an experimental paradigm known to require recruitment of descending inhibitory controls. Anesthetized mice were stimulated with water at 50°C either on their forepaw, hindpaw, or on both the hindpaw plus forepaw concurrently. Wild-type mice, naı̈ve or treated with an NK1 antagonist (RP67580) or its inactive isomer (RP68651), were compared with NK1−/− mice. C-Fos expression at the lumbar laminae I-II level was significantly reduced, whereas it was significantly greater in the raphe magnus and pallidus nuclei in the double stimulation situation in wild-type compared with NK1−/− mice. Blocking the NK1 receptor pharmacologically reproduced, in an enantiomere-selective manner, the data from NK1−/− mice, with no evidence for recruitment of descending inhibition at the lumbar cord level after forepaw stimulation. The present study demonstrates that the NK1 receptor is essential for the full development of noxiously evoked descending inhibition.

Blockade of substance P (neurokinin 1) receptors enhances extracellular serotonin when combined with a selective serotonin reuptake inhibitor: an in vivo microdialysis study in mice

Substance P antagonists of the neurokinin‐1 receptor type (NK1) are gaining growing interest as new antidepressant therapies. It has been postulated that these drugs exert this putative therapeutic effect without direct interactions with serotonin (5‐HT) neurones. Our recent microdialysis experiment performed in NK1 receptor knockout mice suggested evidence of changes in 5‐HT neuronal function ( Froger et al. 2001 ). The aim of the present study was to evaluate the effects of coadministration of the selective 5‐HT reuptake inhibitor (SSRI) paroxetine with a NK1 receptor antagonist (GR205171 or L733060), given either intraperitoneally (i.p.) or locally into the dorsal raphe nucleus, on extracellular levels of 5‐HT ([5‐HT]ext) in the frontal cortex and the dorsal raphe nucleus using in vivo microdialysis in awake, freely moving mice. The systemic or intraraphe administration of a NK1 receptor antagonist did not change basal cortical [5‐HT]ext in mice. A single systemic dose of paroxetine (4 mg/kg; i.p.) resulted in a statistically significant increase in [5‐HT]ext with a larger extent in the dorsal raphe nucleus (+ 138% over basal AUC values), than in the frontal cortex (+ 52% over basal AUC values). Co‐administration of paroxetine (4 mg/kg; i.p.) with the NK1 receptor antagonists, GR205171 (30 mg/kg; i.p.) or L733060 (40 mg/kg; i.p.), potentiated the effects of paroxetine on cortical [5‐HT]ext in wild‐type mice, whereas GR205171 (30 mg/kg; i.p.) had no effect on paroxetine‐induced increase in cortical [5‐HT]ext in NK1 receptor knock‐out mice. When GR205171 (300 µmol/L) was perfused by ‘reverse microdialysis’ into the dorsal raphe nucleus, it potentiated the effects of paroxetine on cortical [5‐HT]ext, and inhibited paroxetine‐induced increase in [5‐HT]ext in the dorsal raphe nucleus. Finally, in mice whose 5‐HT transporters were first blocked by a local perfusion of 1 µmol/L of citalopram into the frontal cortex, a single dose of paroxetine (4 mg/kg i.p.) decreased cortical 5‐HT release, and GR205171 (30 mg/kg i.p.) reversed this effect. The present findings suggest that NK1 receptor antagonists, when combined with a SSRI, augment 5‐HT release by modulating substance P/5–HT interactions in the dorsal raphe nucleus.

Quantification and immunocytochemical characteristics of trigeminal ganglion neurons projecting to the cornea: effect of corneal wounding.

The number and immunocytochemical characteristics of trigeminal ganglion neurons providing sensory innervation to the cornea were studied in the mouse. Corneal neurons were retrogradely labelled with fluorogold placed on the cornea after removal of the epithelium with n‐heptanol. Corneal neurons were counted, sized and characterized immunocytochemically with antisera against substance P (SP), calcitonin gene‐related peptide (CGRP), calbindin, calretinin, and with a monoclonal antibody (RT97) against neurofilament proteins. A total of 258 corneal neurons were counted, most of them located in the ophthalmic division of the trigeminal ganglion. They represent only a small fraction (1.3%) of the population of trigeminal ganglion neurons. More than 70% of corneal neurons were classified as ‘small dark’ according to their cell body area and the absence of immunoreactivity to RT97. A low percentage of corneal neurons, usually large in size, contained calcium binding proteins. Fifty‐eight percent of the corneal neurons were immunoreactive to CGRP, and 20% to SP Corneal wounding with NaOH, which affects stromal nerve trunk, did not modify the total number of corneal neurons or their neuropeptide content. However, this increased the total number of calbindin‐positive and decreased the RT97‐positive neurons. Thus, unlike in other nociceptive neurons, peripheral axotomy did not modify the SP/CGRP content of corneal neurons.