Quantification of neurons in the myenteric plexus: an evaluation of putative pan-neuronal markers

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Abstract

Accurate estimates of the total number of neurons located in the wall of the gut are essential for studies of the enteric nervous system (ENS). Though several stains and antibodies are used routinely as pan-neuronal markers, controversies of relative sensitivity and completeness have been difficult to resolve, at least in part because comparisons often must be made across experiments and laboratories. Therefore, we evaluated the efficacy of four putative pan-neuronal markers for the ENS, under comparable conditions. Neurons in the myenteric plexus of wholemounts taken from the small intestines of Fischer 344 rats were stained using Cuprolinic Blue, anti-HuC/D, anti-protein gene product 9.5, or FluoroGold injections followed by permanent labeling with an antibody to the FluoroGold molecule. All four markers had useful features, but both protein gene product 9.5 and FluoroGold were found to be problematic for obtaining reliable counts. As a result, only neurons labeled with either Cuprolinic Blue or anti-HuC/D were compared quantitatively. Based on counts from permanently labeled tissue, Cuprolinic Blue and HuC/D were similarly effective in labeling all neurons. Because the two protocols have different strengths and weaknesses, Cuprolinic Blue and HuC/D provide a complementary set of labels to study the total neuronal population of the ENS.

Introduction

The enteric nervous system (ENS) consists of a large number of neurons with diverse morphologies and neurochemical phenotypes (Furness, 2000) forming a complex neural network embedded in the wall of the gastrointestinal (GI) tract (Gershon, 1981). This plexus controls and coordinates complex GI functions such as motility and secretion, and evaluations of the neurons are critical to our understanding of the functional changes that occur during injury, disease, and age-related deterioration (e.g. Ardizzone and Porro, 2002; Jost, 1997, Sharkey and Kroese, 2001, Wade, 2002). To achieve a full understanding of how the ENS is affected by such conditions, a stain that specifically and completely labels all enteric neurons is needed.

Karaosmanoglu et al. (1996) have put forth the standard that a marker for a total population of ENS neurons must pass several criteria: first, the marker must be neuron specific (and, for example, not stain glia). Second, the marker must label all neurons. And, finally, the marker must be readily available to all investigators. A number of studies have addressed one or more of these issues and made recommendations as to which of several candidate markers provides universal, complete, and sensitive labeling of the ENS. Four of the most prominent and widely used candidate labels are: antibodies to protein gene product 9.5 (PGP 9.5; Krammer et al., 1993) and the HuC/D protein family (Hu; Lin et al., 2002), the neuronal stain Cuprolinic Blue (CB; Heinicke et al., 1987, Holst and Powley, 1995, Karaosmanoglu et al., 1996), and the tracer FluoroGold (FG; Powley and Berthoud, 1991).

At a first approximation, the four markers mentioned above fulfill the criteria of Karaosmanoglu et al. (1996). All are thought to be (a) neuron specific, (b) complete, and (c) readily available. No one study, however, has directly compared these four widely used markers under matched and standardized conditions. In addition, FG and Hu have only been used as fluorescent markers (e.g. Lin et al., 2002, Powley and Berthoud, 1991), so their utility as permanent labels has not been evaluated.

The goals of the present study, therefore, were (a) to evaluate, for each of the markers, whether the labeling was selective for neurons, (b) to determine, for the different markers, if they were complete, and (c) to determine which of the markers were most useful under some commonly used protocols.

Section snippets

Subjects

Virgin male Fischer 344 rats (3 months of age; n=24) were obtained from the National Institute on Aging colony maintained by Harlan Laboratory (Indianapolis, IN). Upon arrival, rats were group housed (n=3/cage) in polypropylene cages in a room maintained at 22–24 °C on a 12:12 h light:dark cycle. Solid chow (laboratory diet no. 5001; PMI Feeds, Inc., Brentwood, MO) and tap water were available ad libitum. All procedures were conducted in accordance with the NIH Guide for the Care and Use of

Results

Observations on the labeling of the four candidate markers indicated that only two of the labels, namely, CB and Hu, yielded reliable and essentially complete staining. For these two labels, the results of the quantitative trials are summarized in the next section (Section 3.1). Qualitative observations on all four markers examined are then described in the following section (Section 3.2).

Discussion

Counting myenteric neurons permanently labeled with either CB or Hu resulted in similar—though, in the case of two out of three investigators, not identical—estimates of neuronal number or density. In addition, both labels were selective for neurons (see discussion of Hu below for qualification), and complete in their labeling of the entire neuronal population. Conversely, PGP 9.5 and FG were poor markers for studies that require accurate counts of the entire neuronal population. PGP 9.5

Acknowledgements

Grant sponsors: NIH; award numbers: DK27627 and DK61317.

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