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1.
PLoS One ; 6(3): e17670, 2011 Mar 14.
Article in English | MEDLINE | ID: mdl-21423802

ABSTRACT

Neuropathic pain resulting from nerve lesions or dysfunction represents one of the most challenging neurological diseases to treat. A better understanding of the molecular mechanisms responsible for causing these maladaptive responses can help develop novel therapeutic strategies and biomarkers for neuropathic pain. We performed a miRNA expression profiling study of dorsal root ganglion (DRG) tissue from rats four weeks post spinal nerve ligation (SNL), a model of neuropathic pain. TaqMan low density arrays identified 63 miRNAs whose level of expression was significantly altered following SNL surgery. Of these, 59 were downregulated and the ipsilateral L4 DRG, not the injured L5 DRG, showed the most significant downregulation suggesting that miRNA changes in the uninjured afferents may underlie the development and maintenance of neuropathic pain. TargetScan was used to predict mRNA targets for these miRNAs and it was found that the transcripts with multiple predicted target sites belong to neurologically important pathways. By employing different bioinformatic approaches we identified neurite remodeling as a significantly regulated biological pathway, and some of these predictions were confirmed by siRNA knockdown for genes that regulate neurite growth in differentiated Neuro2A cells. In vitro validation for predicted target sites in the 3'-UTR of voltage-gated sodium channel Scn11a, alpha 2/delta1 subunit of voltage-dependent Ca-channel, and purinergic receptor P2rx ligand-gated ion channel 4 using luciferase reporter assays showed that identified miRNAs modulated gene expression significantly. Our results suggest the potential for miRNAs to play a direct role in neuropathic pain.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation , MicroRNAs/genetics , Neuralgia/genetics , Spinal Nerves/metabolism , Spinal Nerves/pathology , Animals , Data Mining , Disease Models, Animal , Enzyme Assays , Ganglia, Spinal/metabolism , Ganglia, Spinal/pathology , Gene Knockdown Techniques , Genes, Reporter , Ligation , Luciferases/metabolism , Male , Mice , MicroRNAs/metabolism , MicroRNAs/standards , Neuralgia/pathology , Quality Control , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Reference Standards , Reproducibility of Results
2.
Mol Pharmacol ; 78(6): 996-1003, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20855465

ABSTRACT

Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.


Subject(s)
Cannabinoid Receptor Modulators/physiology , Endocannabinoids , Monoacylglycerol Lipases/metabolism , Receptor, Cannabinoid, CB1/physiology , Animals , Enzyme Activation/genetics , Enzyme Activation/physiology , Hydrolysis , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Monoacylglycerol Lipases/deficiency , Monoacylglycerol Lipases/physiology , Pain Measurement/methods
3.
J Neurosci ; 30(6): 2017-24, 2010 Feb 10.
Article in English | MEDLINE | ID: mdl-20147530

ABSTRACT

Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-alpha (DAGLalpha) and -beta (DAGLbeta) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show approximately 80% reductions in 2-AG levels in the brain and spinal cord in DAGLalpha(-/-) mice and a 50% reduction in the brain in DAGLbeta(-/-) mice. In contrast, DAGLbeta plays a more important role than DAGLalpha in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGLbeta(-/-) mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABA-mediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGLalpha(-/-) animals and relatively unaffected in DAGLbeta(-/-) animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGLalpha(-/-) and/or DAGLbeta(-/-) mice. These findings provide the first evidence that DAGLalpha is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.


Subject(s)
Brain/metabolism , Cannabinoid Receptor Modulators/physiology , Endocannabinoids , Lipoprotein Lipase/genetics , Animals , Arachidonic Acids/metabolism , Brain/cytology , Glycerides/metabolism , Hippocampus/cytology , Hippocampus/metabolism , Liver/metabolism , Mice , Mice, Knockout , Neurogenesis , Neuronal Plasticity , Signal Transduction , Spinal Cord/metabolism , Synapses/physiology
4.
Brain Res ; 1295: 89-98, 2009 Oct 27.
Article in English | MEDLINE | ID: mdl-19651113

ABSTRACT

Validation of gait analysis has the potential to bridge the gap between data from animal pain models and clinical observations. The goal of these studies was to compare alterations in gait due to inflammation or nerve injury to traditional pain measurements in animals. Pharmacological experiments determined whether gait alterations were related to enhanced nociception, edema, or motor nerve dysfunction. Gait was analyzed using an automated system (DigiGait) after injection of an inflammatory agent (carrageenan; CARR or FCA; Freund's complete adjuvant) or nerve injury (axotomy; AXO, partial sciatic nerve ligation; PSNL, spinal nerve ligation; SNL or chronic constriction injury; CCI). All models caused significant alterations in gait and thermal (inflammatory) or mechanical (nerve injury) hyperalgesia. Both indomethacin and morphine were able to block or reverse thermal hyperalgesia and normalize gait in the CARR model. Indomethacin partially blocked and did not reverse paw edema, suggesting that gait alterations must be primarily driven by enhanced nociception. In nerve injury models, AXO, PSNL, CCI, and SNL caused changes to the largest number of gait indices with the rank order being AXO>PSNL=CCI >> SNL. Gabapentin and duloxetine reversed mechanical hyperalgesia but did not normalize gait in any nerve injury model. Collectively, these data suggest that pain is the primary driver of abnormal gait in models of inflammatory but not nerve injury-related pain and suggests that, in the latter, disruption in gait is due to perturbation to the motor system. Gait may therefore constitute an alternative and potentially clinically relevant measure of pain due to inflammation.


Subject(s)
Gait/drug effects , Inflammation/physiopathology , Pain/physiopathology , Sciatic Nerve/injuries , Amines/pharmacology , Analgesics, Opioid/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Axotomy , Carrageenan , Cyclohexanecarboxylic Acids/pharmacology , Duloxetine Hydrochloride , Edema/chemically induced , Edema/physiopathology , Freund's Adjuvant , Gabapentin , Gait/physiology , Hot Temperature , Hyperalgesia/chemically induced , Hyperalgesia/physiopathology , Indomethacin/pharmacology , Inflammation/chemically induced , Male , Morphine/pharmacology , Neuralgia/physiopathology , Pain/chemically induced , Pain Measurement/drug effects , Pain Threshold/drug effects , Physical Stimulation/adverse effects , Rats , Rats, Sprague-Dawley , Sciatic Nerve/physiopathology , Thiophenes/pharmacology , gamma-Aminobutyric Acid/pharmacology
5.
J Pharmacol Exp Ther ; 322(3): 1294-304, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17586724

ABSTRACT

Here, we have investigated the in vitro pharmacology of a muscarinic agonist, (3R,4R)-3-(3-hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983), and we demonstrated its activity in several models of pain. WAY-132983 had a similar affinity for the five muscarinic receptors (9.4-29.0 nM); however, in calcium mobilization studies it demonstrated moderate selectivity for M(1) (IC(50) = 6.6 nM; E(max) = 65% of 10 muM carbachol-stimulation) over the M(3) (IC(50) = 23 nM; E(max) = 41%) and M(5) receptors (IC(50) = 300 nM; E(max) = 18%). WAY-132983 also activated the M(4) receptor, fully inhibiting forskolin-induced increase in cAMP levels (IC(50) = 10.5 nM); at the M(2) receptor its potency was reduced by 5-fold (IC(50) = 49.8 nM). In vivo, WAY-132983 demonstrated good systemic bioavailability and high brain penetration (>20-fold over plasma levels). In addition, WAY-1329823 produced potent and efficacious antihyperalgesic and antiallodynic effects in rodent models of chemical irritant, chronic inflammatory, neuropathic, and incisional pain. It is noteworthy that efficacy in these models was observed at doses that did not produce analgesia or ataxia. Furthermore, a series of antagonist studies demonstrated that the in vivo activity of WAY-132983 is mediated through activation of muscarinic receptors primarily through the M(4) receptor. The data presented herein suggest that muscarinic agonists, such as WAY-132983, may have a broad therapeutic efficacy for the treatment of pain.


Subject(s)
Bridged-Ring Compounds/pharmacokinetics , Muscarinic Agonists/pharmacology , Pain/prevention & control , Pyrazines/pharmacokinetics , Animals , Biological Availability , Bridged-Ring Compounds/pharmacology , Chronic Disease , Disease Models, Animal , Inflammation , Inhibitory Concentration 50 , Pyrazines/pharmacology , Rats , Receptors, Muscarinic
6.
J Neurophysiol ; 97(5): 3713-21, 2007 May.
Article in English | MEDLINE | ID: mdl-17392420

ABSTRACT

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons, playing an important role in pain processing. We found that the known analgesic loperamide inhibited I(h) channels in rat DRG neurons. Loperamide blocked I(h) in a concentration-dependent manner, with an IC(50) = 4.9 +/- 0.6 and 11.0 +/- 0.5 microM for large- and small-diameter neurons, respectively. Loperamide-induced I(h) inhibition was unrelated to the activation of opioid receptors and was reversible, voltage-dependent, use-independent, and was associated with a negative shift of V(1/2) for I(h) steady-state activation. Loperamide block of I(h) was voltage-dependent, gradually decreasing at more hyperpolarized membrane voltages from 89% at -60 mV to 4% at -120 mV in the presence of 3.7 microM loperamide. The voltage sensitivity of block can be explained by a loperamide-induced shift in the steady-state activation of I(h). Inclusion of 10 microM loperamide into the recording pipette did not affect I(h) voltage for half-maximal activation, activation kinetics, and the peak current amplitude, whereas concurrent application of equimolar external loperamide produced a rapid, reversible I(h) inhibition. The observed loperamide-induced I(h) inhibition was not caused by the activation of peripheral opioid receptors because the broad-spectrum opioid receptor antagonist naloxone did not reverse I(h) inhibition. Therefore we suggest that loperamide inhibits I(h) by direct binding to the extracellular region of the channel. Because I(h) channels are involved in pain processing, loperamide-induced inhibition of I(h) channels could provide an additional molecular mechanism for its analgesic action.


Subject(s)
Analgesics/pharmacology , Ganglia, Spinal/cytology , Loperamide/pharmacology , Neural Inhibition/drug effects , Neurons/drug effects , Potassium Channels/metabolism , Animals , Cells, Cultured , Cyclic Nucleotide-Gated Cation Channels , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Drug Interactions , Electric Stimulation , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Patch-Clamp Techniques/methods , Rats , Rats, Wistar
7.
J Comp Neurol ; 484(2): 144-55, 2005 Apr 04.
Article in English | MEDLINE | ID: mdl-15736227

ABSTRACT

Potassium channels are key determinants of neuronal excitability. We recently identified KChIPs as a family of calcium binding proteins that coassociate and colocalize with Kv4 family potassium channels in mammalian brain (An et al. [2000] Nature 403:553). Here, we used light microscopic immunohistochemistry and multilabel immunofluorescence labeling, together with transmission electron microscopic immunohistochemistry, to examine the subcellular distribution of KChIPs and Kv4 channels in adult rat cerebellum. Light microscopic immunohistochemistry was performed on 40-microm free-floating sections using a diaminobenzidine labeling procedure. Multilabel immunofluorescence staining was performed on free-floating sections and on 1-microm ultrathin cryosections. Electron microscopic immunohistochemistry was performed using an immunoperoxidase pre-embedding labeling procedure. By light microscopy, immunoperoxidase labeling showed that Kv4.2, Kv4.3, and KChIPs 1, 3, and 4 (but not KChIP2) were expressed at high levels in cerebellar granule cells (GCs). Kv4.2 and KChIP1 were highly expressed in GCs in rostral cerebellum, whereas Kv4.3 was more highly expressed in GCs in caudal cerebellum. Immunofluorescence labeling revealed that KChIP1 and Kv4.2 are concentrated in somata of cerebellar granule cells and in synaptic glomeruli that surround synaptophysin-positive mossy fiber axon terminals. Electron microscopic analysis revealed that KChIP1 and Kv4.2 immunoreactivity is concentrated along the plasma membrane of cerebellar granule cell somata and dendrites. In synaptic glomeruli, KChIP1 and Kv4.2 immunoreactivity is concentrated along the granule cell dendritic membrane, but is not concentrated at postsynaptic densities. Taken together, these data suggest that A-type potassium channels containing Kv4.2 and KChIP1, and perhaps also KChIP3 and 4, play a critical role in regulating postsynaptic excitability at the cerebellar mossy-fiber/granule cell synapse.


Subject(s)
Calcium-Binding Proteins/ultrastructure , Cerebellum/chemistry , Cerebellum/metabolism , Cerebellum/ultrastructure , Potassium Channels, Voltage-Gated/ultrastructure , Animals , Calcium-Binding Proteins/analysis , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/metabolism , Kv Channel-Interacting Proteins , Microscopy, Polarization/methods , Potassium Channels, Voltage-Gated/biosynthesis , Potassium Channels, Voltage-Gated/metabolism , Rats , Shal Potassium Channels
8.
J Neurosci ; 24(36): 7903-15, 2004 Sep 08.
Article in English | MEDLINE | ID: mdl-15356203

ABSTRACT

Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs (Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family alpha subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation (An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family alpha subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 alpha subunits in brain membranes, indicating that KChIPs 1-4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.


Subject(s)
Brain Chemistry , Calcium-Binding Proteins/physiology , Potassium Channels, Voltage-Gated/physiology , Potassium Channels/physiology , Repressor Proteins/physiology , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , COS Cells , Chlorocebus aethiops , Corpus Striatum/cytology , Corpus Striatum/metabolism , Dendrites/chemistry , Dendrites/ultrastructure , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Ibotenic Acid/toxicity , Immunoprecipitation , Interneurons/chemistry , Interneurons/physiology , Kv Channel-Interacting Proteins , Mice , Mice, Inbred BALB C , Microscopy, Fluorescence , Neocortex/cytology , Neocortex/metabolism , Neuronal Plasticity , Neurons/chemistry , Neurons/drug effects , Neurons/physiology , Protein Interaction Mapping , Protein Subunits , Rats , Recombinant Fusion Proteins/physiology , Shal Potassium Channels , Synaptic Transmission/physiology , Transfection
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