Interstitial Cells of Cajal Mediate Cholinergic Neurotransmission from Enteric Motor Neurons

A, D, IC-IM and smooth muscle cells are of similar spindle-like shape in the circular muscle layer of the fundus. These cells have distinct ultrastructural features. IC-IM (ic) contain numerous mitochondria (m), smooth and rough endoplasmic reticulum, dense heterochromatic nuclei, and few myofilaments and dense bodies. Smooth muscle with typical ultrastructural features surrounds the IC-IM. IC-IM and enteric neurons with vAChT-LI form intimate contacts as revealed by immunoelectron microscopy. B, An IC-IM within the circular muscle layer (cm) is shown. Areas of close, synaptic-like structures (arrow) exist between vAChT-containing nerve terminals (asterisks) and IC-IM. C, The region denoted by the arrow in B is shown in higher power.

IC-IM and enteric neurons with NOS-LI form intimate contacts as revealed by immunoelectron microscopy.A, An IC-IM (ic) within the circular muscle layer (cm) is shown. IC-IM were readily identified by the large number of mitochondria (m), rough endoplasmic reticulum, and free ribosomes. NOS-LI in enteric nerve terminals is observed as a dense DAB reaction product (asterisk). A, C, A close, synaptic-like region of membrane exists between a NOS-LI nerve terminal and IC-IM (shortarrow; at higher magnification in C). A, B, The IC-IM in A also forms a gap junction with a neighboring smooth muscle cell (longarrow; at higher power in B).

Differences in responses to EFS in wild-type andW/W^v mutant animals.A, EFS (arrowhead; 0.5 msec pulse supramaximal voltage) produced a biphasic electrical response in wild-type muscles, characterized by a rapid EJP. The EJP was followed by an IJP. B, l-NA (100 μm) reduced the IJPs and increased the amplitudes of the EJPs.C, After l-NA, atropine (1 μm) completely blocked the EJPs, suggesting that muscarinic receptors mediated the EJP responses. D, InW/W^v mutant animals, EJPs and IJPs were greatly attenuated. E, F, l-NA had little or no effect on responses to EFS (E), and after l-NA, atropine had no effect (F). G, H, The effects ofl-NA on EJPs and IJPs from experiments on muscles of wild type (n = 17; filledverticalbars) and ofW/W^v mutants (n = 13; openverticalbars) are summarized in G and H,respectively.

Pulses of EFS of different durations (0.1–0.75 msec) altered responses of gastric fundus muscles. A–C, Increasing the duration of pulses (arrowheads) potentiated the amplitude of EJPs in wild-type tissues (A–C for pulse durations of 0.1, 0.3, and 0.5 msec, respectively). D–F, EFS of the same pulse duration had no effect in W/W^v muscles (D–F for pulses of 0.1, 0.3, and 0.5 msec, respectively). G, The electrical responses to EFS as a function of pulse duration in wild type before (filledtriangles) and after (filledcircles) addition ofl-NA (100 μm) are summarized. Responses were greatly diminished in W/W^v muscles using the same stimulus parameters before (opentriangles) and after (opencircles) l-NA.

Mechanical responses to EFS in wild-type and mutant gastric tissues. A–C, Isometric contractions to single pulses of EFS (arrowheads; 0.1, 0.5, and 0.75 msec, respectively) are shown. D–F, Mechanical responses of a W/W^v muscle using the same stimulus parameters are shown. G, Mechanical responses to EFS of wild-type muscles before (filledtriangles) and after (filledcircles) l-NA (100 μm) are summarized. Responses ofW/W^v muscles were greatly attenuated using EFS with the same stimulus parameters. A summary of responses ofW/W^v muscles to EFS before (opentriangles) and after (opencircles) l-NA are shown.

Electrical and mechanical responses of wild-type and W/W^v muscles to exogenous ACh. Application of ACh (arrowheads) tonically depolarized muscles in a concentration-dependent manner. A,C, Responses of wild-type muscles to 0.3 and 1.0 μm, respectively. B,D, Responses of W/W^vmuscles to the same doses. Acetylcholine caused contractions of wild-type and W/W^v muscles.E, The mechanical response of a wild-type muscle to 1.0 μm ACh. F, The response of aW/W^v muscle to the same concentration of ACh. Many W/W^v muscles displayed oscillations in tone superimposed on the tonic contraction. This type of response also occurred, but more rarely in wild-type muscles.G, A summary of the responses of wild-type (filledcircles;n = 5) and W/W^v(opencircles; n = 5) muscles to ACh (0.01–10 μm).

Inhibition of endogenous cholinesterase with neostigmine revealed a slowly developing membrane depolarization in wild-type and W/W^v mutant tissues.A, The electrical responses of wild-type muscles to EFS (arrowheads; single pulses of 0.1, 0.3, and 0.5 msec duration) before (left) and after (middle) l-NA (100 μm) and after l-NA with neostigmine (0.5 μm;right) are shown. l-NA blocked IJPs and potentiated EJPs. In the presence of l-NA, neostigmine increased the amplitude of the fast EJP and revealed a slowly developing, second depolarization (arrows).B, Responses of W/W^vmuscles to EFS were negligible under control conditions (left) and after l-NA (middle). However, neostigmine added afterl-NA revealed a slowly developing membrane depolarization in response to EFS (right) that was similar in detail and time course to the second depolarization response of wild-type muscles (arrows).

Electrical responses of wild-type andW/W^v muscles to EFS.A–D, Multiple pulses of EFS (arrowheads; 1–10 pulses delivered within 100 msec) caused pronounced EJPs and IJPs in wild-type muscles (1–10 pulses as labeled). E–H, EJPs were not observed inW/W^v muscles; however 3–10 pulses (delivered within 100 msec) caused slowly developing hyperpolarization responses in these tissues. l-NA had no effect on the IJPs elicited in W/W^v muscles with 3 or more pulses. I, Data for the effect of l-NA on control and W/W^v mutant tissues are summarized. EJPs elicited in wild-type muscles (filledtriangles) were potentiated by l-NA (100 μm;filledcircles), whereas EJPs were absent in W/W^v muscles (opentriangles) and not revealed by l-NA (opencircles).

[¹⁴C]Choline release from wild-type and W/W^v muscles in response to EFS. A, The typical [¹⁴C]choline overflow from a wild-type fundus muscle in response to EFS is shown. Tissues were stimulated at 40 min intervals for 1 min (5 Hz; 0.1-msec-duration pulses;filledhorizontalbar), and [¹⁴C]choline overflow was assayed from superfusion samples. [¹⁴C]Choline overflow is plotted as a percentage of fractional release with time of collection. Overflow decreased with subsequent stimulations (S₁, discarded; S₂,opencircles; S₃,filledtriangles; S₄,opensquares; S₅,filleddiamonds).B, The overflow of [¹⁴C]choline in response to EFS (opencircles) was inhibited by TTX (1 μm; n = 9 tissues of 3 animals; filledtriangles), and this was reversible after washout (filledcircles). C, D, EFS caused release of similar amounts of [¹⁴C]choline from wild-type (C) orW/W^v (D) muscles (p > 0.05). [¹⁴C]Choline release under control conditions (opencircles, S₂) was not affected by l-NA (100 μm;filledtriangles, S₃) but was slightly increased by neostigmine (0.5 μm;opensquares, S₄).