Ariel R. Ase

Transgenic Expression of a Dominant-Negative ASIC3 Subunit Leads to Increased Sensitivity to Mechanical and Inflammatory Stimuli

Molecular and behavioral evidence suggests that acid-sensing ion channels (ASICs) contribute to pain processing, but an understanding of their precise role remains elusive. Existing ASIC knock-out mouse experiments are complicated by the heteromultimerization of ASIC subunits. Therefore, we have generated transgenic mice that express a dominant-negative form of the ASIC3 subunit that inactivates all native neuronal ASIC-like currents by oligomerization. Using whole-cell patch-clamp recordings, we examined the response properties of acutely isolated dorsal root ganglion neurons to protons (pH 5.0). We found that whereas 33% of the proton-responsive neurons from wild-type mice exhibited an ASIC-like transient response, none of the neurons from the transgenic mice exhibited a transient inward current. Capsaicin-evoked responses mediated by the TRPV1 receptor were unaltered in transgenic mice. Adult male wild-type and transgenic mice were subjected to a battery of behavioral nociceptive assays, including tests of thermal, mechanical, chemical/inflammatory, and muscle pain. The two genotypes were equally sensitive to thermal pain and to thermal hypersensitivity after inflammation. Compared with wild types, however, transgenic mice were more sensitive to a number of modalities, including mechanical pain (von Frey test, tail-clip test), chemical/inflammatory pain (formalin test, 0.6% acetic acid writhing test), mechanical hypersensitivity after zymosan inflammation, and mechanical hypersensitivity after intramuscular injection of hypotonic saline. These data reinforce the hypothesis that ASICs are involved in both mechanical and inflammatory pain, although the increased sensitivity of transgenic mice renders it unlikely that they are direct transducers of nociceptive stimuli.

Altered Serotonin and Dopamine Metabolism in the CNS of Serotonin 5‐HT1A or 5‐HT1B Receptor Knockout Mice

Abstract: Measurements of serotonin (5‐HT), dopamine (DA), andnoradrenaline, and of 5‐HT and DA metabolites, were obtained by HPLC from 16brain regions and the spinal cord of 5‐HT1A or 5‐HT1Bknockout and wild‐type mice of the 129/Sv strain. In 5‐HT1Aknockouts, 5‐HT concentrations were unchanged throughout, but levels of 5‐HTmetabolites were higher than those of the wild type in dorsal/medial raphenuclei, olfactory bulb, substantia nigra, and locus coeruleus. This was takenas an indication of increased 5‐HT turnover, reflecting an augmented basalactivity of midbrain raphe neurons and consequent increase in theirsomatodendritic and axon terminal release of 5‐HT. It provided a likelyexplanation for the increased anxious‐like behavior observed in5‐HT1A knockout mice. Concomitant increases in DA content and/or DAturnover were interpreted as the result of a disinhibition of DA, whereasincreases in noradrenaline concentration in some territories of projection ofthe locus coeruleus could reflect a diminished activity of its neurons. In5‐HT1B knockouts, 5‐HT concentrations were lower than those of thewild type in nucleus accumbens, locus coeruleus, spinal cord, and probablyalso several other territories of 5‐HT innervation. A decrease in DA,associated with increased DA turnover, was measured in nucleus accumbens.These changes in 5‐HT and DA metabolism were consistent with the increasedaggressiveness and the supersensitivity to cocaine reported in5‐HT1B knockout mice. Thus, markedly different alterations in CNSmonoamine metabolism may contribute to the opposite behavioral phenotypes ofthese two knockouts.

Remote Optogenetic Activation and Sensitization of Pain Pathways in Freely Moving Mice

We report a novel model in which remote activation of peripheral nociceptive pathways in transgenic mice is achieved optogenetically, without any external noxious stimulus or injury. Taking advantage of a binary genetic approach, we selectively targeted Nav1.8+ sensory neurons for conditional expression of channelrhodopsin-2 (ChR2) channels. Acute blue light illumination of the skin produced robust nocifensive behaviors, evoked by the remote stimulation of both peptidergic and nonpeptidergic nociceptive fibers as indicated by c-Fos labeling in laminae I and II of the dorsal horn of the spinal cord. A non-nociceptive component also contributes to the observed behaviors, as shown by c-Fos expression in lamina III of the dorsal horn and the expression of ChR2–EYFP in a subpopulation of large-diameter Nav1.8+ dorsal root ganglion neurons. Selective activation of Nav1.8+ afferents in vivo induced central sensitization and conditioned place aversion, thus providing a novel paradigm to investigate plasticity in the pain circuitry. Long-term potentiation was similarly evoked by light activation of the same afferents in isolated spinal cord preparations. These findings demonstrate, for the first time, the optical control of nociception and central sensitization in behaving mammals and enables selective activation of the same class of afferents in both in vivo and ex vivo preparations. Our results provide a proof-of-concept demonstration that optical dissection of the contribution of specific classes of afferents to central sensitization is possible. The high spatiotemporal precision offered by this non-invasive model will facilitate drug development and target validation for pain therapeutics.

Regulation of DOPA Decarboxylase Activity in Brain of Living Rat

Abstract: To test the hypothesis that l‐DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [3H]DOPA. The unidirectional blood‐brain clearance of [3H]DOPA (KD1) was 0.049 ml g−1 min−1. The relative DDC activity (kD3) was 0.26 min−1 in striatum, 0.04 min−1 in hypothalamus, and 0.02 min−1 in hippocampus. In striatum, 3,4‐[3H]dihydroxyphenylacetic acid ([3H]DOPAC) was formed from [3H]DA with a rate constant of 0.013 min−1, [3H]homovanillic acid ([3H]HVA) was formed from [3H]DOPAC at a rate constant of 0.020 min−1, and [3H]HVA was eliminated from brain at a rate constant of 0.037 min−1. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, kD3 was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.

Differential regulation of microglial P2X4 and P2X7 ATP receptors following LPS-induced activation

Activation of microglia has been implicated in many neurological conditions including Alzheimers disease and neuropathic pain. Recent studies provide evidence that P2X ATP receptors on the surface of microglia play a crucial role in initiation of inflammatory cascades. We investigated changes in surface P2X receptors in BV-2 murine microglial cells following their activation by pro-inflammatory bacterial lipopolysaccharides (LPS). mRNA analysis using RT-PCR confirmed the presence of P2X4 and P2X7 as the main P2X subunits. Application of ATP at low (< or =100 microM) and high (> or =1 mM) concentrations, as well as BzATP, activated inward currents in BV-2 cells. Current responses of P2X4 and P2X7 subtypes could be distinguished based on their respective sensitivity to the positive modulator ivermectin and to the antagonist Brilliant Blue G. Treatment of BV-2 cells with LPS leads to a transient increase in ivermectin-sensitive P2X4 currents, while dominant P2X7 currents remain largely unaffected. This increase in P2X4 function was concomitant with higher receptor protein expression, itself related to an upregulation of P2X4 mRNA levels that peaked at 48 h post-LPS treatment. Our data demonstrate that although LPS activation has a minor impact on P2X7 receptors that remain the major ionotropic ATP receptors in microglia, it specifically enhances responses to low ATP concentrations mediated by P2X4 receptors, highlighting the significant contribution of both subtypes to neuroinflammatory mechanisms and pathologies.

Phosphoinositides Regulate P2X4 ATP-Gated Channels through Direct Interactions

P2X receptors are ATP-gated nonselective cation channels highly permeable to calcium that contribute to nociception and inflammatory responses. The P2X4 subtype, upregulated in activated microglia, is thought to play a critical role in the development of tactile allodynia following peripheral nerve injury. Posttranslational regulation of P2X4 function is crucial to the cellular mechanisms of neuropathic pain, however it remains poorly understood. Here, we show that the phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), products of phosphorylation by wortmannin-sensitive phosphatidylinositol 4-kinases and phosphatidylinositol 3-kinases, can modulate the function of native and recombinant P2X4 receptor channels. In BV-2 microglial cells, depleting the intracellular levels of PIP2 and PIP3 with wortmannin significantly decreased P2X4 current amplitude and P2X4-mediated calcium entry measured in patch clamp recordings and ratiometric ion imaging, respectively. Wortmannin-induced depletion of phosphoinositides in Xenopus oocytes decreased the current amplitude of P2X4 responses by converting ATP into a partial agonist. It also decreased their recovery from desensitization and affected their kinetics. Injection of phosphoinositides in wortmannin-treated oocytes reversed these effects and application of PIP2 on excised inside-out macropatches rescued P2X4 currents from rundown. Moreover, we report the direct interaction of phospholipids with the proximal C-terminal domain of P2X4 subunit (Cys360–Val375) using an in vitro binding assay. These results demonstrate novel regulatory roles of the major signaling phosphoinositides PIP2 and PIP3 on P2X4 function through direct channel–lipid interactions.

Regional changes in density of serotonin transporter in the brain of 5-HT1A and 5-HT1B knockout mice, and of serotonin innervation in the 5-HT1B knockout

5‐HT1A knockout (KO) mice display an anxious‐like phenotype, whereas 5‐HT1B KOs are over‐aggressive. To identify serotoninergic correlates of these altered behaviors, autoradiographic measurements of 5‐HT1A and 5‐HT1B serotonin (5‐HT) receptors and transporter (5‐HTT) were obtained using the radioligands [3H]8‐OH‐DPAT, [125I]cyanopindolol and [3H]citalopram, respectively. By comparison to wild‐type, density of 5‐HT1B receptors was unchanged throughout brain in 5‐HT1A KOs, and that of 5‐HT1A receptors in 5‐HT1B KOs. In contrast, decreases in density of 5‐HTT binding were measured in several brain regions of both genotypes. Moreover, 5‐HTT binding density was significantly increased in the amygdalo‐hippocampal nucleus and ventral hippocampus of the 5‐HT1B KOs. Measurements of 5‐HT axon length and number of axon varicosities by quantitative 5‐HT immunocytochemistry revealed proportional increases in the density of 5‐HT innervation in these two regions of 5‐HT1B KOs, whereas none of the decreases in 5‐HTT binding sites were associated with any such changes. Several conclusions could be drawn from these results: (i) 5‐HT1B receptors do not adapt in 5‐HT1A KOs, nor do 5‐HT1A receptors in 5‐HT1B KOs. (ii) 5‐HTT is down‐regulated in several brain regions of 5‐HT1A and 5‐HT1B KO mice. (iii) This down‐regulation could contribute to the anxious‐like phenotype of the 5‐HT1A KOs, by reducing 5‐HT clearance in several territories of 5‐HT innervation. (iv) The 5‐HT hyperinnervation in the amygdalo‐hippocampal nucleus and ventral hippocampus of 5‐HT1B KOs could play a role in their increased aggressiveness, and might also explain their better performance in some cognitive tests. (v) These increases in density of 5‐HT innervation provide the first evidence for a negative control of 5‐HT neuron growth mediated by 5‐HT1B receptors.

Effects of chronic neuroleptic treatments on dopamine D1 and D2 receptors: homogenate binding and autoradiographic studies

The antipsychotic effects of neuroleptics are believed to be mediated via dopamine D2 receptor blockade; however, the anatomical and pharmacological targets of these drugs remain somewhat controversial. The purpose of this study was to examine the effects of chronic clozapine (CLZ) and haloperidol (HAL) treatments on the densities of DA D1 and D2 receptors. Adult male Sprague-Dawley rats (300-350 g) were treated for 21 days with either HAL (1 mg/kg/day, i.p.), CLZ (20 mg/kg/day, i.p.) or saline. Three days after ending the treatments, the brains were removed and used for biochemical assays of tissue DA and metabolites as well as for receptor studies. DA D1 and D2 receptors were labelled with [3H]SCH23390 and [3H]raclopride, respectively, and measured in the neostriatum by binding studies, and in autoradiograms of forebrain sections by quantitative densitometry. The autoradiographic measurements revealed significant increases in the densities of D2 receptors in nucleus accumbens, in the medio-ventral, latero-dorsal and latero-ventral quadrants of the rostral neostriatum, in caudal neostriatum and in globus pallidus of both HAL-(28-44%) and CLZ-treated (15-85%) animals. The HAL-induced up-regulation of D2 receptors in rostral and caudal neostriatum was homogenous, but CLZ produced a more uneven increase, with the highest absolute densities measured in latero-dorsal neostriatum, as well as with changes in the medio-dorsal rostral neostriatum. For D1 receptors, only CLZ and not HAL, produced significant increases in five regions, namely nucleus accumbens (43%) latero-dorsal rostral neostriatum (16%), caudal neostriatum (30%), globus pallidus (67%) and substantia nigra (12%). The observation that CLZ, contrary to HAL, also has an effect on D1 receptor densities may explain the greater therapeutic and selective efficacy with fewer side-effects of this agent, in comparison to other neuroleptics.

Effects of antipsychotic drugs on dopamine and serotonin contents and metabolites, dopamine and serotonin transporters, and serotonin1A receptors

Summary. The effects of neuroleptics have been attributed to dopamine (DA) receptor blockade; however, other neurotransmitters, in particular serotonin (5-HT), have also been implicated. In this study, we examined the effects of clozapine and haloperidol on the distribution of DA and 5-HT transporters, on endogenous DA, 5-HT and their major metabolites, and on 5-HT1A receptors. Adult male Sprague-Dawley rats were treated with either haloperidol (1 mg/kg/day, i.p.), clozapine (20 mg/kg/day, i.p.) or saline for 21 days, and following 3 days of withdrawal, the brains were removed. Tissue levels of DA and 5-HT and their metabolites were measured by high-performance liquid chromatography in 16 brain regions, while quantitative autoradiography with [125I]RTI-121, [3H]citalopram and [3H]8-OH-DPAT was employed to label DA transporters, 5-HT transporters and 5-HT1A receptors, respectively. After haloperidol, densities of 5-HT transporters were increased in the dorsal insular cortex and in the ventral half of caudal neostriatum, while 5-HT1A receptors augmented in cingulate cortex but decreased in the entorhinal area. After clozapine, [3H]citalopram labelling was increased in ventral hippocampus, ventral caudal neostriatum and nucleus raphe dorsalis, but decreased in medio-dorsal and latero-dorsal neostriatum as well as in substantia nigra. Binding of [3H]8-OH-DPAT following clozapine was decreased in frontal, parietal, temporal and entorhinal cortices but increased in the CA3 division of Ammons horn. The changes in 5-HT transporters in nucleus raphe dorsalis and substantia nigra, as well as the 5-HT1A receptor down-regulations caused by clozapine but not by haloperidol, may explain effects obtained with clozapine and other atypical neuroleptics. There were no modifications in densities of DA transporters, nor of tissue DA levels, after the chronic neuroleptic treatments. The results are in line with previous suggestions that a certain degree of tolerance to neuroleptics develops, in spite of profound D1 and D2 receptor changes that persist during the entire chronic treatment with these psychotropic agents.

In Vivo Regulation of DOPA Decarboxylase by Dopamine Receptors in Rat Brain

To test the hypothesis that dopamine (DA) receptors influence cerebral DOPA-decarboxylase (DDC) activity in vivo, we used HPLC to measure the kinetics of the cerebral uptake and metabolism of [3H]DOPA in carbidopa-treated rats, and in rats also treated acutely with a DA receptor antagonist (flupenthixol, 2 mg/kg, intraperitoneally) or a DA receptor agonist (apomorphine, 200 μg/g, subcutaneously). The unidirectional blood-brain clearance of [3H]DOPA (K1DOPA, 0.030 mL g−1 min−1) increased by 50% after flupenthixol. The magnitudes of the relative DDC activity (k3DOPA) in striatum (0.20 min−1), olfactory tubercle (0.11 min−1), and hypothalamus (0.15 min−1) of carbidopa-treated rats were doubled with flupenthixol, but cortical DDC activity was unaffected (0.02 min−1). Apomorphine reduced the magnitude of k3DOPA in striatum by 20%. The rate constant for catabolism of [3H]DA formed in brain (k7′, monoamine oxidase [MAO] activity), which ranged from 0.025 min−1 in striatum to 0.08 min−1 in hypothalamus of carbidopa-treated rats, globally increased 2- to 4-fold after flupenthixol, and decreased to 0.003 min−1 in striatum after apomorphine. These in vivo results confirm the claim that acute blockade of DA receptors with flupenthixol stimulates the synthesis of [3H]DA from [3H]DOPA, and that this [3H]DA is subject to accelerated catabolism. Conversely, activation of the DA receptors with apomorphine inhibits DDC activity and DA catabolism.