Anke Tappe-Theodor

A Molecular Basis of Analgesic Tolerance to Cannabinoids

Clinical usage of cannabinoids in chronic pain states is limited by their central side effects and the pharmacodynamic tolerance that sets in after repeated dosage. Analgesic tolerance to cannabinoids in vivo could be caused by agonist-induced downregulation and intracellular trafficking of cannabinoid receptors, but little is known about the molecular mechanisms involved. We show here that the type 1 cannabinoid receptor (CB1) interacts physically with G-protein-associated sorting protein 1 (GASP1), a protein that sorts receptors in lysosomal compartments destined for degradation. CB1–GASP1 interaction was observed to be required for agonist-induced downregulation of CB1 in spinal neurons ex vivo as well as in vivo. Importantly, uncoupling CB1 from GASP1 in mice in vivo abrogated tolerance toward cannabinoid-induced analgesia. These results suggest that GASP1 is a key regulator of the fate of CB1 after agonist exposure in the nervous system and critically determines analgesic tolerance to cannabinoids.

Presynaptically localized cyclic GMP-dependent protein kinase 1 is a key determinant of spinal synaptic potentiation and pain hypersensitivity.

Electrophysiological and behavioral experiments in mice reveal that a cGMP-dependent kinase amplifies neurotransmitter release from peripheral pain sensors, potentiates spinal synapses, and leads to exaggerated pain.

GABA Blocks Pathological but Not Acute TRPV1 Pain Signals

Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.

Studying ongoing and spontaneous pain in rodents--challenges and opportunities.

The measurement of spontaneous ongoing pain in rodents is a multiplex issue and a subject of extensive and longstanding debate. Considering the need to align available rodent models with clinically relevant forms of pain, it is of prime importance to thoroughly characterize behavioral outcomes in rodents using a portfolio of measurements that are not only stimulus‐dependent but also encompass voluntary behavior in unrestrained animals. Moreover, the temporal course and duration of behavioral tests should be taken into consideration when we plan our studies to measure explicit chronic pain, with a particular emphasis on performing longitudinal studies in rodents. While using rodents as model organisms, it is also worth considering their circadian rhythm and the influence of the test conditions on the behavioral results, which are affected by social paradigms, stress and anxiety. In humans, general wellbeing is closely related to pain perception, which also makes it necessary in rodents to consider modulators as well as readouts of overall wellbeing. Optimizing the above parameters in study design and the new developments that are forthcoming to test the affective motivational components of pain hold promise in solving inconsistencies across studies and improving their broad applicability in translational research. In this review, we critically discuss a variety of behavioral tests that have been developed and reported in recent years, attempt to weigh their benefits and potential limitations, and discuss key requirements and challenges that lie ahead in measuring ongoing pain in rodent models.

Pain in experimental autoimmune encephalitis: a comparative study between different mouse models

BackgroundPain can be one of the most severe symptoms associated with multiple sclerosis (MS) and develops with varying levels and time courses. MS-related pain is difficult to treat, since very little is known about the mechanisms underlying its development. Animal models of experimental autoimmune encephalomyelitis (EAE) mimic many aspects of MS and are well-suited to study underlying pathophysiological mechanisms. Yet, to date very little is known about the sensory abnormalities in different EAE models. We therefore aimed to thoroughly characterize pain behavior of the hindpaw in SJL and C57BL/6 mice immunized with PLP139-151 peptide or MOG35-55 peptide respectively. Moreover, we studied the activity of pain-related molecules and plasticity-related genes in the spinal cord and investigated functional changes in the peripheral nerves using electrophysiology.MethodsWe analyzed thermal and mechanical sensitivity of the hindpaw in both EAE models during the whole disease course. Qualitative and quantitative immunohistochemical analysis of pain-related molecules and plasticity-related genes was performed on spinal cord sections at different timepoints during the disease course. Moreover, we investigated functional changes in the peripheral nerves using electrophysiology.ResultsMice in both EAE models developed thermal hyperalgesia during the chronic phase of the disease. However, whereas SJL mice developed marked mechanical allodynia over the chronic phase of the disease, C57BL/6 mice developed only minor mechanical allodynia over the onset and peak phase of the disease. Interestingly, the magnitude of glial changes in the spinal cord was stronger in SJL mice than in C57BL/6 mice and their time course matched the temporal profile of mechanical hypersensitivity.ConclusionsDiverse EAE models bearing genetic, clinical and histopathological heterogeneity, show different profiles of sensory and pathological changes and thereby enable studying the mechanistic basis and the diversity of changes in pain perception that are associated with distinct types of MS.

Gαq/11 signaling tonically modulates nociceptor function and contributes to activity-dependent sensitization

TOC summary The functional role of Gq/11 G proteins in nociceptors not only spans pathological pain, but, surprisingly, also includes tonic modulation of nociception. ABSTRACT Peripheral injury or inflammation leads to a release of mediators capable of binding to a variety of ion channels and receptors. Among these are the 7‐transmembrane receptors (G protein‐coupled receptors) coupling to Gs, Gi/o, G12/13, or Gq/11 G proteins. Each of the G protein‐coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of individual signaling pathways in vivo has not yet been worked out. The Gq/G11 signaling branch is of particular interest because it leads to the activation of phospholipase C‐β, protein kinase C, the release of calcium from intracellular stores, and it modulates extracellular regulated kinases. To investigate the contribution of the entire Gq/11‐signaling pathway in nociceptors towards regulation of pain, we generated double‐deficient mice lacking Gq/11 selectively in nociceptors using a conditional gene‐targeting approach. We observed that nociceptor‐specific loss of Gq and G11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. Surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in Gq/11 mutant mice, suggesting a novel function for Gq/11 in tonic modulation of acute nociception. Patch‐clamp recordings revealed changes in voltage‐dependent tetrodotoxin‐resistant and tetrodotoxin‐sensitive sodium channels in nociceptors upon a loss of Gq/11, whereas potassium currents remained unchanged. Our results indicate that the functional role of the Gq/G11 branch of G‐protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain.

A novel biological role for the phospholipid lysophosphatidylinositol in nociceptive sensitization via activation of diverse G-protein signalling pathways in sensory nerves in vivo

Summary Lysophosphatidylinositol (LPI) is a novel regulator of peripheral sensory neuron function and pathological pain. The roles of GPCR and non‐GPCR components to the biological function of LPI are delineated. Abstract The rich diversity of lipids and the specific signalling pathways they recruit provides tremendous scope for modulation of biological functions. Lysophosphatidylinositol (LPI) is emerging as a key modulator of cell proliferation, migration, and function, and holds important pathophysiological implications due to its high levels in diseased tissues, such as in cancer. Here we report a novel role for LPI in sensitization of peripheral sensory neurons, which was evident as exaggerated sensitivity to painful and innocuous pressure. Histopathological analyses indicated lack of involvement of myelin pathology and immune cell recruitment by LPI. Using pharmacological and conditional genetic tools in mice, we delineated receptor‐mediated from non‐receptor‐mediated effects of LPI and we observed that GPR55, which functions as an LPI receptor when heterologously expressed in mammalian cells, only partially mediates LPI‐induced actions in the context of pain sensitization in vivo; we demonstrate that, in vivo, LPI functions by activating G&agr;13 as well as G&agr;q/11 arms of G‐protein signalling in sensory neurons. This study thus reports a novel pathophysiological function for LPI and elucidates underlying molecular mechanisms.

Early-onset treadmill training reduces mechanical allodynia and modulates calcitonin gene-related peptide fiber density in lamina III/IV in a mouse model of spinal cord contusion injury.

Abstract Below-level central neuropathic pain (CNP) affects a large proportion of spinal cord injured individuals. To better define the dynamic changes of the spinal cord neural network contributing to the development of CNP after spinal cord injury (SCI), we characterized the morphological and behavioral correlates of CNP in female C57BL/6 mice after a moderate T11 contusion SCI (50 kdyn) and the influence of moderate physical activity. Compared with sham-operated animals, injured mice developed mechanical allodynia 2 weeks post injury when tested with small-diameter von Frey hair filaments (0.16 g and 0.4 g filament), but presented hyporesponsiveness to noxious mechanical stimuli (1.4 g filament). The mechano-sensory alterations lasted up to 35 days post injury, the longest time point examined. The response latency to heat stimuli already decreased significantly 10 days post injury reaching a plateau 2 weeks later. In contrast, injured mice developed remarkable hyposensitivity to cold stimuli. Animals that underwent moderate treadmill training (2 × 15 minutes; 5 d/wk) showed a significant reduction in the response rate to light mechanical stimuli as early as 6 days after training. Calcitonin gene-related peptide (CGRP) labeling in lamina III-IV of the dorsal horn revealed significant increases in CGRP-labeling density in injured animals compared with sham control animals. Importantly, treadmill training reduced CGRP-labeling density by about 50% (P < 0.01), partially reducing the injury-induced increases. Analysis of IB4-labeled nonpeptidergic sensory fibers revealed no differences between experimental groups. Abnormalities in temperature sensation were not influenced by physical activity. Thus, treadmill training partially resolves signs of below-level CNP after SCI and modulates the density of CGRP-labeled fibers.

Voluntary and evoked behavioral correlates in neuropathic pain states under different social housing conditions

Background There is an urgent need to develop and incorporate novel behavioral tests in classically used preclinical pain models. Most rodent studies are based upon stimulus-evoked hindpaw measurements even though chronic pain is usually a day and night experience. Chronic pain is indeed a debilitating condition that influences the sociability and the ability for voluntary tasks, but the relevant behavioral readouts for these aspects are mostly under-represented in the literature. Moreover, we lack standardization in most behavioral paradigms to guarantee reproducibility and ensure adequate discussion between different studies. This concerns not only the combination, application, and duration of particular behavioral tasks but also the effects of different housing conditions implicating social isolation. Results Our aim was to thoroughly characterize the classically used spared nerve injury model for 12 weeks following surgery. We used a portfolio of classical stimulus-evoked response measurements, detailed gait analysis with two different measuring systems (Dynamic weight bearing (DWB) system and CatWalk), as well as observer-independent voluntary wheel running and home cage monitoring (Laboras system). Additionally, we analyzed the effects of social isolation in all behavioral tasks. We found that evoked hypersensitivity temporally matched changes in static gait parameters, whereas some dynamic gait parameters were changed in a time-dependent manner. Interestingly, voluntary wheel running behavior was not affected in spared nerve injury mice but by social isolation. Besides a reduced climbing activity, spared nerve injury mice did not showed tremendous alterations in the home cage activity. Conclusion This is the first longitudinal study providing detailed insights into various voluntary behavioral parameters related to pain and highlights the importance of social environment on spontaneous non-evoked behaviors in a mouse model of chronic neuropathy. Our results provide fundamental considerations for future experimental planning and discussion of pain-related behavioral changes.

Altered surface mGluR5 dynamics provoke synaptic NMDAR dysfunction and cognitive defects in Fmr1 knockout mice

Metabotropic glutamate receptor subtype 5 (mGluR5) is crucially implicated in the pathophysiology of Fragile X Syndrome (FXS); however, its dysfunction at the sub-cellular level, and related synaptic and cognitive phenotypes are unexplored. Here, we probed the consequences of mGluR5/Homer scaffold disruption for mGluR5 cell-surface mobility, synaptic N-methyl-D-aspartate receptor (NMDAR) function, and behavioral phenotypes in the second-generation Fmr1 knockout (KO) mouse. Using single-molecule tracking, we found that mGluR5 was significantly more mobile at synapses in hippocampal Fmr1 KO neurons, causing an increased synaptic surface co-clustering of mGluR5 and NMDAR. This correlated with a reduced amplitude of synaptic NMDAR currents, a lack of their mGluR5-activated long-term depression, and NMDAR/hippocampus dependent cognitive deficits. These synaptic and behavioral phenomena were reversed by knocking down Homer1a in Fmr1 KO mice. Our study provides a mechanistic link between changes of mGluR5 dynamics and pathological phenotypes of FXS, unveiling novel targets for mGluR5-based therapeutics.Dysfunction of mGluR5 has been implicated in Fragile X syndrome. Here, using a single-molecule tracking technique, the authors found an increased lateral mobility of mGluR5 at the synaptic site in Fmr1 KO hippocampal neurons, leading to abnormal NMDAR-mediated synaptic plasticity and cognitive deficits.