The presence of nitric oxide synthase (the enzyme which synthesizes nitric oxide), vasoactive intestinal peptide (VIP) and pituitary adenylyl-cyclase-activating peptide (PACAP) in the rat enteric nervous system between term (E18) and 90 days postpartum (P90) was investigated by immunohistochemistry. Neuronal maturity was ascertained by emanining for the presence of two novel neuron intermediate filament proteins: alpha-internexin, which is transiently expressed in developing neurons, and peripherin, which is expressed in differentiating as well as in mature neurons. The alimentary canal of the foetus at E18, unfed newborn, suckling, weaning and adult rats was studied. Throughout the rat gut the myenteric neurons were recognized by the time the foetal stage was reached and the submucous neurons by the suckling period. alpha-Internexin was present at E18; its levels increased during ageing and were markedly reduced at P30. Peripherin was first detected at birth; its levels increased with ageing and practically almost all of the neurons and nerve fibres were labelled from P30. Nitric oxide synthase was present at E18 and the number of labelled neurons gradually increased with ageing, while both VIP and PACAP could be detected by the end of the suckling period in an equal number of neurons to that in adulthood. These data indicate that in the rat gut the onset of the mature neuronal phenotype is not yet achieved at birth and that the neurochemical differentiation is accomplished during the first month of postnatal life.

The stomach, small and large intestine of fetuses at term, of unfed newborns, of suckling, weaning and of adult rats were studied by a combined light (LM) and electron microscope (EM) examination. Neuron-specific enolase was used as a neuronal marker under LM. Zinc-iodide-osmium (ZIO) impregnation was used for a selective staining of neurons and interstitial cells of Cajal under both LM and EM. A routine EM procedure made it possible to identify the nerve elements and ICC and to evaluate their degree of differentiation. The differentiation of enteric plexuses and ICC was poor at birth and was accomplished during the weaning period. The myenteric plexus differentiation preceded the submucous plexus differentiation; in particular, under both LM and EM myenteric neurons were already recognizable in the fetus, while the submucous neurons by day 0 under EM and by day 7 under LM. The ICC were poorly differentiated at birth and acquired the adult morphology during the suckling period. Nerve endings contacting ICC were differentiated before ICC differentiation. The ZIO uptake by both nerve elements and ICC correlated with the establishment of their differentiated features. In conclusion, the present findings confirmed that differentiation of ICC and enteric plexuses is microenvironment dependent, since their differentiative steps are interrelated and correlated with diet changes. ZIO impregnation under EM enabled a distinction to be made between a ‘morphological’ and a ‘functional’ differentiation, and revealed that the former is achieved during the suckling period and the latter by the weaning period. It can be suggested that during the postnatal developmental stages ICC and neuronal functions might be different from those in adulthood.

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(a) the right colon contains a lower number of neurons and of VIP- and NOS-positive ones than the left colon, and (b) VIP- and NOS-positive fibers are differently distributed in the inner and outer portions of the circular muscle.

The organization of the cytoskeleton has been studied during mouse differentiation in cells of the myenteric neuronal lineage. The entire gut was examined starting from day 12.5 of embryonic life (E12.5) until birth (P0). Immunocytochemistry was performed to evaluate the expression of five of the most represented neurofilaments proteins (the low, NF-L, medium, NF-M, and heavy, NF-H, molecular weight subunits, alpha-internexin and peripherin) and of two of the microtubule-associated proteins (MAPI and MAP2a+2b). In parallel, the appearance in the differentiating myenteric neurons of filamentous and microtubular structures and their intracytoplasmatic distribution were observed under the electron microscope. A differential immunohistochemical expression of the structural proteins was found. Immature cells expressed alpha-internexin, peripherin, NF-M and MAP1 by day E12.5; alpha-internexin expression was strong in these cells, but gradually decreased with age and was practically absent in adulthood. Conversely, the expression of the other three proteins increased with cell differentiation and was still present in adulthood. NF-L and NF-H expression appeared later, by day E16.5, and was weak for the entire pre- and postnatal life. MAP2a+2b was never expressed. Under the electron microscope, at day E12.5 the cytoskeleton was already organized in filamentous and microtubular structures. At this age neurofilaments were few and mainly located in the cell processes, and microtubules were numerous and mainly assembled in the neuritic growth cones, together with synaptic vesicles. With ageing, neurofilaments and microtubules were ubiquitous in the neuron. Data obtained demonstrate that cytoskeletal proteins gradually accumulate in the cells of the neuronal lineage in parallel with the organization of the cytoskeletal structures, which in turn mediate important neural events by the earliest stages of murine embryonic life, including growth of nerve processes and initiation of axonal transport.