12. - 16.11.2022 | Neuroscience 2022
San Diego Convention Center
111 W Harbor Dr, 92101
San Diego, CA, USA
Go to the Conference website here.
Meet the Nanion team
Exhibiting a booth (#2607) and presenting posters
additional information to follow.
Presenter: Andrea Brüggemann
Session Title: High-Throughput Patch-Clamp Approaches
Time slot: 1:00 PM - 5:00 PM
Title: Ion channel currents endogenously expressed in Neuro2A cells recorded using automated patch clamp
Neuro2A cells are a mouse neuroblastoma cell line used extensively to investigate neuronal differentiation, axonal growth and cell signalling pathways . They are also used as an expression system for studying ion channels. Neuro2A cells have been shown to endogenously express NaV channels, predominantly NaV1.7 but also NaV1.2, 1.3 and 1.4, mechanosensitive channels Piezo 1, purinergic receptors and glutamate receptors.
Using automated patch clamp (APC), we recorded different ion channels endogenously expressed in Neuro2A cells. On medium and high throughput APC devices, we recorded voltage-gated sodium channels that were blocked by TTX, tetracaine and lidocaine, with an IC50 of 4.1 ± 0.8 nM (n = 8), 9.9 ± 1.9 µM (n = 8) and 713.6 ± 57.3 (n=14), respectively. The Vhalf of activation was -19.4 ± 0.6 mV (n = 8), although there was some variation between cells. We also recorded Piezo1-mediated responses in Neuro2A cells activated by Yoda1. In addition, we recorded a heat-activated response to external solution heated to 42°C. This heat-activated response could be attributed to TRPV3 or TRPV4 but not TRPV1 as ligand-gated responses to 2-APB and GSK1016790A were observed but no response to the TRPV1 ligand, capsaicin.
In conclusion, Neuro2A cells can be used on APC devices with success rates of 60-80% for >1 GOhm seals and are a suitable cell type for investigating endogenous NaV currents, as well as Piezo1 and some TRP channels.
Presenter: Alison Obergrussberger
Session Title: Touch Reception and Mechanosensitive Channels
Time slot: 08:00 AM - 12:00 PM
Title: Mechanical activation of Piezo1 by shear stress using high throughput automated patch clamp
Piezo channels are mechanosensitive cation channels that play important roles in biological functions including touch, proprioception, shear stress, stretch sensation and blood pressure regulation. Piezo1 channels have been shown to sense mechanical stimuli in central nervous system vasculature and are expressed in retina and cortex capillaries (Harraz, et al, 2022). Piezo1 is also important for promoting vascular pathfinding in a variety of organs including brain (Liu, et al, 2020), and loss of function mutations of Piezo1 impair endothelial pathfinding. Interestingly, overactivation of Piezo1 by Yoda-1 impairs neuronal myelination causing neuronal damage, whereas inhibition of Piezo1 has a neuroprotective effect (Velasco-Estevez, 2020).
A significant challenge in Piezo1 channel drug development using automated patch clamp assay recordings is evoking highly reproducible Piezo 1 current amplitudes using pseudo-physiological mechanical stimulation. Here we show that optimization of pipetting parameters coupled with modification of the NPC-384 chip of the SyncroPatch 384 lead to Piezo1-mediated currents activated by mechanical stimulation. Data from mouse and human Piezo1 channels expressed in HEK293 cells activated by either mechanical or chemical stimuli will be shown, as well as the combination of both methods. Using this approach we were able to show that very specific modification of the Yoda-1 molecule permits the development of new Piezo1 agonists with improved physico-chemical properties. These new tool compounds should improve Piezo1 channel modulation in experimental physiological models.
In this way, mechanical stimulation of Piezo1 channels using a high throughput planer patch clamp system could be demonstrated. The possibility of comparing and combining mechanical and chemical stimulation in a high throughput patch clamp assay facilitates the biophysical and pharmacological characterization of Piezo channels and thereby provides an important experimental tool for studying Piezo channel biology in CNS function.
Presenter: Vincent Truong (Anatomic)
Title: Human Induced Pluripotent Stem Cell-Derived Nociceptors on an Automated Patch Clamp System for High Throughput Pain Drug Discovery
There is still an unmet need for novel non-addictive pain analgesics as the opioid epidemic continues. The ability to functionally screen target compounds on human nociceptors in high throughput would increase both the efficiency and pace of preclinical pain drug discovery. We have previously demonstrated that human nociceptors can be generated in an accelerated, scalable method from human induced pluripotent stem cells (hiPSCs). These hiPSC-derived nonciceptors (RealDRG™) nociception via manual patch clamp. In this study, we have further developed on a novel dissociation method to electrophysiologically interrogate multiple ion channels in RealDRG™ cultures on the Nanion SyncroPatch 384 automated patch clamp system at 14, 21, 28 days in culture in high throughput 384 well format. The expression and properties of voltage-gated sodium (Nav) and potassium ion channels (Kv), transient receptor potential vanilloid 1 (TRPV1), GABA, and P2X ligand-gated ion channels, along with excitability properties in current clamp mode (resting membrane potential, spontaneous and evoked action potentials) were all explored. The percentage of cells with at least one evoked action potential increased from 42% to 77% over the course of maturation, though success rates decreased from 56% to 35% as the cells matured. There was also an increase in voltage-gated sodium (Nav) and potassium (Kv) currents as time progressed with success rates ranging from 94-98% and 93-100%, respectfully. Most neurons had tetrodotoxin-resistant (TTXr) sodium currents, with the trends of increasing number of cells with current and fraction of TTXr current per cell. A ligand puff protocol was developed to examine ligand-gated GABAR and P2X ion channels and responses to ATP and GABA currents were observed. Together, these findings demonstrate the ability to functionally screen multiple different targets in human nociceptors in a high throughput system.
About the Society for Neuroscience (SfN)
Founded in 1969, the Society for Neuroscience (SfN) now has more than 36,000 members in more than 95 countries. Year-round programming includes the publishing of two highly regarded scientific journals, JNeurosci and eNeuro; professional development resources and career training through Neuronline¸ the Society’s home for learning and discussion; science advocacy and public policy engagement including annual Capitol Hill Day; and a variety of engaging public outreach efforts, led by the expanding and interactive collection of public-facing resources on BrainFacts.org.
Approximately 107 staff committed to SfN’s mission are employed at its headquarters building in downtown Washington, D.C., which the Society has owned since 2006. SfN’s annual meeting regularly attracts more than 30,000 attendees, representing 80 countries; 536 exhibiting companies; and close to 300 journalists.