A multiparametric activity profiling platform for neuron disease phenotyping and drug screening.

Patient stem cell-derived models enable imaging of complex disease phenotypes and the development of scalable drug discovery platforms. Current preclinical methods for assessing cellular activity do not, however, capture the full intricacies of disease-induced disturbances and instead typically focus on a single parameter, which impairs both the understanding of disease and the discovery of effective therapeutics. Here, we describe a cloud-based image processing and analysis platform that captures the intricate activity profile revealed by GCaMP fluorescence recordings of intracellular calcium changes and enables the discovery of molecules that correct 153 parameters that define the amyotrophic lateral sclerosis motor neuron disease phenotype. In a high-throughput screen, we identified compounds that revert the multiparametric disease profile to that found in healthy cells, a novel and robust measure of therapeutic potential quite distinct from unidimensional screening. This platform can guide the development of therapeutics that counteract the multifaceted pathological features of diseased cellular activity.

Convergent neural representations of experimentally-induced acute pain in healthy volunteers: A large-scale fMRI meta-analysis.

Characterizing a reliable, pain-related neural signature is critical for translational applications. Many prior fMRI studies have examined acute nociceptive pain-related brain activation in healthy participants. However, synthesizing these data to identify convergent patterns of activation can be challenging due to the heterogeneity of experimental designs and samples. To address this challenge, we conducted a comprehensive meta-analysis of fMRI studies of stimulus-induced pain in healthy participants. Following pre-registration, two independent reviewers evaluated 4,927 abstracts returned from a search of 8 databases, with 222 fMRI experiments meeting inclusion criteria. We analyzed these experiments using Activation Likelihood Estimation with rigorous type I error control (voxel height p < 0.001, cluster p < 0.05 FWE-corrected) and found a convergent, largely bilateral pattern of pain-related activation in the secondary somatosensory cortex, insula, midcingulate cortex, and thalamus. Notably, these regions were consistently recruited regardless of stimulation technique, location of induction, and participant sex. These findings suggest a highly-conserved core set of pain-related brain areas, encouraging applications as a biomarker for novel therapeutics targeting acute nociceptive pain.

Breaking barriers to novel analgesic drug development.

Acute and chronic pain complaints, although common, are generally poorly served by existing therapies. This unmet clinical need reflects a failure to develop novel classes of analgesics with superior efficacy, diminished adverse effects and a lower abuse liability than those currently available. Reasons for this include the heterogeneity of clinical pain conditions, the complexity and diversity of underlying pathophysiological mechanisms, and the unreliability of some preclinical pain models. However, recent advances in our understanding of the neurobiology of pain are beginning to offer opportunities for developing novel therapeutic strategies and revisiting existing targets, including modulating ion channels, enzymes and G-protein-coupled receptors.

A three-dimensional human neural cell culture model of Alzheimer's disease.

Alzheimer's disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-ß plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer's disease posits that the excessive accumulation of amyloid-ß peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer's disease (FAD) mutations exhibit amyloid-ß-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer's disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer's disease patients have shown elevated levels of toxic amyloid-ß species and phosphorylated tau but did not demonstrate amyloid-ß plaques or neurofibrillary tangles. Here we report that FAD mutations in ß-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-ß, including amyloid-ß plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-ß generation with ß- or γ-secretase inhibitors not only decreased amyloid-ß pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-ß-mediated tau phosphorylation. We have successfully recapitulated amyloid-ß and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer's disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers.

Most local anaesthetics used clinically are relatively hydrophobic molecules that gain access to their blocking site on the sodium channel by diffusing into or through the cell membrane. These anaesthetics block sodium channels and thereby the excitability of all neurons, not just sensory neurons. We tested the possibility of selectively blocking the excitability of primary sensory nociceptor (pain-sensing) neurons by introducing the charged, membrane-impermeant lidocaine derivative QX-314 through the pore of the noxious-heat-sensitive TRPV1 channel. Here we show that charged sodium-channel blockers can be targeted into nociceptors by the application of TRPV1 agonists to produce a pain-specific local anaesthesia. QX-314 applied externally had no effect on the activity of sodium channels in small sensory neurons when applied alone, but when applied in the presence of the TRPV1 agonist capsaicin, QX-314 blocked sodium channels and inhibited excitability. Inhibition by co-applied QX-314 and capsaicin was restricted to neurons expressing TRPV1. Injection of QX-314 together with capsaicin into rat hindpaws produced a long-lasting (more than 2 h) increase in mechanical and thermal nociceptive thresholds. Long-lasting decreases in pain sensitivity were also seen with regional injection of QX-314 and capsaicin near the sciatic nerve; however, in contrast to the effect of lidocaine, the application of QX-314 and capsaicin together was not accompanied by motor or tactile deficits.

Patients with chest pain and occult gastroesophageal reflux demonstrate visceral pain hypersensitivity which may be partially responsive to acid suppression.

OBJECTIVES: Mechanisms of chest pain in gastroesophageal reflux disease (GERD) are poorly understood. The recent demonstration in healthy subjects that lower esophageal acid exposure induces pain hypersensitivity within the non-acid-exposed upper esophagus (secondary allodynia) raises the possibility that an increase in spinal neuronal excitability (i.e., central sensitization) contributes to chest pain in GERD. The aim of this study was to determine whether in patients with unexplained chest pain, acid reflux contributes to esophageal pain hypersensitivity. METHODS: In 14 patients with chest pain and GERD and 8 healthy volunteers, electrical pain thresholds (PT) were recorded from the upper esophagus before, and then repeatedly for 90 min after either hydrochloric acid (0.15 M) or saline (0.15 M) infusion into the lower esophagus. Six patients underwent a repeat study after 6 wk of high-dose proton pump inhibitor (PPI) therapy. RESULTS: GERD patients had lower resting upper esophageal PT than in healthy subjects (40.8 +/- 9 mA and 70.4 +/- 11 mA, respectively; p= 0.018). Acid infusion reduced PT in the non-acid-exposed upper esophagus in healthy subjects, but not in the patients (area under curve [AUC] - 304 +/- 333 and 786 +/- 464; p= 0.03, respectively). Following PPI therapy, resting PT increased (34.65 +/- 13.4 to 40.5 +/- 12.5 mA; p= 0.03), and a reduction in PT now occurred in acid infusion (AUC - 369 +/- 321; p= 0.03). CONCLUSIONS: Patients with unexplained chest pain and occult GERD have esophageal pain hypersensitivity that is PPI responsive. The increase in resting PT and secondary allodynia only following PPI therapy suggests that pain hypersensitivity in these GERD patients may partially be the result of central sensitization.

Noxious stimuli induce an N-methyl-D-aspartate receptor-dependent hypersensitivity of the flexion withdrawal reflex to touch: implications for the treatment of mechanical allodynia.

Primary C-fiber afferents can induce a state of increased excitability in spinal cord neurons amplifying their responsiveness to noxious and innocuous stimuli--the phenomenon of central sensitization. We have examined whether the hypersensitivity to low-intensity stimuli (mechanical allodynia) evoked by C-afferent conditioning inputs is NMDA receptor dependent. The enhancement by C-afferent conditioning stimuli of the normally low or absent cutaneous touch-evoked responses of posterior biceps femoris/semitendinosus flexor motoneurons in the decerebrate-spinal rat has been used as a model of touch-evoked allodynia. Three different noxious conditioning stimuli (electrical stimulation of the sural nerve at C-fiber strength, cutaneous application of mustard oil and the intramuscular (i.m.) injection of bradykinin) enhanced touch-evoked responses and decreased the threshold of the flexor reflex to mechanical stimulation. Intrathecal (i.t.) injections of NMDA (20 pmol) produced a similar effect. A non-competitive NMDA antagonist MK 801, administered prior to the conditioning input at doses that did not modify the baseline reflex, significantly attenuated both the enhancement of touch-evoked responses and the threshold decrease following the noxious conditioning stimuli. Treatment with MK 801 some time after the conditioning input when the mechanical hypersensitivity is fully established, also reduced the increased touch-evoked responses. The reduction in threshold and the increase in touch responsiveness induced by cutaneous and muscle noxious C-fiber conditioning stimuli in the rat spinal cord are, therefore, both prevented and reversed by NMDA receptor antagonism. NMDA antagonists may be potentially useful, therefore, in treating postinjury pain hypersensitivity.

Sensitization of high mechanothreshold superficial dorsal horn and flexor motor neurones following chemosensitive primary afferent activation.

Nociceptive primary afferents have the capacity to induce a state of increased excitability or central sensitization in dorsal horn neurones. This contributes to the mechanical hypersensitivity (allodynia) which occurs after peripheral tissue injury where low-mechanothreshold primary afferent activation begins to elicit pain. The relative susceptibility of dorsal horn cells with an apparent exclusive nociceptive input (nociceptive-specific (NS) or high-threshold (HT) cells) and those with a convergent input from low- and high-threshold mechanoreceptors (wide-dynamic-range (WDR) or multireceptive neurones) to sensitivity changes has been disputed. We have examined whether high-mechanothreshold neurones in the superficial dorsal and the ventral horn can modify their sensitivity following cutaneous application of the chemical irritant mustard oil. This produced both a prolonged reduction in the mechanical threshold of the cutaneous flexion withdrawal reflex, recorded from semitendinosus alpha-motor neurones, and an increase in the activity evoked in these neurones by low-intensity touch stimuli to the glabrous skin. Eight NS or HT only cells, defined in terms of their cutaneous mechanoreceptive field properties, were recorded in the superficial dorsal horn before and after cutaneous application of mustard oil. Mustard oil was applied outside of the mechanical receptive field of the cells and produced a transient increase in action potential discharge in 4 cells but increased the mechanoreceptive field size in all cells for 30-60 min. Mechanical thresholds declined in 6 cells to levels associated with low-threshold (LT) and WDR cells, and this was accompanied by recruitment of a novel brush/touch response in 5 cells. The responses evoked by graded electrical stimulation of the sural nerve were tested in 5 cells. Only 1 cell failed to show any change after mustard oil. In 3 cells, an increase in the response to A-fibre afferents occurred, a novel A-fibre response was recruited in 2 cells and the C-fibre response increased in 2 cells. Cells in the superficial dorsal horn of the rat spinal cord that are normally NS can begin, therefore, to respond to LT primary afferent mechanoreceptors after an increase in central excitability produced by activation of peripheral chemoreceptors. Sensitization of these, as well as of WDR cells, may contribute to the generation of post-injury mechanical pain and reflex hypersensitivity.

The induction and maintenance of central sensitization is dependent on N-methyl-D-aspartic acid receptor activation; implications for the treatment of post-injury pain hypersensitivity states.

Repetitive stimulation of small diameter primary afferent fibres produces a progressive increase in action potential discharge (windup) and a prolonged increase in the excitability of neurones in the spinal cord following the stimulus. Previous studies have demonstrated that windup is the consequence of the temporal summation of slow synaptic potentials and that the slow potentials and windup are reduced by pretreatment with N-methyl-D-aspartic acid (NMDA) antagonists. We have now examined whether primary afferent induced hypersensitivity states in flexor motoneurones are also dependent on the activation of NMDA receptors and whether windup is a possible trigger for the production of the central hypersensitivity. Both a non-competitive (MK-801) and a competitive (D-CPP) NMDA antagonist, at doses that did not modify the baseline reflex, reduced the facilitation of the flexor reflex produced by either brief electrical stimulation of the sural nerve (1 Hz for 20 sec at C-fibre strength), or by the cutaneous application of the chemical irritant mustard oil. These antagonists also prevented windup from occurring in the motoneurones. When the the MK-801 and the D-CPP were administered once a state of central facilitation had been induced by prior treatment with mustard oil, they returned the facilitated reflex to its pretreatment level. These results indicate that NMDA receptors are involved in the induction and maintenance of the central sensitization produced by high threshold primary afferent inputs. Because central sensitization is likely to contribute to the post-injury pain hypersensitivity states in man, these data have a bearing both on the potential role of NMDA antagonists for pre-emptive analgesia and for treating established pain states.