Synaptic cell adhesion molecule Cdh6 identifies a class of sensory neurons with novel functions in colonic motility

Abstract

Intrinsic sensory neurons are an essential part of the enteric nervous system (ENS) and play a crucial role in gastrointestinal tract motility and digestion. Neuronal subtypes in the ENS have been distinguished by their electrophysiological properties, morphology, and expression of characteristic markers, notably neurotransmitters and neuropeptides. Here we investigated synaptic cell adhesion molecules as novel cell type markers in the ENS. Our work identifies two Type II classic cadherins, Cdh6 and Cdh8, specific to sensory neurons in the mouse colon. We show that Cdh6+ neurons demonstrate all other distinguishing classifications of enteric sensory neurons including marker expression of Calcb and Nmu, Dogiel type II morphology and AH-type electrophysiology and I _H current. Optogenetic activation of Cdh6+ sensory neurons in distal colon evokes retrograde colonic motor complexes (CMCs), while pharmacologic blockade of rhythmicity-associated current I _H disrupts the spontaneous generation of CMCs. These findings provide the first demonstration of selective activation of a single neurochemical and functional class of enteric neurons, and demonstrate a functional and critical role for sensory neurons in the generation of CMCs.

Fig. 1.. Cdh6 expression overlaps with IPAN markers Calcb and Nmu.

(A, B) Representative images of jejunum (A) and distal colon (B) myenteric plexus labeled with HuC/D (IHC) (blue) and Cdh6 (RNA) (red). (C) Proportion of total HuC/D neurons positive for Cdh6 (jejunum, n = 9; distal colon, n = 9). (D, E) As in (A, B) for HuC/D (IHC) (blue), Cdh6 (RNA) (red), and Cdh8 (RNA) (green). (F) Proportion of Cdh6+ neurons positive for Cdh8 (jejunum, n = 8; distal colon, n = 8). (G) Proportion of Cdh8+ neurons positive for Cdh6 (jejunum, n = 8; distal colon, n = 8). (H) Proportion of total HuC/D neurons positive for Cdh8 (jejunum, n = 8; distal colon, n = 8). (I, J) As in (A, B) for HuC/D (IHC) (blue), Calcb (RNA) (red), and Nmu (RNA) (green). (K) Proportion of Nmu+ neurons positive for Calcb (jejunum, n =5; distal colon, n = 5). (L) Proportion of Calcb+ neurons positive for Nmu (jejunum, n = 5; distal colon, n = 5). (M, N) As in (A, B) for HuC/D (IHC) (blue), Cdh6 (RNA) (red), and Nmu (RNA) (green). (O) Proportion of Cdh6+ neurons positive for Nmu (jejunum, n = 7; distal colon, n = 8). (P) Proportion of Nmu+ neurons positive for Cdh6 (jejunum, n = 7; distal colon, n = 8). (Q) Proportion of total HuC/D neurons positive for Nmu (jejunum, n = 7; distal colon, n = 8). (R, S) As in (A, B) for HuC/D (IHC) (blue), Cdh6 (RNA) (red), and Calcb (RNA) (green). (T) Proportion of Cdh6+ neurons positive for Calcb (jejunum, n = 9; distal colon, n = 7). (U) Proportion of Calcb+ neurons positive for Cdh6 (jejunum, n = 9; distal colon, n = 7). (V) Proportion of total HuC/D neurons positive for Calcb (jejunum, n = 9; distal colon, n = 9). (W, X) Schematic of marker overlap in jejunum (W) and distal colon (X). Scale bar represents 100 μm for (A, B, I, J, M, N, R, S), 50 μm for (D, E). All charts (mean ± SEM). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Fig. 2.. Hb9:GFP+ is expressed in a small proportion of Cdh6+ colon myenteric neurons.

(A, B) Representative images of Hb9:GFP+ distal colon myenteric plexus labeled with HuC/D (IHC) (magenta) and GFP (green). (C) Proportion of total distal colon HuC/D neurons positive for GFP (n = 3). (D) Proportion of distal colon Cdh6+ neurons positive for eGFP (n = 3). (E) Proportion of distal colon eGFP+ neurons positive for Cdh6 (n=3). (F-H) Representative images of Hb9:GFP+ distal colon myenteric plexus labeled with HuC/D (IHC) (blue), Cdh6 (RNA) (red), and eGFP (RNA) (green). Scale bar represents 200 μm for (A, B), 50 μm for (E-G). All charts (mean ± SEM).

Fig. 3.. Hb9:GFP+ distal colon neurons have AH electrophysiological characteristics.

(A) IR videomicroscopy image of an Hb9:GFP distal colon neuron that presented a large soma located in a ganglion (scale bar, 10 μm). (B) Current-clamp recordings of the same neuron in (A) obtained in response to application of current pulse (bottom traces) of −50 pA, and + 10 pA. Note the presence of a sag and a post-hyperpolarization rebound depolarization. (C-E) Box-and-whisker plots of cellular properties of recorded neurons. (C) resting membrane potential (RMP), (D) capacitance (Cm), and (E) membrane resistance (Rm) (N = 5). (F) Averaged traces of the first spike (rheobase action potential) after a depolarization step of 1s. (J) Averaged derivative traces of the first spike (rheobase action potential). An inflection on the repolarizing phase is observed in the first derivative (arrow). (G-I, K-N) Box-and-whisker plots of electrophysiological properties of recorded neurons; rheobase action potential (AP, G-I) (G) current threshold, (H) half-width, (I) amplitude, (K) afterhyperpolarization (AHP), and (L) threshold. (M, N) non-AP properties sag (mV) and rebound (mV). (O) H and T currents in recorded neurons. Top: example of currents obtained from voltage protocol. Bottom: 500 ms hyperpolarizations ranging from −90 to −45 for 500 ms followed by depolarizing to −40 mV. Hyperpolarizations evoked slowly activating inward current (H-Current, arrow), followed by a transient inward current upon post-conditioning step to −30mV (T- current, arrow) Largest T and H currents were obtained with the most hyperpolarized potentials (red trace). (P) Normalized peak I_T plotted versus holding potential to obtain the I/Imax curve (N = 3/3). Scale bar represents 10 μm for (A).

Fig. 4.. Hb9:GFP+ distal colon neurons have circumferential branching projections.

(A-C) Representative images of Hb9:GFP+ distal colon myenteric plexus labeled with HuC/D (blue), streptavidin (red), and GFP (green). (D, E) Tracings of Hb9:GFP+ distal colon neurons filled with biocytin during whole cell patch clamp recording. (F) Image of patched and filled Hb9:GFP+ distal colon neuron traced in (E). (G, H) Inset of (F). Scale bar represents 40 μm for (A-C), 200 μm for (D-F), 100 μm for (G, H).

Fig. 5.. Cdh6^CreER+/ tdTomato+ neurons have Dogiel type II morphology.

(A, B) Representative images of Cdh6^CreER+;tdTomato+ distal colon myenteric plexus labeled with HuC/D (IHC) (magenta) and tdTomato (IHC) (green). (C) Inset of (A). (D) Dimensions of tdTomato+ neurons (major and minor axes) (N = 73; n = 3). (E) Representative image of Cdh6^CreER+;tdTomato+ distal colon myenteric plexus labeled with HuC/D (IHC) (blue), tdTomato (IHC) (red), and Cdh6 (RNA) (green). Arrowheads indicate Cdh6+ / tdTomato+ cells; arrowhead, Cdh6+ / tdTomato-negative cell. (F) Proportion of Cdh6+ distal colon neurons positive for tdTomato (n = 6). (G) Proportion of total HuC/D neurons positive for tdTomato (proximal colon, n = 5; mid colon, n = 5; distal colon, n = 10). Scale bar represents 100 μm for all images. All charts (mean ± SEM).

Fig. 6.. Optogenetic stimulation of distal colonic Cdh6+ neurons evokes CMCs, while pharmacologic blockade of I_H abolishes spontaneous CMCs

(A, B) Representative images of Cdh6^CreER+;ChR2-eYFP+ distal colon myenteric plexus labeled with HuC/D (magenta) and GFP (green). (C) Proportion of total distal colon HuC/D neurons positive for ChR2-eYFP (n = 6). (D) Representative force traces. Blue bars indicate timing of light stimulation. LEDs placed distal to distal hook. (E) CMC intervals recorded from force traces. Evoked (blue) intervals represent the time from the prior spontaneous CMC before stimulation to the evoked CMC following stimulation. Control (grey) intervals represent the time between the spontaneous CMC prior to stimulation and the previous spontaneous CMC (n = 5). Paired t test, one tailed. (F) CMC peak amplitude recorded from force traces. Evoked (blue) indicates the evoked CMC following stimulation. Grey (control) indicates the spontaneous CMC prior to stimulation (n = 5). Paired t test, two tailed. (G) CMC AUC (area under the curve). Evoked (blue) and control (grey) as in (F) (n = 5). Paired t test, two tailed. (H) CMC duration. Evoked (blue) and control (grey) as in (F) (n = 5). Paired t test, two tailed. (I) Representative force traces. Hex indicates addition of 300 μM hexamethonium. Blue bars indicate timing of light stimulation. LEDs placed distal to distal hook (n = 5/5). (J) Representative force traces on tethered pellets. First arrowhead indicates addition of 10 μM ZD7288. Second arrowhead indicates washout in Krebs. Third arrowhead indicates addition of 1 μM TTX. ZD7288 abolished CMCs in both proximal and distal colon (n = 6/6, p = 0.0022, Fisher’s exact test). Washout in Krebs restored CMCs in both proximal and distal colon (n = 6/6, p = 0.0022, Fisher’s exact test). (K) As in (J). First arrowhead indicates addition of 2 mM CsCl. Second arrowhead indicates washout in Krebs. Third arrowhead indicates addition of 1 μM TTX. Typical CMC production was impaired or altered by CsCl (proximal colon, n = 5/6, p = 0.0152; distal colon, n = 6/6, p = 0.0022, Fisher’s exact test): increased frequency (proximal colon, n = 5/6, p = 0.0152; distal colon, n = 6/6, p = 0.0022, Fisher’s exact test), decreased in amplitude (proximal colon, n = 5/6, p = 0.0152; distal colon, n = 4/6, p = 0.0606, Fisher’s exact test); retrograde force (proximal colon, n = 2/6; distal colon, n = 2/6). Scale bar represents 100 μm for (A, B). * p<0.05; ** p<0.01; *** p<0.001.