Correlation of electrophysiology, shape and synaptic properties of myenteric AH neurons of the guinea pig distal colon

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Abstract

Well-defined correlations between morphology, electrophysiological properties and the types of synaptic inputs received are established for myenteric neurons in the guinea pig ileum. However, in the distal colon, the correlations between AH electrophysiological properties, presence of fast excitatory post-synaptic potentials (EPSPs) and neuronal shape have been inadequately resolved and it is unknown whether any colon neurons receive synaptic inputs that generate sustained excitation. In this work, we have used intracellular recording, dye filling via the recording electrode, and immunohistochemistry to classify distal colon neurons. Neurons (24 of 168) had Dogiel type II morphology and 42% of these were dendritic type II neurons, compared to about 10% in the ileum. All Dogiel type II neurons had AH electrophysiological properties, including a prolonged post-spike after-hyperpolarization (AHP). None of these received fast excitatory post-synaptic potentials, 11 of 22 tested exhibited sustained slow post-synaptic excitation (SSPE) in response to 1 Hz pre-synaptic stimulation and 13 of 15 tested were immunoreactive for calbindin. Neurons (127) had Dogiel type I, filamentous or other uniaxonal cell shape and S type electrophysiology. Neurons of this group had fast excitatory post-synaptic responses to stimulation of synaptic inputs, but did not exhibit a prolonged post-spike after-hyperpolarization or sustained slow post-synaptic excitation. Another group of neurons (17) had both AH electrophysiological characteristics and fast excitatory post-synaptic potentials. These neurons had Dogiel type I, filamentous or other uniaxonal shapes, but none had Dogiel type II morphology and none showed sustained slow post-synaptic excitation.

It is concluded that Dogiel type II neurons are all AH neurons and are probably intrinsic sensory neurons that could be involved in long-term changes in excitability in the colon. All other neurons are monoaxonal; these are motor neurons and interneurons, and most are S neurons, electrophysiologically. A small number of monoaxonal neurons display AH electrophysiology and also receive fast excitatory synaptic inputs. These include motor and interneurons, but not sensory neurons.

Introduction

There has been steady progress in defining the types of neurons in the enteric nervous system and their places in enteric nerve circuits. A significant advance was to combine electrophysiological characterization with morphology and chemistry by using marker dyes to fill neurons that had been recorded from and subsequently to examine their shapes and immunohistochemical labelling Hodgkiss and Lees, 1983, Bornstein et al., 1984, Iyer et al., 1988, Messenger et al., 1994, Clerc et al., 1998, Lomax et al., 1999, Tamura et al., 2001.

Morphological classification of enteric neurons is based on the 1899 system of Dogiel (1899). One of the types he defined, neurons with large smooth-surfaced oval cell bodies and several long processes, are known as Dogiel type II neurons. In the guinea pig small intestine, these neurons are well defined by a number of electrophysiological and chemical features. They have AH electrophysiological characteristics Iyer et al., 1988, Clerc et al., 1998. That is, they have broad action potentials that are partly tetrodotoxin resistant, and they have prolonged after-hyperpolarizations (late AHPs) following single soma action potentials. In the ileum, these neurons very rarely exhibit fast excitatory post-synaptic potentials (EPSPs), and, when they are observed, fast EPSPs are of small amplitude Hirst et al., 1974, Iyer et al., 1988, Lees et al., 1992, Bornstein et al., 1994, Evans et al., 1994, Song et al., 1997. Stimulation of interganglionic connectives for extended periods (usually more than 1 min) at 1 Hz evokes sustained slow post-synaptic excitation (SSPE) in Dogiel type II but not in other neurons in the small intestine Clerc et al., 1999, Alex et al., 2002. The SSPE is associated with a sustained increase in excitability of the neurons, accompanied by a membrane depolarization and increase in input resistance which lasts for up to several hours Clerc et al., 1999, Alex et al., 2001. Until now, the SSPE has only been reported in the ileum. Dogiel type II neurons of the ileum, but not neurons of other shapes, are immunoreactive for calbindin (Iyer et al., 1988).

Two subpopulations of Dogiel type II cells, dendritic and adendritic, were described in the myenteric plexus of the porcine ileum (Stach, 1989), guinea pig ileum (Brookes et al., 1995) and guinea pig distal colon (Lomax et al., 1999). Functional differences between these two morphological subgroups of Dogiel type II neurons have not been found Brookes et al., 1995, Lomax et al., 1999, although the dendritic neurons have long anally projecting axons (Brookes et al., 1995).

All other neurons in the ileum have uniaxonal morphology and receive prominent fast excitatory synaptic input. Electrophysiologically, these are S neurons Hirst et al., 1974, Bornstein et al., 1994. A small subpopulation of uniaxonal neurons, the filamentous interneurons in the ileum and duodenum, have some properties in common with AH neurons; they have a Ca2+ component of the action potential and a delayed post action potential hyperpolarization (late AHP), although these features are less prominent than they are in AH neurons, and the AHP is sometimes only observed after a volley of action potentials Song et al., 1997, Clerc et al., 1998. Filamentous interneurons exhibit fast EPSPs of large amplitude. It has been suggested that some discrepancies in the literature have resulted from misidentification of filamentous interneurons as AH/Dogiel type II neurons (Brookes, 2001).

There has been less extensive investigation in other regions. In the distal colon of the guinea pig, the studies of Wade and Wood, 1988a, Wade and Wood, 1988b suggest the presence of fast synaptic transmission in type 2/AH neurons in the myenteric plexus. These authors did not correlate synaptic properties of neurons with their morphology. Correlations were made in two subsequent studies. Lomax et al. (1999) reported fast EPSPs in 3 of 29 Dogiel type II neurons and Tamura et al. (2001) reported that they were present in fewer than 3 of 37 Dogiel type II neurons. Both reports indicated that AH electrophysiology occurred in non-Dogiel type II neurons, but the shapes of these neurons were only partly described.

The major purpose of the present study was to determine the relationships between AH electrophysiological characteristics, types of synaptic input (especially fast EPSPs and SSPEs), neurochemistry and morphological subtype for Dogiel type II neurons of the distal colon. We had the further aim to investigate which neurons, other than Dogiel type II neurons, exhibit late AHPs and SSPEs.

Section snippets

Materials and methods

All experiments were performed on segments of distal colon removed from guinea pigs (150–275 g) after they were stunned by a blow to the head and killed by cutting the carotid arteries and severing the spinal cord. The animals were from the inbred Hartley strain colony of the Department of Anatomy and Cell Biology at the University of Melbourne. The University of Melbourne Animal Experimentation Ethics Committee approved all procedures. The segments (2–3 cm) were taken between 2 and 5 cm oral

Results

A total of 196 neurons were characterized electrophysiologically and then identified after intracellular injection of biocytin. Most of the neurons had sufficiently well-filled cell bodies and processes to be classified morphologically and the shapes and projections of 168 cells were determined after diaminobenzidine staining. Labelled cells were identified as multiaxonal Dogiel type II neurons (n=24, 14% of adequately filled neurons) and uniaxonal neurons (n=144) which included the following

Relation between Dogiel type II morphology and AH electrophysiology

Investigations of the electrophysiological properties of morphologically identified neurons, primarily in the small intestine of the guinea pig, led to the idea that AH electrophysiological properties corresponded to Dogiel type II morphology Iyer et al., 1988, Bornstein et al., 1994. This apparent correspondence was questioned when a prolonged AHP following the action potential was observed in somatostatin-immunoreactive filamentous descending interneurons of the guinea pig ileum (Song et al.,

Conclusion

The current study clarifies the relationships between electrophysiological properties, types of synaptic inputs received and morphologies of neurons in the myenteric ganglia of the distal colon. Neurons with Dogiel type II morphology are AH neurons, electrophysiologically. Activation of their synaptic inputs never elicited fast EPSPs, but could evoke sustained excitation. On the basis of their similarity to neurons in the ileum, they are suggested to be intrinsic sensory neurons. The prolonged

Acknowledgements

These studies were funded by the National Health and Medical Research Council (Australia). We thank Melanie Coffey and Varsha Lal for their excellent assistance with the immunohistochemical studies and preparation of the manuscript, especially the figures.

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