High-Throughput Controlled Mechanical Stimulation and Functional Imaging In Vivo

AbstractUnderstanding mechanosensation and other sensory behavior in genetic model systems such as C. elegans is relevant to many human diseases. These studies conventionally require immobilization by glue and manual delivery of stimuli, leading to low experimental throughput and high variability. Here we present a microfluidic platform that delivers precise mechanical stimuli robustly. The system can be easily used in conjunction with functional imaging and optical interrogation techniques, as well as other capabilities such as sorting or more sophisticated fluid delivery schemes. The platform is fully automated, thereby greatly enhancing the throughput and robustness of experiments. We show that behavior of the well-known gentle and harsh touch neurons and their receptive fields can be recapitulated in our system. Using calcium dynamics as a readout, we demonstrate the ability to perform a drug screen in vivo. Furthermore, using an integrated chip platform that can deliver both mechanical and chemical stimuli, we examine sensory integration in interneurons in response to multimodal sensory inputs. We envision that this system will be able to greatly accelerate the discovery of genes and molecules involved in mechanosensation and multimodal sensory behavior, as well as the discovery of therapeutics for related diseases.

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In vivo responses of cutaneous C-mechanosensitive neurons in mouse to punctate chemical stimuli that elicit itch and nociceptive sensations in humans

Journal of Neurophysiology ◽

10.1152/jn.00801.2011 ◽

2012 ◽

Vol 107 (1) ◽

pp. 357-363 ◽

Cited By ~ 32

Author(s):

C. Ma ◽

H. Nie ◽

Q. Gu ◽

P. Sikand ◽

R. H. LaMotte

Keyword(s):

Time Course ◽

Discharge Rate ◽

Receptive Fields ◽

Mechanical Stimuli ◽

Nociceptive Neurons ◽

Electrophysiological Recordings ◽

The Mean ◽

Chemical Stimuli ◽

Temporal Profiles

Native cowhage spicules, and heat-inactivated spicules containing histamine or capsaicin, evoke similar sensations of itch and nociceptive sensations in humans. In ongoing studies of the peripheral neural mechanisms of chemical itch and pain in the mouse, extracellular electrophysiological recordings were obtained, in vivo, from the cell bodies of mechanosensitive nociceptive neurons in response to spicule stimuli delivered to their cutaneous receptive fields (RFs) on the distal hindlimb. A total of 43 mechanosensitive, cutaneous, nociceptive neurons with axonal conduction velocities in the C-fiber range (C-nociceptors) were classified as CM if responsive to noxious mechanical stimuli, such as pinch, or CMH if responsive to noxious mechanical and heat stimuli (51°C, 5 s). The tips of native cowhage spicules, or heat-inactivated spicules containing histamine or capsaicin, were applied to the RF. Heat-inactivated spicules containing no chemical produced only a transient response occurring during insertion. Of the 43 mechanosensitive nociceptors recorded, 20 of the 25 CMHs responded to capsaicin, and of these, 13 also responded to cowhage and/or histamine. In contrast, none of the 18 CMs responded to any of the chemical stimuli. The time course of the mean discharge rate of CMHs was similar in response to each type of spicule and generally similar, although reaching a peak earlier, to the temporal profiles of itch and nociceptive sensations evoked by the same stimuli in humans. These findings are consistent with the hypothesis that the itch and nociceptive sensations evoked by these punctuate chemical stimuli are mediated at least in part by the activity of mechanoheat-sensitive C-nociceptors. In contrast, activity in mechanosensitive C-nociceptors that do not respond to heat or to pruritic chemicals is hypothesized as contributing to pain but not to itch.

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Automated and controlled mechanical stimulation and functional imaging in vivo in C. elegans

Lab on a Chip ◽

10.1039/c7lc00465f ◽

2017 ◽

Vol 17 (15) ◽

pp. 2609-2618 ◽

Cited By ~ 30

Author(s):

Yongmin Cho ◽

Daniel A. Porto ◽

Hyundoo Hwang ◽

Laura J. Grundy ◽

William R. Schafer ◽

...

Keyword(s):

Functional Imaging ◽

Mechanical Stimulation ◽

Mechanical Stimuli ◽

Microfluidic Platform ◽

C Elegans ◽

Neural Imaging ◽

Wide Range

A new automated microfluidic platform can deliver a wide range of mechanical stimuli for functional neural imaging inC. elegans.

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Caenorhabditis elegans as an in vivo Model to Assess Amphetamine Tolerance

Brain Behavior and Evolution ◽

10.1159/000514858 ◽

2021 ◽

pp. 1-9

Author(s):

Dayana Torres Valladares ◽

Sirisha Kudumala ◽

Murad Hossain ◽

Lucia Carvelli

Keyword(s):

Caenorhabditis Elegans ◽

Molecular Mechanisms ◽

Drugs Of Abuse ◽

Genetic Model ◽

In Vivo Model ◽

C Elegans ◽

Hyperactivity Disorder ◽

In Vitro Data

Amphetamine is a potent psychostimulant also used to treat attention deficit/hyperactivity disorder and narcolepsy. In vivo and in vitro data have demonstrated that amphetamine increases the amount of extra synaptic dopamine by both inhibiting reuptake and promoting efflux of dopamine through the dopamine transporter. Previous studies have shown that chronic use of amphetamine causes tolerance to the drug. Thus, since the molecular mechanisms underlying tolerance to amphetamine are still unknown, an animal model to identify the neurochemical mechanisms associated with drug tolerance is greatly needed. Here we took advantage of a unique behavior caused by amphetamine in <i>Caenorhabditis elegans</i> to investigate whether this simple, but powerful, genetic model develops tolerance following repeated exposure to amphetamine. We found that at least 3 treatments with 0.5 mM amphetamine were necessary to see a reduction in the amphetamine-induced behavior and, thus, to promote tolerance. Moreover, we found that, after intervals of 60/90 minutes between treatments, animals were more likely to exhibit tolerance than animals that underwent 10-minute intervals between treatments. Taken together, our results show that <i>C. elegans</i> is a suitable system to study tolerance to drugs of abuse such as amphetamines.

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Progressive recruitment of distal MEC-4 channels determines touch response strength in C. elegans

10.1101/587014 ◽

2019 ◽

Cited By ~ 1

Author(s):

S. Katta ◽

A. Sanzeni ◽

A. Das ◽

M. Vergassola ◽

M.B. Goodman

Keyword(s):

Temporal Dynamics ◽

Mechanical Strain ◽

Tissue Mechanics ◽

Mechanical Stimuli ◽

Patch Clamp Recording ◽

Open Probability ◽

C Elegans ◽

Somatosensory Neurons ◽

Touch Response

AbstractTouch deforms, or strains, the skin beyond the immediate point of contact. The spatiotemporal nature of the touch-induced strain fields depend on the mechanical properties of the skin and the tissues below. Somatosensory neurons that sense touch branch out within the skin and rely on a set of mechano-electrical transduction channels distributed within their dendrites to detect mechanical stimuli. Here, we sought to understand how tissue mechanics shape touch-induced mechanical strain across the skin over time and how individual channels located in different regions of the strain field contribute to the overall touch response. We leveraged C. elegans’ touch receptor neurons (TRNs) as a simple model amenable to in vivo whole-cell patch clamp recording and an integrated experimental-computational approach to dissect the mechanisms underlying the spatial and temporal dynamics that we observed. Consistent with the idea that strain is produced at a distance, we show that delivering strong stimuli outside the anatomical extent of the neuron is sufficient to evoke MRCs. The amplitude and kinetics of the MRCs depended on both stimulus displacement and speed. Finally, we found that the main factor responsible for touch sensitivity is the recruitment of progressively more distant channels by stronger stimuli, rather than modulation of channel open probability. This principle may generalize to somatosensory neurons with more complex morphologies.SummaryThrough experiment and simulation, Katta et al. reveal that pushing faster and deeper recruits more and more distant mechano-electrical transduction channels during touch. The net result is a dynamic receptive field whose size and shape depends on tissue mechanics, stimulus parameters, and channel distribution within sensory neurons.

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In vivo analysis of FANCD2 recruitment at meiotic DNA breaks in Caenorhabditis elegans

Scientific Reports ◽

10.1038/s41598-019-57096-1 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 2

Author(s):

Marcello Germoglio ◽

Anna Valenti ◽

Ines Gallo ◽

Chiara Forenza ◽

Pamela Santonicola ◽

...

Keyword(s):

Dna Repair ◽

Caenorhabditis Elegans ◽

Genome Stability ◽

Genetic Interaction ◽

Model Systems ◽

Dna Breaks ◽

Strand Breaks ◽

C Elegans ◽

In Vivo Analysis

AbstractFanconi Anemia is a rare genetic disease associated with DNA repair defects, congenital abnormalities and infertility. Most of FA pathway is evolutionary conserved, allowing dissection and mechanistic studies in simpler model systems such as Caenorhabditis elegans. In the present study, we employed C. elegans to better understand the role of FA group D2 (FANCD2) protein in vivo, a key player in promoting genome stability. We report that localization of FCD-2/FANCD2 is dynamic during meiotic prophase I and requires its heterodimeric partner FNCI-1/FANCI. Strikingly, we found that FCD-2 recruitment depends on SPO-11-induced double-strand breaks (DSBs) but not RAD-51-mediated strand invasion. Furthermore, exposure to DNA damage-inducing agents boosts FCD-2 recruitment on the chromatin. Finally, analysis of genetic interaction between FCD-2 and BRC-1 (the C. elegans orthologue of mammalian BRCA1) supports a role for these proteins in different DSB repair pathways. Collectively, we showed a direct involvement of FCD-2 at DSBs and speculate on its function in driving meiotic DNA repair.

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Correction: automated and controlled mechanical stimulation and functional imaging in vivo in C. elegans

Lab on a Chip ◽

10.1039/c7lc90104f ◽

2017 ◽

Vol 17 (22) ◽

pp. 3935-3935

Author(s):

Yongmin Cho ◽

Daniel A. Porto ◽

Hyundoo Hwang ◽

Laura J. Grundy ◽

William R. Schafer ◽

...

Keyword(s):

Functional Imaging ◽

Mechanical Stimulation ◽

C Elegans

Correction for ‘Automated and controlled mechanical stimulation and functional imaging in vivo in C. elegans’ by Yongmin Cho et al., Lab Chip, 2017, 17, 2609–2618.

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Leech Mechanosensation

Oxford Research Encyclopedia of Neuroscience ◽

10.1093/acrefore/9780190264086.013.179 ◽

2017 ◽

Cited By ~ 3

Author(s):

Brian D. Burrell

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Sensory Neurons ◽

Receptive Fields ◽

Model Systems ◽

Sensory Cells ◽

Mechanical Stimuli ◽

Nociceptive Neurons ◽

Mechanosensory Neurons ◽

The Central Nervous System

The medicinal leech (Hirudo verbana) is an annelid (segmented worm) and one of the classic model systems in neuroscience. It has been used in research for over 50 years and was one of the first animals in which intracellular recordings of mechanosensory neurons were carried out. Remarkably, the leech has three main classes of mechanosensory neurons that exhibit many of the same properties found in vertebrates. The most sensitive of these neurons are the touch cells, which are rapidly adapting neurons that detect low-intensity mechanical stimuli. Next are the pressure cells, which are slow-adapting sensory neurons that respond to higher intensity, sustained mechanostimulation. Finally, there are nociceptive neurons, which have the highest threshold and respond to potentially damaging mechanostimuli, such as a pinch. As observed in mammals, the leech has separate mechanosensitive and polymodal nociceptors, the latter responding to mechanical, thermal, and chemical stimuli. The cell bodies for all three types of mechanosensitive neurons are found in the central nervous system where they are arranged as bilateral pairs. Each neuron extends processes to the skin where they form discrete receptive fields. In the touch and pressure cells, these receptive fields are arranged along the dorsal-ventral axis. For the mechano-only and polymodal nociceptive neurons, the peripheral receptive fields overlap with the mechano-only nociceptor, which also innervates the gut. The leech also has a type of mechanosensitive cell located in the periphery that responds to vibrations in the water and is used, in part, to detect potential prey nearby. In the central nervous system, the touch, pressure, and nociceptive cells all form synaptic connections with a variety of motor neurons, interneurons, and even each other, using glutamate as the neurotransmitter. Synaptic transmission by these cells can be modulated by a variety of activity-dependent processes as well as the influence of neuromodulatory transmitters, such as serotonin. The output of these sensory neurons can also be modulated by conduction block, a process in which action potentials fail to propagate to all the synaptic release sites, decreasing synaptic output. Activity in these sensory neurons leads to the initiation of a number of different motor behaviors involved in locomotion, such as swimming and crawling, as well as behaviors designed to recoil from aversive/noxious stimuli, such as local bending and shortening. In the case of local bending, the leech is able to bend in the appropriate direction away from the offending stimuli. It does so through a combination of which mechanosensory cell receptive fields have been activated and the relative activation of multiple sensory cells decoded by a layer of downstream interneurons.

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Warm-Sensitive Afferent Splanchnic C-Fiber Units In Vitro

Journal of Neurophysiology ◽

10.1152/jn.1997.77.6.2989 ◽

1997 ◽

Vol 77 (6) ◽

pp. 2989-3002 ◽

Cited By ~ 28

Author(s):

David W. Adelson ◽

Jen Yu Wei ◽

Lawrence Kruger

Keyword(s):

Receptive Fields ◽

Sense Organ ◽

Cardiovascular Responses ◽

Peak Temperature ◽

Mechanical Stimuli ◽

Tonic Component ◽

Brown Adipose ◽

C Fiber

Adelson, David W., Jen Yu Wei, and Lawrence Kruger. Warm-sensitive afferent splanchnic C-fiber units in vitro. J. Neurophysiol. 77: 2989–3002, 1997. Receptive fields of 41 slowly conducting sensory fibers were located using a thermal (warm) search stimulus in an in vitro splanchnic nerve-mesentery preparation. Warm-sensitive receptive fields were punctate and were densest in the region surrounding the prevertebral ganglia, an area with prominent deposits of brown adipose tissue, where the abdominal aorta branches into the major trunks supplying the abdominal viscera. Impulse activity was recorded while applying a warm stimulus to identified receptive fields (RFs). The warm stimulus consisted of a warming ramp (10–15°C in 1–2 s to a 42–49°C peak temperature) followed by a 10- to 30-s period during which the RF was maintained at this peak temperature (plateau phase). Eighty percent (33/41) of warm-sensitive units responded to warming with discharge comprising both a phasic and a tonic component (slowly adapting warm-sensitive, or SA-W, units). The remainder (8/41) responded with only phasic discharge (rapidly adapting warm-sensitive, or RA-W, units). Units' adaptation characteristics were consistent from trial to trial and when applying stimuli from different positions. Fifty percent of SA-W units (8/16) and 17% of RA-W units (1/6) were activated by transient exposure to 9–90 nM bradykinin (BK). Twenty-seven percent (9/33) of SA-W units and 12%(1/8) of RA-W units were activated by probing their RF with von Frey hairs with bending forces <10 mN (∼1 g equivalent mass). An additional five SA-W units tested were activated by strong mechanical stimuli (compression with a metal probe or firm stretching). No BK-responsive warm-sensitive units were activated by von Frey probing <10 mN, but two (both SA-W) responded to strong mechanical stimuli. In six SA-W units and one RA-W unit, the number of impulses evoked by warming ∼5 min after exposure to BK was >2 SD greater than the mean pre-BK response, indicating sensitization. This sensitization was transient, the response to warming returning to within one standard deviation of the pretrial mean or less over the course of the next 5–10 min. Changes in background activity, mechanical sensitivity, BK sensitivity, and BK-induced sensitization were noted in various splanchnic units over the course of prolonged observations, suggesting that these indices may not reliably distinguish unit type, but instead may indicate the functional state of the sense organ. Splanchnic neurons responsive to the intense warming used in the present in vitro experiments may participate in the cardiovascular responses observed in vivo in heat-stressed rats. The dense distribution of warm-receptive fields in the vicinity of the celiac-superior mesenteric ganglionic complex is consistent with the localization of splanchnic thermosensitive units previously noted in vivo in the rabbit.

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Sequence and transmembrane topology of MEC-4, an ion channel subunit required for mechanotransduction in Caenorhabditis elegans.

The Journal of Cell Biology ◽

10.1083/jcb.133.5.1071 ◽

1996 ◽

Vol 133 (5) ◽

pp. 1071-1081 ◽

Cited By ~ 78

Author(s):

C C Lai ◽

K Hong ◽

M Kinnell ◽

M Chalfie ◽

M Driscoll

Keyword(s):

Caenorhabditis Elegans ◽

Ion Channel ◽

Critical Role ◽

Comparative Sequence Analysis ◽

Mechanical Stimuli ◽

Transmembrane Topology ◽

C Elegans ◽

Carboxy Terminal

The process by which mechanical stimuli are converted into cellular responses is poorly understood, in part because key molecules in this mode of signal transduction, the mechanically gated ion channels, have eluded cloning efforts. The Caenorhabditis elegans mec-4 gene encodes a subunit of a candidate mechanosensitive ion channel that plays a critical role in touch reception. Comparative sequence analysis of C. elegans and Caenorhabditis briggsae mec-4 genes was used to initiate molecular studies that establish MEC-4 as a 768-amino acid protein that includes two hydrophobic domains theoretically capable of spanning a lipid bilayer. Immunoprecipitation of in vitro translated mec-4 protein with domain-specific anti-MEC-4 antibodies and in vivo characterization of a series of mec-4lacZ fusion proteins both support the hypothesis that MEC-4 crosses the membrane twice. The MEC-4 amino- and carboxy-terminal domains are situated in the cytoplasm and a large domain, which includes three Cys-rich regions, is extracellular. Definition of transmembrane topology defines regions that might interact with the extracellular matrix or cytoskeleton to mediate mechanical signaling.

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Comparing Caenorhabditis elegans gentle and harsh touch response behavior using a multiplexed hydraulic microfluidic device

10.1101/162990 ◽

2017 ◽

Author(s):

Patrick D. McClanahan ◽

Joyce H. Xu ◽

Christopher Fang-Yen

Keyword(s):

Caenorhabditis Elegans ◽

Microfluidic Device ◽

Receptive Fields ◽

The Body ◽

Mechanical Stimuli ◽

C Elegans ◽

Response Characteristics ◽

Touch Receptor Neurons ◽

Touch Response ◽

Receptor Neurons

AbstractThe roundworm Caenorhabditis elegans is an important model system for understanding the genetics and physiology of touch. Classical assays for C. elegans touch, which involve manually touching the animal with a probe and observing its response, are limited by their low throughput and qualitative nature. We developed a microfluidic device in which several dozen animals are subject to spatially localized mechanical stimuli with variable amplitude. The device contains 64 sinusoidal channels through which worms crawl, and hydraulic valves that deliver touch stimuli to the worms. We used this assay to characterize the behavioral responses to gentle touch stimuli and the less well studied harsh (nociceptive) touch stimuli. First, we measured the relative response thresholds of gentle and harsh touch. Next, we quantified differences in the receptive fields between wild type worms and a mutant with non-functioning posterior touch receptor neurons. We showed that under gentle touch the receptive field of the anterior touch receptor neurons extends into the posterior half of the body. Finally, we found that the behavioral response to gentle touch does not depend on the locomotion of the animal immediately prior to the stimulus, but does depend on the location of the previous touch. Responses to harsh touch, on the other hand, did not depend on either previous velocity or stimulus location. Differences in gentle and harsh touch response characteristics may reflect the different innervation of the respective mechanosensory cells. Our assay will facilitate studies of mechanosensation, sensory adaptation, and nociception.

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