Gate control of sensory neurotransmission in peripheral ganglia by proprioceptive sensory neurons.

Melzak and Wall's gate control theory proposed that innocuous input into the dorsal horn of the spinal cord represses pain-inducing nociceptive input. Here we show that input from proprioceptive parvalbumin-expressing sensory neurons tonically represses nociceptor activation within dorsal root ganglia. Deletion of parvalbumin-positive sensory neurons leads to enhanced nociceptor activity measured with GCaMP3, increased input into wide dynamic range neurons of the spinal cord and increased acute and spontaneous pain behaviour, as well as potentiated innocuous sensation. Parvalbumin-positive sensory neurons express the enzymes and transporters necessary to produce vesicular GABA that is known to be released from depolarized somata. These observations support the view that gate control mechanisms occur peripherally within dorsal root ganglia.

Pregabalin Silences Oxaliplatin-Activated Sensory Neurons to Relieve Cold Allodynia.

Oxaliplatin is a platinum-based chemotherapeutic agent that causes cold and mechanical allodynia in up to 90% of patients. Silent Nav1.8-positive nociceptive cold sensors have been shown to be unmasked by oxaliplatin, and this event has been causally linked to the development of cold allodynia. We examined the effects of pregabalin on oxaliplatin-evoked unmasking of cold sensitive neurons using mice expressing GCaMP-3 in all sensory neurons. Intravenous injection of pregabalin significantly ameliorates cold allodynia, while decreasing the number of cold sensitive neurons by altering their excitability and temperature thresholds. The silenced neurons are predominantly medium/large mechano-cold sensitive neurons, corresponding to the "silent" cold sensors activated during neuropathy. Deletion of α2δ1 subunits abolished the effects of pregabalin on both cold allodynia and the silencing of sensory neurons. Thus, these results define a novel, peripheral inhibitory effect of pregabalin on the excitability of "silent" cold-sensing neurons in a model of oxaliplatin-dependent cold allodynia.

Calcium imaging for analgesic drug discovery.

Somatosensation and pain are complex phenomena involving a rangeofspecialised cell types forming different circuits within the peripheral and central nervous systems. In recent decades, advances in the investigation of these networks, as well as their function in sensation, resulted from the constant evolution of electrophysiology and imaging techniques to allow the observation of cellular activity at the population level both in vitro and in vivo. Genetically encoded indicators of neuronal activity, combined with recent advances in DNA engineering and modern microscopy, offer powerful tools to dissect and visualise the activity of specific neuronal subpopulations with high spatial and temporal resolution. In recent years various groups developed in vivo imaging techniques to image calcium transients in the dorsal root ganglia, the spinal cord and the brain of anesthetised and awake, behaving animals to address fundamental questions in both the physiology and pathophysiology of somatosensation and pain. This approach, besides giving unprecedented details on the circuitry of innocuous and painful sensation, can be a very powerful tool for pharmacological research, from the characterisation of new potential drugs to the discovery of new, druggable targets within specific neuronal subpopulations. Here we summarise recent developments in calcium imaging for pain research, discuss technical challenges and advances, and examine the potential positive impact of this technique in early preclinical phases of the analgesic drug discovery process.

Silent cold-sensing neurons contribute to cold allodynia in neuropathic pain.

Patients with neuropathic pain often experience innocuous cooling as excruciating pain. The cell and molecular basis of this cold allodynia is little understood. We used in vivo calcium imaging of sensory ganglia to investigate how the activity of peripheral cold-sensing neurons was altered in three mouse models of neuropathic pain: oxaliplatin-induced neuropathy, partial sciatic nerve ligation, and ciguatera poisoning. In control mice, cold-sensing neurons were few in number and small in size. In neuropathic animals with cold allodynia, a set of normally silent large diameter neurons became sensitive to cooling. Many of these silent cold-sensing neurons responded to noxious mechanical stimuli and expressed the nociceptor markers Nav1.8 and CGRPα. Ablating neurons expressing Nav1.8 resulted in diminished cold allodynia. The silent cold-sensing neurons could also be activated by cooling in control mice through blockade of Kv1 voltage-gated potassium channels. Thus, silent cold-sensing neurons are unmasked in diverse neuropathic pain states and cold allodynia results from peripheral sensitization caused by altered nociceptor excitability.

Tools for analysis and conditional deletion of subsets of sensory neurons.

Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons.    Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Na v1.8 Cre and then crossed to CGRP CreER (Calca), Th CreERT2, Tmem45b Cre, Tmem233 Cre, Ntng1 Cre and TrkB CreER (Ntrk2) lines. Pain behavioural assays included Hargreaves', hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Na v1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.

3D Free-Standing Ordered Graphene Network Geometrically Regulates Neuronal Growth and Network Formation.

The control of cell-microenvironment interactions plays a pivotal role in constructing specific scaffolds for tissue engineering. Here, we fabricated a 3D free-standing ordered graphene (3D-OG) network with a precisely defined pattern. When primary cortical cells are cultured on 3D-OG scaffolds, they form well-defined 3D connections. Astrocytes have a more ramified shape similar to that seen in vivo because of the nanosized ripples and wrinkles on the surface of graphene skeleton. Neurons have axons and dendrites aligned along the graphene skeleton, allowing the formation of neuronal networks with highly controlled connections. Neuronal networks have higher electrical activity with functional signaling over a long distance along the graphene skeleton. Our study, for the first time, investigated the geometrical cues on ordered neuronal growth and network formation with the support of graphene in 3D, which therefore advanced the development of customized scaffolds for brain-machine interfaces or neuroprosthetic devices.

Single-shot, dual-mode, water-immersion microscopy platform for biological applications.

A single-shot water-immersion digital holographic microscope combined with broadband (white light) illumination mode is presented. This double imaging platform allows conventional incoherent visualization with phase holographic imaging of inspected samples. The holographic architecture is implemented at the image space (that is, after passing the microscope lens), thus reducing the sensitivity of the system to vibrations and/or thermal changes in comparison to regular interferometers. Because of the off-axis holographic recording principle, quantitative phase images of live biosamples can be recorded in a single camera snapshot at full-field geometry without any moving parts. And, the use of water-immersion imaging lenses maximizes the achievable resolution limit. This dual-mode microscope platform is first calibrated using microbeads, then applied to the characterization of fixed cells (neuroblastoma, breast cancer, and hippocampal neuronal cells) and, finally, validated for visualization of dynamic living cells (hippocampal neurons).

Sodium channels.

In 2000, with the completion of the human genome project, nine related channels were found to comprise the complete voltage-gated sodium gene family and they were renamed NaV1.1-NaV1.9. This millennial event reflected the extraordinary impact of molecular genetics on our understanding of electrical signalling in the nervous system. In this review, studies of animal electricity from the time of Galvani to the present day are described. The seminal experiments and models of Hodgkin and Huxley coupled with the discovery of the structure of DNA, the genetic code and the application of molecular genetics have resulted in an appreciation of the extraordinary diversity of sodium channels and their surprisingly broad repertoire of functions. In the present era, unsuspected roles for sodium channels in a huge range of pathologies have become apparent.

An improved method for growing neurons: Comparison with standard protocols.

BACKGROUND: Since different culturing parameters - such as media composition or cell density - lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons - both in vivo and in vitro - is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes. NEW METHOD: Our culturing protocol is based on a novel serum-free preparation of astrocyte - conditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods Neurobasal/B27- and FBS- based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1, 7, 14 and 60days in vitro (DIV). RESULTS: ACM-based cultures gave the best results for all tested criteria, i.e. growth cone's size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27 cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. COMPARISON WITH EXISTING METHOD(S): ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. CONCLUSIONS: We propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures.

Combining FRET and optical tweezers to study RhoGTPases spatio-temporal dynamics upon local stimulation.

Local stimulation with optical tweezers has been used to mimic natural stimuli that occur in biological processes such as cell migration or differentiation. Carriers (beads and lipid vesicles) with sizes down to 30 nm can be manipulated with a high spatial and temporal resolution: they are positioned with a sub-micrometric precision on a specific cell compartment and the beginning of the stimulation can be triggered with millisecond precision. RhoGTPases are a Ras-related family of proteins that regulate many different functions including cell polarity, microtubule dynamics and membrane transport pathways. Here we combine local stimulation with FRET microscopy to study RhoGTPases spatial and temporal activation following guidance cue local stimulation. We used two different vectors for local delivery: silica micro-beads and micro-sized lipid vesicles. The experimental methods associated with neuronal growth cone local stimulation are discussed in detail, as well as the analysis methods. Here we present a protocol that enables to study neuronal growth cone cytoskeleton rearrangements in response to a gradient of molecules in a way that better mimics physiological conditions, and it can be similarly applied to each secreted molecule involved in cell signaling.

Actin Waves Do Not Boost Neurite Outgrowth in the Early Stages of Neuron Maturation.

During neurite development, Actin Waves (AWs) emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC). Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration.

Cdc42 and RhoA reveal different spatio-temporal dynamics upon local stimulation with Semaphorin-3A.

Small RhoGTPases, such as Cdc42 and RhoA, are key players in integrating external cues and intracellular signaling pathways that regulate growth cone (GC) motility. Indeed, Cdc42 is involved in actin polymerization and filopodia formation, whereas RhoA induces GC collapse and neurite retraction through actomyosin contraction. In this study we employed Förster Resonance Energy Transfer (FRET) microscopy to study the spatio-temporal dynamics of Cdc42 and RhoA in GCs in response to local Semaphorin-3A (Sema3A) stimulation obtained with lipid vesicles filled with Sema3A and positioned near the selected GC using optical tweezers. We found that Cdc42 and RhoA were activated at the leading edge of NG108-15 neuroblastoma cells during spontaneous cycles of protrusion and retraction, respectively. The release of Sema3A brought to a progressive activation of RhoA within 30 s from the stimulus in the central region of the GC that collapsed and retracted. In contrast, the same stimulation evoked waves of Cdc42 activation propagating away from the stimulated region. A more localized stimulation obtained with Sema3A coated beads placed on the GC, led to Cdc42 active waves that propagated in a retrograde manner with a mean period of 70 s, and followed by GC retraction. Therefore, Sema3A activates both Cdc42 and RhoA with a complex and different spatial-temporal dynamics.

Fetal mesenchymal stromal cells from cryopreserved human chorionic villi: cytogenetic and molecular analysis of genome stability in long-term cultures.

BACKGROUND AIMS: First-trimester chorionic villi (CV) are an attractive source of human mesenchymal stromal cells (hMSC) for possible applications in cellular therapy and regenerative medicine. Human MSC from CV were monitored for genetic stability in long-term cultures. METHODS: We set up a good manufacturing practice cryopreservation procedure for small amounts of native CV samples. After isolation, hMSC were in vitro cultured and analyzed for biological end points. Genome stability at different passages of expansion was explored by karyotype, genome-wide array-comparative genomic hybridization and microsatellite genotyping. RESULTS: Growth curve analysis revealed a high proliferative potential of CV-derived cells. Immunophenotyping showed expression of typical MSC markers and absence of hematopoietic markers. Analysis of multilineage potential demonstrated efficient differentiation into adipocytes, osteocytes, chondrocytes and induction of neuro-glial commitment. In angiogenic experiments, differentiation in endothelial cells was detected by in vitro Matrigel assay after vascular endothelial growth factor stimulation. Data obtained from karyotyping, array-comparative genomic hybridization and microsatellite genotyping comparing early with late DNA passages did not show any genomic variation at least up to passage 10. Aneuploid clones appeared in four of 14 cases at latest passages, immediately before culture growth arrest. CONCLUSIONS: Our findings indicate that hCV-MSC are genetically stable in long-term cultures at least up to passage 10 and that it is possible to achieve clinically relevant amounts of hCV-MSC even after few stages of expansion. Genome abnormalities at higher passages can occasionally occur and are always associated with spontaneous growth arrest. Under these circumstances, hCV-MSC could be suitable for therapeutic purposes.