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The cell population and distribution of NADPH-diaphorase positive and NOS immunoreactive intramural ganglion cells were examined on stretched whole-mount preparations of the guinea pig urinary bladder which was divided into 3 regions: base, body and dome. The results showed that the highest frequency both of NADPH-d and NOS positive neurons was observed in the bladder base. Cell counts in the whole bladder showed that the number of NADPH-d positive neurons was much more than that of NOS immunoreactive neurons. Using neuron specific enolase (NSE) positive neurons as a reference (100%), NADPH-d positive neurons accounted for 84% while NOS immunoreactive neurons only made up 45% of the total neuronal population. These results, along with previous studies on the function of nitric oxide, suggest that nitric oxide may be involved in the relaxation activity in the bladder base during micturition. The significant difference in the number of NADPH-d positive and NOS immunoreactive neurons suggests that the localisation of one enzyme does not necessarily reflect the presence of the other.

The present study examined the existence of catecholamine-, corticotropin-releasing factor (CRF)- and neurotensin (NT)-containing neurons in the external cuneate nucleus (ECN) of the gerbil using single label pre-embedding immunocytochemistry in an attempt to shed light on the increasing evidence for autonomic involvement of the ECN. Peroxidase immunoreactivity of phenylethanolamine-N-methyl-transferase (PNMT), CRF or NT was identified in the heterogeneous population of the ECN neurons characterized by a deeply infolded nucleus. The label was localized in their somata, dendrites, myelinated axons and axon terminals. The immunolabelled dendrites were contacted by spherical (S) and flattened (F) types of presynaptic boutons containing spherical and flattened synaptic vesicles, respectively. The PNMT-labelled dendrites, however, were postsynaptic to an additional type of axon terminals containing pleomorphic (P) synaptic vesicles. Among the immunoreactive axon terminals, the PNMT-labelled boutons consisted of two types: S and F; in the CRF- and NT-labelled axon terminals, only the S type was observed. The catecholamine-containing ECN neurons differed from the CRF- and NT-immunoreactive neurons in their synaptic organization. The latter two were considered to be of the same cell population because of their similarities in ultrastructural features and synaptic relations. In view of a high frequency (48% for PNMT, 50% for CRF and 46% for NT) of the F-typed boutons associated with the three categories of immunolabelled neurons in the ECN, it is possible that they are under considerable inhibitory control. The presence of catecholamine, CRF and NT in the ECN suggests that the nucleus may be involved in the integration of proprioception-, exercise- or stress-evoked autonomic responses.

Using acetylcholinesterase histochemical and choline acetyltransferase immunocytochemical localization methods, this study has provided conclusive evidence for the existence of cholinergic neurons in the external cuneate nucleus of gerbils. By light microscopy, both acetylcholinesterase and choline acetyltransferase labelling was confined to the rostral portion of the external cuneate nucleus. Ultrastructurally, acetylcholinesterase reaction products were found in the nuclear envelope, cisternae of rough endoplasmic reticulum and Golgi saccules of some somata and large dendrites as well as in the membranes of small dendrites, myelinated axons and axon terminals. These neuronal elements were also stained for choline acetyltransferase; immunoreactivity was associated with nuclear pores, nuclear envelope, perikaryal membrane and all the membranous structures within the cytoplasm. Of the total choline acetyltransferase-labelled neuronal profiles analysed, 79% were myelinated axons, 15% dendrites, 4% somata and 2% axon terminals. The immunostained axon terminals consisted of two types containing either round (Rd type; 62.5%) or pleomorphic (Pd type; 37.5%) vesicles. Both were associated directly with choline acetyltransferase-positive dendrites. In contrast to the paucity of choline acetyltransferase-labelled axon terminals, numerous choline acetyltransferase-positive myelinated axons were present. It may thus be hypothesized that most, if not all, of the external cuneate nucleus cholinergic neurons are projection cells; such cells may give rise to axonal collaterals which synapse onto their own dendrites for possible feedback control. Choline acetyltransferase-positive dendrites were contacted by numerous unlabelled presynaptic boutons, 60% of which contained round or spherical synaptic vesicles (Rd boutons) and 40% flattened vesicles (Fd boutons), suggesting that these neurons are under strong inhibitory control. The preferential concentration of cholinergic components in the rostral external cuneate nucleus may be significant in the light of the highly organized somatotopy in the external cuneate nucleus and its extensive efferent projections to medullary autonomic-related nuclei. Our results suggest that the cholinergic neurons may be involved in somatoautonomic integration.

With retrograde tracing using fluorogold injection into the superior cervical ganglion and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, the present comparative study revealed that the retrogradely labelled neurons in n. intermediolateralis pars funicularis (ILf) and n. intermediolateralis pars principalis (ILp) of the autonomic region in the upper thoracic cord exhibited a much stronger reactivity for NADPH-diaphorase in Wistar-Kyoto (WKY) rats than those in spontaneously hypertensive rats (SHR). It was found that in ILf in WKY rats, 77.62% of the fluorogold-labelled neurons were NADPH-d positive, while in SHR, only 56.43% of the labelled neurons were NADPH-d positive. The frequency distribution of NADPH-d positive retrogradely labelled neurons was significantly reduced in ILf of the spinal cord of SHR (U-test: P < 0.01). In ILp in WKY rats, 65.25% of fluorogold-labelled neurons were NADPH-d positive in WKY rats, while in SHR, only 56.28% of the labelled neurons were NADPH-d positive. Although the difference (P > 0.05) in the frequency of NADPH-d positive neurons in ILp between the two strains of rats was not significant, the reductions in SHR seemed considerable. Examination of the preganglionic sympathetic trunk and the superior cervical ganglion between SHR and WKY rats revealed that virtually all the NADPH-d positive fibers were derived from the sympathetic preganglionic neurons. In SHR, the NADPH-d positive fibers were not as intensely stained as those of WKY rats. This preliminary results suggest that nitric oxide, as an inhibitory neurotransmitter, may be implicated in the onset of hypertension.

The present study examined the synaptic organization of cuneocerebellar neurons and their relationships with the primary afferents in the gerbil external cuneate nucleus following multiple injections of horseradish peroxidase over a widespread area in the cerebellum in conjunction with a simultaneous injection of horseradish peroxidase into the cervical or brachial nerve plexus. The external cuneate nucleus is topographically organized: the rostral portion receiving the primary afferents from the cervical plexus and the caudal portion primary afferents from the brachial plexus. This study attempted to correlate the synaptology with the topography and different cytoarchitecture in these two specific regions in the external cuneate nucleus. Ultrastructurally, the profiles of horseradish peroxidase-labelled cuneocerebellar neurons could be divided into three types, namely, small, medium and large on the basis of their cross-sectional areas. Axon terminals which formed axosomatic synapses could be classified into: round (Rs type; 22.2%), pleomorphic (Ps type; 55.6%) and flattened (Fs type; 22.2%) vesicle boutons. The horseradish peroxidase-labelled dendritic elements of the cuneocerebellar neurons were postsynaptic to a greater number of axon terminals which were also classified into Rd (77.5%), Pd (18.8%) and Fd (3.7%) type boutons. Some of the Rd boutons making direct synaptic contacts with the cuneocerebellar neurons originated from primary afferents since they were simultaneously labelled by transganglionic transport of horseradish peroxidase. In the rostral external cuneate nucleus, synapses on cuneocerebellar neurons were more frequent on their primary dendrites as compared with those on the primary dendrites of the caudal cuneocerebellar neurons. The latter, on the other hand, showed more synapses on their distal dendrites. This may have functional implications with regard to the afferent inputs to cuneocerebellar neurons in the rostral and caudal external cuneate nucleus.

Sympathetic preganglionic neurons in the intermediolateral nucleus of the thoracic spinal cord of the adult rat which innervate the superior cervical ganglion (SPN-scg) were identified by means of retrograde transport of cholera subunit B-conjugated horseradish peroxidase and fluorogold. In horizontal sections of the spinal cord, the SPN-scg were observed to be arranged in clusters which displayed a characteristic triangular configuration. Within this triangle, the cells showed no preferential orientation for their long axes were oriented obliquely, transversely or longitudinally. The dendrites arising from these clusters were oriented either longitudinally, medially, or laterally. The medially-oriented dendrites formed a subependymal plexus and some have been observed to cross the midline to the opposite side. The most significant finding was the presence of the white matter dendritic plexus which was formed by the laterally-directed bundles of dendrites. The present findings thus suggested that SPN-scg may be regulated by means of two circuits: the classical (medial) core circuit and a paralateral circuit which may convey supraspinal afferent inputs to the SPN-scg.

Comparative ultrastructural study of the sympathetic preganglionic neurons that innervated the superior cervical ganglion (SPN-scg) was made between spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats. After injection of choleragen subunit-B horseradish peroxidase (CB-HRP) into the superior cervical ganglion, three types of the sympathetic preganglionic neurons were identified according to their ultrastructural features. In both SHR and WKY rats, the neurons in the autonomic region of T1-T3 segments of the spinal cord showed a characteristic distribution pattern in which the Type I neurons were located more laterally in n. intermediolateralis pars funicularistic (ILf), and n. intermediolateralis pars principalis (ILp), Type III neurons more medially in n. intercalatus spinalis (IC), n. intercalatus pars paraependymalis (ICpe) and Type II neurons in n. intermediolateralis pars principalis (Ilp) as well as in n. intercalatus spinalis (IC). Of the three types of neurons, there was not noticeable ultrastructural difference in Type II and Type III neurons between the SHR and WKY rats. Some differences, however, were observed in Type I neurons between the two animals stains. In SHR, the nucleus of Type I neurons displayed many deep indentations and a greater number of profiles of Golgi apparatus. Three types of the axon terminal were found to make synaptic contacts with the labelled Type I neurons. Some proportional changes of the different axon terminals were observed between those of SHR and WKY rats. The results of this study suggest that the suppressive effect on the activity of the sympathetic preganglionic neurons in SHR may be attenuated which would result in an unbalanced activities of some of neurotransmitters on the sympathetic preganglionic neurons thereby leading to the onset of hypertension. The increase in the number of nuclear indentations and an increased activity in the Golgi complex may reflect an increase in the synthesis of some of the neurotransmitters or neuromodulators in the sympathetic preganglionic neurons or their axon terminals. The characteristic distribution patterns of the three types of neurons suggest that, of the three types of SPN-scg, only the Type I neurons may be implicated in the regulation of the cardiovascular system.