Optogenetic activation of mechanically insensitive afferents in mouse colorectum reveals chemosensitivity

Abstract

Feng B, Joyce SC, Gebhart GF. Optogenetic activation of mechanically insensitive afferents in mouse colorectum reveals chemosensitivity. Am J Physiol Gastrointest Liver Physiol 310: G790-G798, 2016. First published February 25, 2016 doi:10.1152/ajpgi.00430.2015.-The sensory innervation of the distal colorectum includes mechanically insensitive afferents (MIAs; ϳ25%), which acquire mechanosensitivity in persistent visceral hypersensitivity and thus generate de novo input to the central nervous system. We utilized an optogenetic approach to bypass the process of transduction (generator potential) and focus on transformation (spike initiation) at colorectal MIA sensory terminals, which is otherwise not possible in typical functional studies. From channelrhodopsin2-expressing mice (driven by Advillin-Cre), the distal colorectum with attached pelvic nerve was harvested for ex vivo single-fiber recordings. Afferent receptive fields (RFs) were identified by electrical stimulation and tested for response to mechanical stimuli (probing, stroking, and stretch), and afferents were classified as either MIAs or mechanosensitive afferents (MSAs). All MIA and MSA RFs were subsequently stimulated optically and MIAs were also tested for activation/ sensitization with inflammatory soup (IS), acidic hypertonic solution (AHS), and/or bile salts (BS). Responses to pulsed optical stimuli (1-10 Hz) were comparable between MSAs and MIAs whereas 43% of MIAs compared with 86% of MSAs responded tonically to stepped optical stimuli. Tonic-spiking MIAs responded preferentially to AHS (an osmotic stimulus) whereas non-tonicspiking MIAs responded to IS (an inflammatory stimulus). A significant proportion of MIAs were also sensitized by BS. These results reveal transformation as a critical factor underlying the differences between MIAs (osmosensors vs. inflammatory sensors), revealing a previously unappreciated heterogeneity of MIA endings. The current study draws attention to the sensory encoding of MIA nerve endings that likely contribute to afferent sensitization and thus have important roles in visceral pain. channelrhodopsin2; single fiber; silent afferents; irritable bowel syndrome; afferent sensitization; inflammatory soup SENSORY AFFERENTS INNERVATING the viscera have drawn significant research interest as clinical and preclinical evidence indicates that persistent afferent drive (e.g., afferent sensitization) contributes to long-lasting organ hypersensitivity, discomfort, and pain, hallmark complaints of patients with visceral pain disorders such as irritable bowel syndrome (IBS) (29,39,40) and bladder pain syndrome (27) for which a pathobiology is not apparent. Using an ex vivo colorectumnerve preparation together with different animal models of persistent colorectal hypersensitivity, we and others have documented long-term sensitization of mechanosensitive colorectal afferents that contribute to prolonged colorectal hypersensitivity (1, 13, 14).In addition, the colorectum is also innervated by a population of "silent" or "sleeping"...

Optical recording reveals topological distribution of functionally classified colorectal afferent neurons in intact lumbosacral DRG

“…From our prior single‐fiber recording of pelvic nerves (Feng et al. , ,, , ; Tanaka et al. ; La et al.…”

“…Keeping the colorectum in tubular shape prevents the characterization of mechanically‐insensitive afferents (MIAs), which were previously identified and characterized by us with focal electrical stimulation to the receptive fields in the flattened colorectum (Feng and Gebhart ; Feng et al. ). A recent study indicates those MIAs appear to express nicotinic acetylcholine receptor subunit alpha 3 (Chrna3) (Prato et al.…”

“…The high‐threshold (HT) muscular afferents that encode noxious colorectal distension (>25 mmHg) are putative nociceptors to evoke visceral pain (Feng et al. ). In the current study, the proportion of HT‐muscular afferents in all stretch‐sensitive afferents (25.8%) is in agreement with the range of 14–25% as reported by prior studies via single‐fiber recordings (Sengupta and Gebhart ; Feng et al.…”

“…The colorectum with attached S1, L6, and L5 spinal nerve and DRG was carefully dissected via blunt dissection, and transferred to a tissue chamber superfused with 32–34°C Krebs solution (in mmol/L: 117.9 NaCl, 4.7 KCl, 25 NaHCO 3 , 1.3 NaH 2 PO 4 , 1.2 MgSO 4∙ 7H 2 O, 2.5 CaCl 2 , 11.1 d ‐Glucose, 2 butyrate, 20 acetate, 0.004 nifedipine and 0.003 indomethacin) bubbled with carbogen (95% O 2 , 5% CO 2 ), consistent with our prior ex vivo studies on colorectal afferents (Feng and Gebhart ; Feng et al. ). The colorectum was cannulated and connected to a custom‐built distending device consisting of computer‐controlled fluid valves and hydrostatic pressure columns of 15, 30, 45, and 60 mmHg filled with phosphate buffered saline (PBS).…”

Visceral pain – Novel approaches for optogenetic control of spinal afferents

“…Recent studies have demonstrated the effectiveness of optogenetics in the selective activation of specific classes of visceral afferents in vitro (Feng et al, 2016). These studies showed that different functional classes of spinal afferents from the mouse large intestine can be identified by applying focal pulses of blue light to isolated segments of distal colon in vitro (Feng et al, 2016).…”