C K Tan

Ultrastructural localization of acetylcholinesterase and choline acetyltransferase in oligodendrocytes, glioblasts and vascular endothelial cells in the external cuneate nucleus of the gerbil.

This study reports the reactivities of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in some of the nonneuronal elements in the external cuneate nucleus (ECN) of gerbils. AChE reaction products were localized in some oligodendrocytes in their cisternae of rough endoplasmic reticulum, nuclear envelope and Golgi saccules. The basal lamina lining the capillary endothelia also displayed AChE reactivity. In ChAT immunocytochemistry, the reaction products were found to be associated with the vascular basal lamina as well as the endothelial plasma membrane facing the lumen. The most remarkable finding was the localization of ChAT immunoreactivity in some oligodendrocytes and occasional glioblasts (small glial precursor cells containing a thin rim of cytoplasm surrounding an irregular nucleus with homogeneous chromatin materials). The ChAT-positive oligodendrocytes consisted of two types, medium-dense and dark cells, either associated with blood vessels or ChAT-stained neuronal elements. It is suggested from these new findings that at least some of the oligodendrocytes and glioblasts in the ECN of gerbils may be involved in the synthesis, storage, release and degradation of acetylcholine.

Efferent connections from the external cuneate nucleus to the medulla oblongata in the gerbil

The present study revealed the efferent projections from the external cuneate nucleus (ECN) to various medullary nuclei in the gerbil as demonstrated in fresh living brainstem slices by using in vitro anterogradely tracing with the dextran-tetramethyl-rhodamine-biotin. The tracer-labelled ECN axon terminals were observed (1) in most of the vital autonomic-related nuclei: the nucleus solitary tractus, nucleus ambiguus, rostroventrolateral reticular nucleus and C2 adrenergic area, (2) in the reticular formation: the medullary, parvocellular, intermediate, gigantocellular, dorsal paragigantocellular and lateral paragigantocellular reticular nuclei and medullary linear nucleus, and (3) in sensory nuclei: the cuneate nucleus, spinal trigeminal nuclei caudalis and interpolaris, paratrigeminal nucleus, medial and spinal vestibular nuclei, inferior olive and prepositus hypoglossal nucleus. These new findings are discussed in relation to possible roles of the ECN in cardiovascular, respiratory and sensorimotor controls.

The distribution of NADPH-d in the central grey region (lamina X) of rat upper thoracic spinal cord.

SummaryThe distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the central grey region (lamina X of Rexed) of the rat upper thoracic cord was examined by LM and EM. Numerous NADPH-d positive neuronal somata and fibres were present in the subependymal areas of the central grey region at levels T1–T3. Most of the neurons were located dorsal to the central canal in horizontal sections through this region. Many medially-directed NADPH-d positive fibres arising from neurons in n. intermediolateralis pars principalis, n. intercalatus spinalis and longitudinally-directed NADPH-d positive fibres arising from neurons in n. intercalatus pars paraependymalis formed a subependymal plexus. In horizontal sections through the central canal, some NADPH-d positive nerve fibres appeared to traverse the ependyma to enter and run along the central canal. By EM, NADPH-d reaction products were localized on the nuclear membrane, outer mitochondrial membrane and Golgi apparatus of both neurons and ependymal cells and in some axon terminals containing pleomorphic and round agranular synaptic vesicles. Present results suggest that besides the traditional monoamine-, amino acid- and peptidecontaining axon terminals, the central grey region also contains fibres in which nitric oxide is utilized as a neurotransmitter or neuromodulator. The finding of NADPH-d positive fibres in the central canal suggests that nitric oxide may be released into the cerebrospinal fluid. Since some of the ependymal cells were NADPH-d positive, it is suggested that they may be involved in the modulation of nitric oxide levels in the cerebrospinal fluid.

A comparative study of NADPH-diaphorase in the sympathetic preganglionic neurons of the upper thoracic cord between spontaneously hypertensive rats and Wistar-Kyoto rats

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.

A light-microscopic study of the intermediolateral nucleus following injection of CB-HRP and fluorogold into the superior cervical ganglion of the rat

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.

Ultrastructural study of external cuneothalamic neurons and their synaptic relationships with primary afferents in the gerbil

The present study examined the synaptic organization of external cuneothalamic neurons and their relationships with primary afferents in the gerbil external cuneate nucleus (ECN) following an injection of horseradish peroxidase (HRP) into the anterodorsal cap of the ventrobasal thalamus in conjunction with a simultaneous injection of HRP into the contralateral brachial and cervical nerve plexuses. The thalamus‐projecting neurons have been shown to be confined to the intermediate portion of the caudal half of the ECN at the light microscopic level (Lan et al., 1994c). In this study, HRP‐labelled external cuneothalamic neurons were ultrastructurally characterized by their relatively small‐sized soma bearing a variable number of somal spines. Their nucleus had a slightly indented contour with an eccentric nucleolus. The HRP‐labelled somata were postsynaptic to many axon terminals, which were classified into round (Rs type; 53.0%), pleomorphic (Ps type; 32.7%), and flattened (Fs type; 14.3%) vesicle‐containing boutons. The HRP‐labelled dendritic elements were postsynaptic to a greater number of axon terminals, which were also classified into the round (Rd; 64.7%), pleomorphic (Pd; 25.2%), and flattened (Fd; 10.1%) type boutons. These presynaptic axonal boutons tended to synapse on distal and secondary dendrites of external cuneothalamic neurons. In the present simultaneous HRP labelling study, some of the primary afferent terminals made direct synaptic contacts with the dendrites of the external cuneothalamic neurons. In view of the multiple inputs onto the external cuneothalamic neurons, impinging particularly on their somata and secondary dendrites, it is suggested that the proprioceptive information reaching these neurons is intensively modulated and integrated before transmission ultimately to the cerebral sensorimotor cortex.

Localisation of substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat: a light- and electron-microscopic study

The present study describes substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat. About 60% of neurons in the monkey ciliary ganglion and 40% in the cat ciliary ganglion were substance P-like immunoreactive, ranging from faint to moderate staining. Substance P-like immunoreactivity was located in cell bodies, dendritic profiles and axons. In the monkey, substance P-like immunoreactive pericellular arborisations were associated with about 0.5%–3% of the ganglion cells, which were either negatively, faintly or moderately stained. An electron-microscopic study demonstrated the presence of either substance P-like immunoreactive positive or negative axon terminals synapsing or closely associated with positive dendritic profiles in both the monkey and cat ciliary ganglia. The results suggest that substance P plays an important role in the ciliary ganglion, perhaps as a modulator or transmitter.

ULTRASTRUCTURAL IDENTIFICATION OF CHOLINERGIC NEURONS IN THE EXTERNAL CUNEATE NUCLEUS OF THE GERBIL : ACETYLCHOLINESTERASE HISTOCHEMISTRY AND CHOLINE ACETYLTRANSFERASE IMMUNOCYTOCHEMISTRY

SummaryUsing 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.

An ultrastructural study of cuneocerebellar neurons and primary afferent terminals in the external cuneate nucleus of gerbils as revealed by retrograde and transganglionic transport of horseradish peroxidase

SummaryThe 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.

The ciliary ganglion of the cat: a light and electron microscopic study

The ciliary ganglia of eight healthy adult cats were studied by light and electron microscopy. The ganglion, measuring about 2 mm in length, was consistently found to be attached to the branch from the oculomotor nerve supplying the inferior oblique muscle. The number of neurons varied from 2773 to 3794 after applying Abercrombies correction. The mean of average somal diameter of the neurons was 36.5 μm (SD = 5.0 μm) and the mean of somal cross-sectional area was 904.2 μm2 (SD = 262.8 μm2). The mean of average nuclear diameter was 13.9 urn (SD = 1.8 μm) and the mean of nuclear cross-sectional area was 142.2 μm2 (SD = 37.1 μm2). The mean of the aspect ratios of the soma and nucleus were 1.2 (SD = 0.1) and 1.1 (SD = 0.1) respectively. The frequency distributions of these parameters were all unimodal. Under the light microscope, the Nissl granules in the neurons were prominent and were distributed peripherally, perinuclearly or randomly in the cytoplasm. Under the electron microscope, the rough endoplasmic reticulum showed a similar pattern of distribution in the cytoplasm. In some neurons, glycogen-like granules were present; these were either distributed randomly throughout the cell, or aligned in single rows in relation to sub-surface cisterns and between the cisterns of smooth and rough endoplasmic reticulum. Most of the dendrites were short protrusions from the cell body; some contained glycogen-like granules. Occasionally, the dendritic protrusions were electron-dense. All the synapses encountered were axodendritic. In most axon terminals, the synaptic vesicles were spherical and measured 30–50 nm in diameter; in some, they were flattened, measuring 50 nm by 20 nm. Some axon terminals containing either spherical or flattened synaptic vesicles also contained large dense-cored vesicles that measured 80–100 nm, while their dense core measured 40–60 nm.