High Relevance Combined with High Resolution: Advantages of in vivo Two-Photon Microscopy for Drug Discovery

Despite breath-taking technological progress, modern pharmaceutical industry suffers from high attrition rates. In fact, only one in twenty lead compounds identified in non-clinical development passes through clinical trials and to market. The unacceptably low predictive power of many preclinical models stems, in part, from the lack of relevance and/or poor resolution of imaging techniques employed in drug development. Thus, in vivo imaging typically does not yield sufficient spatial resolution, whereas in vitro microscopy methods lack pathophysiological relevance. At present, only the in vivo two-photon microscopy (IV2PM) combines the crucial advantages of both imaging and microscopy, as it introduces the nanometer-scale spatial resolution of in vitro microscopy into the highly relevant context of in vivo imaging. In the present concise review, we discuss the most exciting applications of this game-changing technique in academic research of the past decade, and provide an outlook on the future role of IV2PM in drug discovery and development.Non peer reviewe

Emerging Role of (Endo)Cannabinoids in Migraine

In this mini-review, we summarize recent discoveries and present new hypotheses on the role of cannabinoids in controlling trigeminal nociceptive system underlying migraine pain. Individual sections of this review cover key aspects of this topic, such as: (i) the current knowledge on the endocannabinoid system (ECS) with emphasis on expression of its components in migraine related structures; (ii) distinguishing peripheral from central site of action of cannabinoids, (iii) proposed mechanisms of migraine pain and control of nociceptive traffic by cannabinoids at the level of meninges and in brainstem, (iv) therapeutic targeting in migraine of monoacylglycerol lipase and fatty acid amide hydrolase, enzymes which control the level of endocannabinoids; (v) dual (possibly opposing) actions of cannabinoids via anti-nociceptive CB1 and CB2 and pro-nociceptive TRPV1 receptors. We explore the cannabinoid-mediated mechanisms in the frame of the Clinical Endocannabinoid Deficiency (CECD) hypothesis, which implies reduced tone of endocannabinoids in migraine patients. We further discuss the control of cortical excitability by cannabinoids via inhibition of cortical spreading depression (CSD) underlying the migraine aura. Finally, we present our view on perspectives of Cannabis-derived (extracted or synthetized marijuana components) or novel endocannabinoid therapeutics in migraine treatment.Peer reviewe

Dynamic visualization of membrane-inserted fraction of pHluorin-tagged channels using repetitive acidification technique

<p>Abstract</p> <p>Background</p> <p>Changes in neuronal excitability, synaptic efficacy and generally in cell signaling often result from insertion of key molecules into plasma membrane (PM). Many of the techniques used for monitoring PM insertion lack either spatial or temporal resolution.</p> <p>Results</p> <p>We improved the imaging method based on time-lapse total internal reflection fluorescence (TIRF) microscopy and pHluorin tagging by supplementing it with a repetitive extracellular acidification protocol. We illustrate the applicability of this method by showing that brief activation of NMDA receptors ("chemical LTP") in cultured hippocampal neurons induced a persistent PM insertion of glutamate receptors containing the pHluorin-tagged GluR-A(flip) subunits.</p> <p>Conclusion</p> <p>The repetitive acidification technique provides a more accurate way of monitoring the PM-inserted fraction of fluorescently tagged molecules and offers a good temporal and spatial resolution.</p

K\u3csub\u3eV\u3c/sub\u3e4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxin binding provide a new tool for studies of channel expression and localization

We report the first successful insertion of an engineered, high-affinity alpha-bungarotoxin (Bgtx) binding site into a voltage-gated ion channel, KV4.2, using a short, intra-protein embedded sequence (GGWRYYESSLEPYPDGG), derived from a previously described mimotope peptide, HAP. A major benefit to this approach is the ability to live-image the distribution and fate of functional channels on the plasma membrane surface. The Bgtx binding sequence was introduced into the putative extracellular loop between the S1 and S2 transmembrane domains of KV4.2. Following co-expression with KChIP3 in tsA201 cells, S1-S2 HAP-tagged channels express at levels comparable to wild-type KV4.2, and their activation and inactivation kinetics are minimally altered under most conditions. Binding assays, as well as live staining of surface-expressed KV4.2 channels with fluorescent-Bgtx, readily demonstrate specific binding of Bgtx to HAP-tagged KV4.2 expressed on the surface of tsA201 cells. Similar live-imaging results were obtained with HAP-tagged KV4.2 transfected into hippocampal neurons in primary culture suggesting applicability for future in vivo studies. Furthermore, the activation kinetics of S1-S2-tagged KV4.2 channels are minimally affected by the binding of Bgtx, suggesting a limited role if any for the S1-S2 loop in voltage sensing or gating associated conformational changes. Successful functional insertion of the HAP sequence into the S1-S2 linker of KV4.2 suggests that other related channels may similarly be amenable to this tagging strategy

Unusually Strong Temperature Dependence of P2X3 Receptor Traffic to the Plasma Membrane

ATP-gated P2X3 receptors are expressed by nociceptive neurons and participate in transduction of pain. Responsiveness of P2X3 receptors is strongly reduced at low temperatures, suggesting a role for these receptors in analgesic effects of cooling. Since sustained responsiveness depends on receptor trafficking to the plasma membrane, we employed total internal reflection fluorescence (TIRF) microscopy to highlight perimembrane pool of DsRed-tagged P2X3 receptors and studied the effects of temperature on perimembrane turnover of P2X3-DsRed. Patch-clamp recordings confirmed membrane expression of functional, rapidly desensitizing P2X3-DsRed receptors. By combining TIRF microscopy with the technique of fluorescence recovery after photobleaching (FRAP), we measured the rate of perimembrane turnover of P2X3-DsRed receptors expressed in hippocampal neurons. At room temperature, the P2X3-DsRed perimembrane turnover as measured by TIRF–FRAP had a time constant of ∼2 min. At 29°C, receptor turnover was strongly accelerated (0.6 min), yielding an extremely high temperature dependence coefficient Q10 ∼4.5. In comparison, AMPA receptor turnover measured with TIRF–FRAP was only moderately sensitive to temperature (Q10 ∼1.5). The traffic inhibitor Brefeldin A selectively decelerated P2X3-DsRed receptor turnover at 29°C, but had no effect at 21°C (Q10 ∼1.0). This indicates that receptor traffic to plasma membrane is the key temperature-sensitive component of P2X3 turnover. The selective inhibitor of the RhoA kinase Y27632 significantly decreased the temperature dependence of P2X3-DsRed receptor turnover (Q10 ∼2.0). In summary, the RhoA kinase-dependent membrane trafficking of P2X3 receptors to plasma membrane has an exceptionally high sensitivity to temperature. These findings suggest an important role of P2X3 receptor turnover in hypothermia-associated analgesia

Cognitive Neuroscience of Pain

Non peer reviewe

In vivo Two-Photon Imaging of Anesthesia-Specific Alterations in Microglial Surveillance and Photodamage-Directed Motility in Mouse Cortex

Two-photon imaging of fluorescently labeled microglia in vivo provides a direct approach to measure motility of microglial processes as a readout of microglial function that is crucial in the context of neurodegenerative diseases, as well as to understand the neuroinflammatory response to implanted substrates and brain-computer interfaces. In this longitudinal study, we quantified surveilling and photodamage-directed microglial processes motility in both acute and chronic cranial window preparations and compared the motility under isoflurane and ketamine anesthesia to an awake condition in the same animal. The isoflurane anesthesia increased the length of surveilling microglial processes in both acute and chronic preparations, while ketamine increased the number of microglial branches in acute preparation only. In chronic (but not acute) preparation, the extension of microglial processes toward the laser-ablated microglial cell was faster under isoflurane (but not ketamine) anesthesia than in awake mice, indicating distinct effects of anesthetics and of preparation type. These data reveal potentiating effects of isoflurane on microglial response to damage, and provide a framework for comparison and optimal selection of experimental conditions for quantitative analysis of microglial function using two-photon microscopy in vivo

In vivo Two-Photon Imaging of Anesthesia-Specific Alterations in Microglial Surveillance and Photodamage-Directed Motility in Mouse Cortex

Two-photon imaging of fluorescently labeled microglia in vivo provides a direct approach to measure motility of microglial processes as a readout of microglial function that is crucial in the context of neurodegenerative diseases, as well as to understand the neuroinflammatory response to implanted substrates and brain-computer interfaces. In this longitudinal study, we quantified surveilling and photodamage-directed microglial processes motility in both acute and chronic cranial window preparations and compared the motility under isoflurane and ketamine anesthesia to an awake condition in the same animal. The isoflurane anesthesia increased the length of surveilling microglial processes in both acute and chronic preparations, while ketamine increased the number of microglial branches in acute preparation only. In chronic (but not acute) preparation, the extension of microglial processes toward the laser-ablated microglial cell was faster under isoflurane (but not ketamine) anesthesia than in awake mice, indicating distinct effects of anesthetics and of preparation type. These data reveal potentiating effects of isoflurane on microglial response to damage, and provide a framework for comparison and optimal selection of experimental conditions for quantitative analysis of microglial function using two-photon microscopy in vivo.Peer reviewe

Exocytotic release of ATP from cultured astrocytes.

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared

In vivo Calcium Imaging of Evoked Calcium Waves in the Embryonic Cortex

The dynamics of intracellular calcium fluxes are instrumental in the proliferation, differentiation, and migration of neuronal cells. Knowledge thus far of the relationship between these calcium changes and physiological processes in the developing brain has derived principally from ex vivo and in vitro experiments. Here, we present a new method to image intracellular calcium flux in the cerebral cortex of live rodent embryos, whilst attached to the dam through the umbilical cord. Using this approach we demonstrate induction of calcium waves by laser stimulation. These waves are sensitive to ATP-receptor blockade and are significantly increased by pharmacological facilitation of intracellular-calcium release. This approach is the closest to physiological conditions yet achieved for imaging of calcium in the embryonic brain and as such opens new avenues for the study of prenatal brain development. Furthermore, the developed method could open the possibilities of preclinical translational studies in embryos particularly important for developmentally related diseases such as schizophrenia and autism.Peer reviewe