From Understanding Cellular Function to Novel Drug Discovery: The Role of Planar Patch-Clamp Array Chip Technology

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions – including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells

Gender-differences of in vitro colonic motility after chemo- and radiotherapy in humans.

Background: The aim of the present in vitro study was to investigate, in different genders, motor responses in surgical colonic specimens from patients with rectal cancer undergoing and not undergoing chemotherapy with capecitabine and radiotherapy. Methods: This in vitro study was conducted from October 2015 to August 2017 at the Experimental Pharmacology Laboratory at the National Institute “S. de Bellis” after collecting samples at the Department of Surgery. Segments of sigmoid colon were obtained from 15 patients (Male (M)/Female (F) = 8/7; control group, CG) operated on for elective colorectal resection for rectal cancer without obstruction and 14 patients (M/F = 7/7; study group, SG) operated on for elective colorectal resection for rectal cancer who also received chemotherapy, based on capecitabine twice daily, and radiotherapy. Isometric tension was measured on colonic circular muscle strips exposed to increasing carbachol or histamine concentrations to obtain concentration-response curves. The motor responses to electrically evoked stimulation were also investigated. Results: In males, carbachol and histamine caused concentration-dependent contractions in the CG and SG. An increased sensitivity and a higher response to carbachol and histamine were observed in SG than CG (P < 0.01). On the contrary, in females, the response to carbachol was not significantly different in CG from the SG and the maximal responses to carbachol were greater in CG than in SG (P < 0.001). The same applied to histamine for half-maximal effective concentrations and maximal response in that they were not significantly different in CG from the SG. Electrically evoked contractions were significantly more pronounced in males, especially in the SG (P < 0.05). Conclusions: This preliminary in vitro study has shown gender differences in motor responses of colonic circular muscle strips in patients who had received chemotherapy with capecitabine and radiotherapy

Neurochemical characterization and distribution of enteric GABAergic neurons and nerve fibres in the human colon

GABA, somatostatin and enkephalin are neurotransmitters of enteric interneurons and comprise part of the intrinsic neural circuits regulating peristalsis. Within the relaxation phase of reflex peristalsis, nitric oxide (NO) is released by inhibitory motor neurons and perhaps enteric interneurons as well. Previously, we identified by GABA transaminase (GABA-T) immunohistochemistry, a subpopulation of GABAergic interneurons in the human colon which also contain NO synthase activity and hence produce NO. In this study, we have examined further the capacity for cotransmission within the GABAegic innervation in human colon. The expression of two important neuropeptides within GABAergic neurons was determined by combined double-labelled immunocytochemistry using antibodies for GABA-T, enkephalin and somatostatin, together with the demonstration of NO synthase-related NADPH diaphorase staining in cryosectioned colon. Both neuropeptides were found in GABAergic neurons of the colon. The evidence presented herein confirms the colocalization of NO synthase activity and GABA-T immunoreactivity in subpopulations of enteric neurons and further allows the neurochemical classification of GABAergic neurons of the human colon into three subsets: (i) neurons colocalizing somatostatin-like immunoreactivity representing about 40% of the GABAergic neurons, (ii) neurons colocalizing enkephalin-like immunoreactivity, about 9% of the GABAergic neurons and (iii) neurons colocalizing NO synthase activity, about 23% of the GABAegic neurons. This division of GABAergic interneurons into distinct subpopulations of neuropeptide or NO synthase containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and the pattern of transmitter release during reflex peristalsis. (C) 1998 Elsevier Science B.V