Optogenetic activation of mechanically insensitive afferents in mouse colorectum reveals chemosensitivity

Transgenic mice.

As described previously(41), male heterozygous Advillin-Cre mice were crossed with homozygous Ai32 female mice (C57BL/6 background). Ai32 mice carry the Channelrhodopsin-2 gene “ChR2(H134R)-EYFP” in the Gt(ROSA)26Sor locus, which is preceded by a LoxP-flanked STOP cassette to prevent its expression. By crossing Ai32 mice with Advillin-Cre mice, the Cre-expressing cell population will have the STOP cassette trimmed, resulting in expression of ChR2-EYFP. Male mice carrying both heterozygous Cre and ChR2-EYFP alleles (Cre^+/−ChR2^+/−) were used for all experiments with optical stimulation. Some additional experiments were done on male C57BL/6 mice (Taconic, Hudson, NY) to assess responses of MIAs to chemical stimuli.

Immunohistochemistry.

As previously described (14), mice (Cre^+/−ChR2^+/−) were killed via CO₂ inhalation. The distal colon and L6 dorsal root ganglion (DRG) were harvested and fixed with 4% paraformaldehyde in 0.16 M phosphate buffer containing 14% picric acid (Sigma-Aldrich). After cryoprotection in 20% sucrose, fixed tissue was embedded in OCT compound (Sakura Finetek, Tokyo, Japan), frozen, and sectioned at 20 μm. Tissue sections were incubated with a goat antibody against YFP (1:1,000; Abcam, Cambridge, MA) and the signals were further amplified by Alexa Fluor 488-conjugated anti-goat IgG (1:200; Molecular Probes, Eugene, OR).

In vitro mouse colorectum-nerve preparation.

As described previously (8, 11), male mice (Cre^+/−ChR2^+/−) 6- to 8-wk old and 20–30 g were killed via CO₂ inhalation followed by exsanguination after perforating the right atrium. The distal colorectum (∼2 cm) along with PN was dissected in ice-cold Kreb's solution (in mM: 117.9 NaCl, 4.7 KCl, 25 NaHCO₃, 1.3 NaH₂PO₄, 1.2 MgSO₄7H₂O, 2.5 CaCl₂, 11.1 d-glucose, 2 butyrate, 20 acetate, 0.004 nifedipine, and 0.003 indomethacin bubbled with carbogen (95% O₂-5% CO₂). The distal colorectum was transferred to a tissue chamber superfused with 32–34°C Krebs solution, opened longitudinally, and pinned flat mucosal side up. The attached PN was placed in the adjacent recording chamber filled with mineral oil (Fisher Scientific) and teased into fine bundles of ∼10-μm thick for single-fiber electrophysiological recordings by a platinum-iridium recording electrode (FHC, Bowdoin, ME).

Afferent identification and classification.

A round-tipped concentric electrode (external diameter: 0.55 mm; internal diameter: 0.125 mm; FHC) was placed perpendicular to the colorectal surface to electrically excite afferent endings (0.5-ms current pulse duration at 0.3 Hz) (8). The tip of the electrode surveys the entire colorectal surface in ∼1.5-mm steps at a suprathreshold stimulus intensity (10 mA) sufficient to excite all afferent endings within a, ∼2-mm radius. The afferent receptive field (RF) was identified as the site of activation requiring minimum stimulus intensity (i.e., stimulus threshold). After location by electrical stimulation, each RF was tested with three distinct mechanical stimuli: probing with calibrated von Frey-like nylon monofilaments (0.4 and 1-g force), mucosal stroking with calibrated nylon filaments (10-mg force), and circumferential stretch (0–170 mN at 5 mN/s, Aurora Scientific, Ontario, Canada).

Optical stimulation of colorectal afferents.

Optical stimulation was applied using a 473-nm Laser (Laserglow Technologies, Toronto, Canada) focused to a point ∼1 mm in diameter on the colorectal mucosal surface, an area comparable to a typical afferent RF. Stepped light stimulation (10 s, constant intensity) was applied to the RF, followed by 5 trains of 20 light pulses (20-ms wide) in ascending frequency (1, 2, 3, 5, and 10 Hz). Light intensity was 6 mW/mm², well in the saturation range for afferent activation (41). Spike2 software was used to control the timing of the optical stimuli (Cambridge Electronic Design, Cambridge, UK).

Chemical stimulation of MIAs.

MIAs that responded to optical stimulation were tested by sequential exposure of their afferent endings to an acidic hypertonic solution (AHS) and an inflammatory soup (IS), each of which has been used previously on colorectal afferent endings (7, 8, 12, 20, 21). Brass tubing of 1-cm high × 4-mm square, which has been shown to be chemically inert to AHS, IS, and bile salts (BS), was placed over the receptive ending on the mucosal surface, the Krebs was solution removed, and 150 μl of AHS solution were applied directly to the receptive ending for 5 min. Subsequently, the solution and the tubing were removed and responses monitored for up to 30 min. The endings were then tested with IS using the same protocol followed by tests of responses to mechanical probing (1 g) and stepped optical stimulation. Electrical stimulation was repeated at the end of testing to verify viability of the afferent ending. Additional MIAs harvested from naïve C57BL/6 mice were tested by exposing their endings to IS, AHS, and a BS solution.

AHS (pH 6.0, 800 mosM) was prepared by adding d-mannitol and hydrochloric acid (1 N) to freshly oxygenated Krebs solution on the day of an experiment. IS was prepared in aliquots of 20 μl by combining bradykinin, serotonin, and histamine dissolved in distilled water with prostaglandin E₂ dissolved in dimethylsulfoxide, frozen, and stored at −20°C. On the day of an experiment, an aliquot was diluted to 10 μM (each mediator) in freshly oxygenated Krebs solution and adjusted to pH 6.0 by hydrochloric acid. BS solution (0.5%) were prepared by dissolving the salts in freshly oxygenated Krebs solution (8). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Data recording and analysis.

Electrical signals from afferent endings were filtered (0.3–10 kHz), differentially amplified (×10,000; DAM 80; World Precision Instruments, New Haven, CT), digitized at 20 kHz using a 1401 interface (Cambridge Electronic Design), stored on a PC, and monitored online by an audio monitor. Action potentials were discriminated based on principal component analysis using Spike2 software (Cambridge Electronic Design). Data are presented as means ± SE. One- or two-way ANOVA and Bonferroni post hoc pairwise multiple comparisons were performed with repeated measures as appropriate using SigmaPlot v11.0 (Systat Software, San Jose, CA). Differences in means and proportions were assessed by t-tests and χ²-tests, respectively. P < 0.05 was considered significant.

Optogenetic activation of colorectal afferents.

As shown in Fig. 1, colorectal afferents in the PN innervation in male Cre^+/−ChR2^+/− mice were localized by electrical stimulation of their receptive endings and classified into one MIA class and four mechanosensitive afferent (MSA) classes: muscular, muscular-mucosal, mucosal, and those responding only to blunt probing (formerly termed “serosal”) afferents. MIAs do not respond to any of the three mechanical stimuli (punctate probing, mucosal stroking, or circumferential stretch). In contrast, all four MSA classes respond to blunt probing of their RF; in addition, muscular and muscular-mucosal afferents also respond to circumferential stretch and mucosal and muscular-mucosal afferents also respond to mucosal stroking. The terms “mucosal” and “muscular” are suggestive of histological localization of RFs in the colorectum, for which histological support exists (36). However, they have not yet been extensively validated by studies that couple functional characterization with afferent ending localization. Colorectal afferents that respond only to punctate probing of their RF were previously denoted “serosal” afferents, suggestive of a RF in the serosa. However, a recent morphological study of the mouse colorectum failed to identify sensory endings in the colorectal serosa (36), prompting the use of “probing only” to represent this class of afferent here.

Confirming our previous findings (41), ChR2 fused with a YFP protein, detected in male Cre^+/−ChR2^+/− mice using a YFP antibody, was expressed in PN primary afferent neurons in both the L6 dorsal root ganglion (Fig. 2A) and colorectum (Fig. 2B). Sixty-three colorectal afferents were identified from 14 male Cre^+/−ChR2^+/− mice and the proportions of the five afferent classes (Fig. 2C) were comparable to those reported previously from naïve C57BL/6 mice(8) (χ²-test, P = 0.12). Optical stimulation was equally effective in exciting MIAs and MSAs, activating >79% of RFs in both groups (Fig. 2D). Laser-responsive (L-R) afferents had significantly slower mean conduction velocities than their laser nonresponsive (L-NR) counterparts in MSA groups (t-test, P = 0.02) as shown in Fig. 2E. Laser-responsive MIAs and MSAs have comparable mean conduction velocities (t-test, P = 0.75).

Responses of MSAs and MIAs to optical stimuli.

As shown in Fig. 3A, after identification of afferent RFs by electrical stimulation, colorectal afferents were further tested with stepped (10-s duration) and pulsed (20-ms duration, 1 Hz) laser stimulation of their RFs on the colorectum. Consistent with our previous findings in naïve C57BL/6 mice, MIAs in the present study were found to have a significantly higher threshold for activation by electrical stimulation than MSAs (Fig. 3B, t-test, P = 0.004). As displayed in Fig. 3C, 57% of MIAs responded to a stepped laser stimulus with a single spike (single-spiking; e.g., Fig. 3A, top trace), a proportion significantly greater than of MSAs (14%, Fisher's exact, P = 0.015). The majority of MSAs (86%) were tonic-spiking, responding to a stepped laser stimulus with multiple spikes (e.g., Fig. 3A, bottom trace). We stimulated 16 single-spiking afferents (4 MIAs and 12 MSAs) at three different light intensities (threshold, 5 times threshold, and 6 mW/mm²) which uniformly evoked single spiking responses from all afferents, validating the optical intensity of 6 mW/mm² for testing the stepped response. When stimulated for 20 pulses at 1 Hz, pulsed laser stimulation evoked a comparable number of action potentials in MIA and MSA groups (Fig. 3D, t-test, P = 0.66). Comparing MSA classes with MIAs stimulated with a range of frequency pulses (1, 2, 3, 5, and 10 Hz, 20 pulses each; Fig. 4A), low-frequency stimulation reliably evoked action potentials from all classes of colorectal afferents with >70% activation by laser pulses. The proportions of activation to pulsed laser stimulation were reduced significantly and to an equivalent extent in MIAs and all four classes of MSAs to <30% as the stimulus frequency increased progressively to 10 Hz (Fig. 4B, two-way repeated-measures ANOVA, F_4,49 = 0.667, P = 0.618).

Responses of MIAs to chemical stimuli.

Differential responses to stepped laser stimuli provided direct evidence for heterogeneity within the MIA population. We hypothesized that tonic-spiking MIAs are regular sensors of another stimulus modality, capable of informing the CNS about the quality of that modality, whereas single-spiking MIAs are putative nociceptors that require sensitization to spike tonically. We thus exposed individual MIA RFs to AHS (an osmotic stimulus; 5-min application) and, after a 30-min washout, IS (an inflammatory/sensitizing stimulus; 5-min application), followed by punctate probing (1 g) of the RF and repeated stepped laser stimulation. Eleven laser-responsive (6 single-spiking and 5 tonic-spiking) MIAs from Cre^+/−ChR2^+/− mice were tested in this manner. Figure 5A illustrates characteristic responses of single- and tonic-spiking MIAs, and Fig. 5, B and C, summarize sthe results. Single-spiking MIAs (5/6) were activated by IS, but not AHS whereas 5/5 tonic-spiking MIAs responded to AHS, two of which also responded to IS (Fig. 5C). Virtually all MIAs (10/11) were sensitized by exposure to AHS and/or IS and acquired mechanosensitivity (responded to blunt probing), and all single-spiking MIAs became tonic spiking to stepped laser stimuli (6/6) after exposure to chemical stimuli. Results presented in Fig. 5, B and C, collectively suggest the segregation of MIAs into two subgroups, one capable of spiking tonically and responsive to AHS whereas the other being single-spiking and responsive to IS.

Responses to chemical stimuli were tested in a larger population of MIAs from C57BL/6 control mice. As illustrated by the typical example in Fig. 6A, MIA endings were exposed to either AHS, IS, or a solution of BS for 5 min to assess direct activation; activation latency (time to response) and duration of response were quantified for comparison between chemical applications. Similar to findings from Cre^+/−ChR2^+/− mice, about half the MIAs tested (56%, 9/16) were activated directly by AHS, and 7 of the 16 MIAs tested (44%) acquired mechanosensitivity (Fig. 6B). About one-third of MIAs tested (37%, 15/41) were directly activated by IS and 68% acquired mechanosensitivity. BS activated a comparatively smaller proportion of MIAs (22%, 2/9), but most MIAs acquired mechanosensitivity afterwards (67%, 6/9). BS had the longest activation latency among the three chemical stimuli (Fig. 6C, F_2,23 = 3.7, P = 0.04, post hoc comparison, P = 0.037 vs. AHS), and activation by AHS had the longest duration (Fig. 6D, F_2,23 = 13.5, P < 0.001, post hoc comparison P < 0.05 vs. IS or BS).