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Nitrergic (NO) neurons play crucial inhibitory roles in the control of gut motility. Variations in the density of these neurons within the gastrointestinal tract (GI) may provide useful functional information, but, most surveys available have employed limited and/or highly localized samples. It remains unclear to what extent (a) NO neurons are concentrated disproportionately in particular GI regions, or (b) variations in NO cell number merely reflect changes in overall myenteric neuron density. This experiment surveyed the distributions of neuronal nitric oxide synthase-positive (NOS+) and other myenteric neurons in the GI tract, using immunohistochemical and Cuprolinic blue counterstaining techniques. Adjustable sampling grids superimposed on wholemounts were used to investigate the topographic patterns in the stomach (90 sampling sites; 45 per side) and proximal duodenum (63 loci). We present four major findings: First, variations were detected in the number of NOS+ neurons in specific regions of the stomach (e.g., corpus > antrum approximately equal to forestomach) and along both longitudinal (oral > anal) and circumferential (mesenteric > antimesenteric) axes in the duodenum. Second, the variations in NOS+ neuronal counts within each organ covaried with the total number of myenteric neurons at different locations (stomach, r=0.77; duodenum, r=0.59), suggesting that local myenteric plexus density is a factor determining NOS+ cell concentrations. Third, in contrast to such a principle of covariation within each organ, NOS+ neurons constituted a consistently smaller proportion of gastric (20%) than of duodenal (28%) myenteric plexus neurons, suggesting that a second principle controls the characteristic percentages of the myenteric plexus that express NOS in different organs. Fourth, the regional samples were used to extrapolate the overall number of NOS+ and total myenteric cells in the rat stomach (43,000; 217,000) and first 3.5 cm of the small intestine (29,000; 103,000). These results, taken together, also suggest that the surveying protocol used is capable of detecting subtle differences in cellular distributions, thus providing a practical strategy for investigating patterns of chemical phenotypes within the GI tract.

The dorsal motor nucleus of the vagus (DMNX) contains neurons with different projections and discrete functions, but little success has been achieved in distinguishing the cells cytoarchitectonically. The present experiment employed multivariate analytical techniques to evaluate DMNX neuronal morphology. Male Sprague-Dawley rats (n = 77) were perfused, and the brainstems were stained en bloc with a Golgi-Cox protocol. DMNX neurons in each of three planes (coronal, sagittal, and horizontal; total sample = 607) were digitized. Three-dimensional features quantified included dendritic length, number of segments, spine density, number of primary dendrites, dendritic orientation, and soma form factor. Cluster analyses of six independent samples of 100+ neurons and of three composite replicate pools of 200+ neurons consistently identified similar sets of four distinct neuronal profiles. One profile (spinous, limited dendrites, small somata) appears to correspond to the interneuron population of the DMNX. In contrast, the other three distinctive profiles (e.g., one is multipolar, with large dendritic fields and large somata) are different types of preganglionic neurons. Each of the four types of neurons is found throughout the DMNX, suggesting that the individual columnar subnuclei and other postulated vagal motorneuron pools are composed of all types of neurons. Within individual motor pools, ensembles of the different neuronal types must cooperatively organize different functions and project to different effectors within a target organ. By extension, specializations of the preganglionic motor pools are more likely to result from their afferent inputs, peripheral target tissues, neurochemistry, or physiological features rather than from any unique morphological profiles.

Potential effects of environmental rearing conditions on the brains of farm animals have not been examined experimentally, with the exception of one report for pig somatosensory cortex. The goal of the present experiment was to determine whether different developmental environments in use in agricultural production units affect neuronal morphology in the pig cerebral cortex. Littermate female pigs (gilts) were cross-fostered at birth and reared in either an indoor (n = 6) or outdoor (n = 6) production unit for 8 weeks. Additional littermates (n = 6) were sacrificed at 3 days of age to provide a developmental reference point. Brains were fixed by perfusion and stained by the Golgi-Cox method. The primary somatosensory, auditory and visual cortices were sectioned at 170 microns, and layer IV stellate neurons (n = 492) were digitized and 3-dimensionally reconstructed. Measurements of dendritic length, membrane surface area, total number of segments, number of 1st- through 7th-order dendrites, spine density, soma area, and soma form factor were taken. In auditory cortex neurons, outdoor pigs compared to indoor pigs had (a) significantly more primary dendrites, (b) significantly greater spine density, and (c) trends of increases both in number of 2nd- and 3rd-order dendrites and in total dendritic length. In visual cortex neurons, indoor pigs had significantly more 7th-order dendrites, whereas in all three cortical areas, the indoor animals had more 5th-order dendrites. Multiple morphological differences occurred in stellate cell populations between the three sensory areas of the Week 8 pigs. Also, within different cortical areas, dendritic morphology changed substantially from 3 days to 8 weeks of age. Further investigations are needed to determine which environmental factors are critical in producing the observed changes in brain morphology and whether other brain effects may be produced by varying developmental environments.

This report details a method for the use of Cuprolinic Blue (quinolinic phthalocyanine) as a counterstain for immunoperoxidase Phaseolus vulgaris (PHA-L)-labeled fibers in gastrointestinal wholemounts. Cuprolinic Blue (in the presence of high MgCl2 concentration) forms a particularly stable blue chromophore with single-stranded RNA in neuronal Nissl substance and nucleoli. In contrast to the non-specific staining provided by traditional neuronal dyes. Cuprolinic Blue produces prominent enteric neuron staining with minimal coloration of the muscle and connective tissue surrounding the ENS plexuses. However, this specific staining pattern is not obtained when the dye is used as a neuronal counterstain for immunoperoxidase-labeled material unless it is applied before the DAB chromagen. The counterstaining of gut wholemounts is optimized by using 0.5% Cuprolinic Blue at 37 degrees C. A lower concentration of the dye and temperature is useful for the concomitant delineation of the PHA-L immunoperoxidase-labeled injection sites in brainstem sections. The protocols outlined for Cuprolinic Blue counterstaining of both enteric and central nervous system material are also appropriate for the localization of other immunocytochemically identified antigens.

The motor outflow of the autonomic nervous system (ANS) is differentiated into two major divisions, parasympathetic (PSNS) and sympathetic (SNS). Both are organized hierarchically into pre- and postganglionic levels, but classically the two divisions have been assumed to differ in their ratios of pre- to postganglionic neurons. The PSNS been characterized as having lower ('one-to-few') ratios, whereas the SNS has been described as possessing higher ('one-to-many') ratios. These patterns have been assumed to measure differing divergences of the outflows. In this review, a ratio of pre- to postganglionic neurons is called a ratio index, and the idea that the PSNS and SNS have characteristically different ratio indexes and divergences is called the ratio rule. The putative differences in the ratio indexes of the two divisions - as well as Fulton's influential proposal that they form one of the bases of contrasting functional capacities of the PSNS and SNS - have been widely accepted for nearly for nearly three quarters of a century. A survey of the original observations yielding the concept of the ratio rule as well as the more recent estimates of pre- and postganglionic numbers, however, challenges both the generality and the adequacy of the ratio rule and indexes. The originally formulated differences between the PSNS and SNS represent an overgeneralization since they were based on observations of only two ganglia, the ciliary ganglion in the PSNS and the superior cervical ganglion in the SNS. Furthermore, these original estimates were based on limited samples and were subject to a number of counting artifacts. A survey of the literature suggests that ratio indexes vary much more within each ANS division than they do between the two divisions. When ganglia other than the ciliary and superior cervical are examined, the two divisions of the ANS have broad, largely overlapping ranges of ratio indexes. Additionally, other PSNS-SNS pairs can be found in which the relative sizes of their respective indexes are completely contrary to the ratio rule. For a given ganglion, there are substantial differences in the ratio index between species, between individuals of the same species, and between stages of development in the same species. Furthermore, both divisions of the ANS have wide and largely overlapping ranges of physiological effects varying from specific to diffuse, from local to widespread. Finally, the ratio index measure ignores the degree of convergence found in different ganglia, and it is insensitive to the fact that many ganglia have multiple functionally distinct motor neuron pools, each with separate inputs varying in their degrees of divergence and/or convergence. Thus ratio indexes do not differentiate the PSNS from the SNS, and conclusions based on such putative distinctions are questionable.

To determine the degree of variation of neuronal morphology both within and between the subnuclei of the dorsal motor nucleus of the vagus (dmnX), structural features of the preganglionic neurons of each of the five primary subnuclei in the rat dmnX were characterized quantitatively. Each of the columnar subnuclei was separately labeled by application of the retrograde tracer fast blue to its corresponding subdiaphragmatic vagal branch. Fixed brain slices of 100 microns thickness were then prepared in coronal, sagittal, and horizontal orientations. Next, randomly selected fast blue labeled neurons (n = 1,256) were injected with Lucifer yellow, drawn with camera lucida, and digitized. For each cell, three features of the perikaryon and twelve of the dendritic tree were measured. Dorsal motor nucleus neurons with up to eight primary dendrites, 30 dendritic segments, and seventh order dendritic branches were observed. Throughout the dmnX, the dendrites of preganglionic neurons were preferentially oriented in the horizontal plane. Consistent with an organizing role for the columnar subnuclei, most dendrites remained within their column of origin. However, between 5 and 30% of the neurons in each of the columns projected dendrites into adjacent dmnX subnuclei or other brainstem nuclei, including the nucleus of the solitary tract (NTS). The cyto- and dendroarchitectural analyses revealed systematic gradations in morphology, although they did not support the idea that the dmnX was composed of multiple distinct preganglionic types. The most parsimonious interpretation of the data is that dmnX motorneurons are variants of a single prototype, with dendrites varying widely in length and degree of ramification. The extent of an individual preganglionic neuron's dendritic field was predicted by three factors: the cell's rostrocaudal position within the dmnX, its location within a transverse plane (i.e., its coronal position within or ectopic to the dmnX), and its subnucleus of origin. Neurons at rostral and midlongitudinal levels of each column had more extensive dendritic arbors than those at caudal levels. Ectopic neurons had more extensive dendritic fields than similar cells in the corresponding columns; in fact, of all vagal preganglionic neurons, ectopics had the most extensive dendritic fields. Somata and dendrites of celiac column neurons were more extensive than those of hepatic and gastric column cells. These differential regional distributions of vagal preganglionics suggest that their structure and function are correlated.

Vagal preganglionic neurons innervate myenteric ganglia. These autonomic efferents are distributed so densely within the ganglia that it has been impractical to track individual vagal axons through the myenteric plexus with tracer labeling. To evaluate whether vagal efferent axons evidence selectivity, particularly for nitrergic or non-nitrergic myenteric neurons within the plexus, we limited the numbers and volumes of brainstem dextran biotin tracer injections per animal. Reduced labeling and the use of immunohistochemistry generated cases in which some individual axons could be distinguished and traced in three dimensions (Neurolucida) within and among successive (up to 46) myenteric ganglia. In the myenteric plexus of all stomach regions, the majority (~86%) of vagal efferents were organized into two distinct subtypes. One subtype (~24% of dextran-labeled efferents, designated "primarily nitrergic") selectively contacted and linked--both within and between ganglia--nitric oxide synthase positive (nNOS+) neurons into presumptive motor modules. A second subtype (~62% of efferents, designated "primarily non-nitrergic") appeared to selectively contact and link--both within and between ganglia--non-nitrergic enteric neurons into a second type of effector ensemble. A third candidate type (~14% of labeled preganglionics), appeared to lack "nitrergic selectivity" and to contact both nNOS+ and nNOS-enteric neurons. In addition to the quantitative assessment of the efferent axons in stomach, qualitative observations of the proximal duodenum indicated similar selective vagal efferent projections, in proportions comparable with those evaluated in the stomach. Limited injections of tracer, three-dimensional (3-D) tracing of individual axons, and histochemistry of myenteric neurons might distinguish additional efferent phenotypes. [ABSTRACT FROM AUTHOR]

To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC-IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran-labelled diaminobenzidin (DAB)-stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC-IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC-IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC-IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC-IM processes, indicating communication between ICC and IMA via synapse-like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA-ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach’s plexus were encountered. [ABSTRACT FROM AUTHOR]