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The aim of this study was to investigate reactive changes of astrocytes and Müller glial cells in rats subjected to kainate treatment, which leads to neuronal degeneration in the ganglion cell layer and the inner border of the inner nuclear layer as confirmed by labelling with Fluoro-Jade B, a marker for degenerating neurons and fibres. Both the astrocytes and the Müller glial cells reacted vigorously to kainate injection as shown by their up-regulated expression of nestin, glial fibrillary acidic protein and glutamine synthetase. A major finding was the induced expression of nestin together with glial fibrillary acidic protein beginning at 1 day post-injection of kainate. The marked nestin expression appeared to be most intense at 1 day and was sustained till 2 weeks as compared with the untreated/normal retina. Western blotting analysis confirmed a marked increase in expression of nestin, glial fibrillary acidic protein and glutamine synthetase as compared with untreated/normal retina. Double labelling study revealed that astrocytes and Müller glial cells expressed the radial glia marker nestin, and incorporated bromodeoxyuridine to re-enter into their cell cycle. The induced expression of these proteins in astrocytes and Müller glial cells indicated an induction of gliotic responses and de-differentiation that may be associated with regenerative efforts after kainate-induced injury. Indeed, with the acquisition of an immature molecular profile as manifested by the induced expression of brain lipid-binding protein and doublecortin in astrocytes and Müller glial cells, the potential of these cells to de-differentiate in retinal neurodegeneration is greatly amplified.

Sleep disorders are a form of stress associated with increased sympathetic activity, and they are a risk factor for the occurrence of cardiovascular disease. Given that nitric oxide (NO) may play an inhibitory role in the regulation of sympathetic tone, this study set out to determine the NO synthase (NOS) reactivity in the primary cardiovascular afferent neurons (i.e. nodose neurons) following total sleep deprivation (TSD). TSD was performed by the disc-on-water method. Following 5 days of TSD, all experimental animals were investigated for quantitative nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d, a co-factor of NOS) histochemistry, neuronal NOS immunohistochemistry and neuronal NOS activity assay. In order to evaluate the endogenous metabolic activity of nodose neurons, cytochrome oxidase (COX) reactivity was further tested. All the above-mentioned reactivities were objectively assessed by computerized image analysis. The clinical significance of the reported changes was demonstrated by alterations of mean arterial blood pressure (MAP). The results indicated that in normal untreated rats, numerous NADPH-d/NOS- and COX-reactive neurons were found in the nodose ganglion (NG). Following TSD, however, both the labelling and staining intensity of NADPH-d/NOS as well as COX reactivity were drastically reduced in the NG compared with normal untreated ganglions. MAP was significantly higher in TSD rats (136+/-4 mmHg) than in normal untreated rats (123+/-2 mmHg). NO may serve as an important sympathoinhibition messenger released by the NG neurons, and decrease of NOS immunoexpression following TSD may account for the decrease in NOS content. In association with the reduction of NOS activity, a defect in NOS expression in the primary cardiovascular afferent neurons would enhance clinical hypertension, which might serve as a potential risk factor in the development of TSD-relevant cardiovascular disturbances.

Previous studies have shown the existence of a sympathetic component in some cranial nerves including the hypoglossal nerve. In this study, the horseradish peroxidase (HRP) tract-tracing retrograde technique and experimental degeneration method were used to elucidate the possible neuroanatomical relationship between the superior cervical ganglion (SCG) and the hypoglossal nerve of hamsters. About 10% of the SCG principal neurons were HRP positive following the tracer application to the trunk of hypoglossal nerve. Most of the HRP-labelled neurons were multipolar and were randomly distributed in the ganglion. When HRP was injected into the medial branch of the hypoglossal nerve, some of the SCG neurons were labelled, but they were not detected when HRP was injected into the lateral branch. The present findings suggest that postganglionic sympathetic fibres from the SCG may travel along the hypoglossal nerve trunk via its medial branch to terminate in visceral targets such as the intralingual glands. By electron microscopy, the HRP reaction product was localised in the neuronal somata and numerous unmyelinated fibres in the SCG. In addition, HRP-labelled axon profiles considered to be the collateral branches of the principal neurons contained numerous clear round and a few dense core vesicles. Besides the above, some HRP-labelled small myelinated fibres, considered to be visceral afferents, were also present. Results of experimental degeneration following the severance of the hypoglossal nerve showed the presence of degenerating neuronal elements both in the hypoglossal nucleus and the SCG. This confirms that the hypoglossal nerve contains sympathetic component from the SCG which may be involved in regulation of the autonomic function of the tongue.

This study examined NADPH-d and nNOS expression in the SCG of hamsters. By light microscopy, numerous NADPH-d/NOS positive processes were widely distributed in the ganglion. Ultrastructurally, the NADPH-d reaction product was associated with the membranous organelles of neuronal soma, dendrites, myelinated fibres, small granular cells, and axon profiles bearing agranular vesicles. The NOS immunoreaction product, on the other hand, was localised in the cytoplasm of principal neurons and dendrites. Some of the NADPH-d/NOS labelled processes formed junctional contacts including synapses or zonulae adherentia. Compared with the neurons, the nonneuronal cells in the ganglion, namely, macrophages, satellite cells and endothelial cells were labelled by NADPH-d but devoid of nNOS immunoreaction product. The results suggest that the NADPH-d/NOS positive fibres in the SCG originate not only from the projecting fibres of the lateral horns of thoracic spinal cord, but also from the principal neurons and small granular cells; some may represent visceral afferent fibres. Electron microscopic morphometry has shown that about 67% of the principal neurons contain NADPH-d reaction product, and that the majority were small to medium sized neurons based on cross-sectional areas in image analysis. On the basis of the present morphological study, it is concluded NO is produced by some local neurons and possibly some nonneuronal cells in the SCG as well as some fibres of extrinsic origin. In this connection, NO may serve either as a neurotransmitter or neuromodulator.

The distribution of glycine-immunoreactive (glycine-IR) neurons and their associated axon terminals in the rat cuneate nucleus was studied using antiglycine postembedding immunoperoxidase labelling and immunogold staining, respectively. The immunoperoxidase-labelled glycine-IR neurons were widely distributed in the entire rostrocaudal extent of the nucleus. They made up 30.8% (9671/31368) of the neurons surveyed. Quantitative evaluation showed that the percentage of glycine-IR neurons in the caudal level was significantly higher than that in the middle and rostral levels. The glycine-IR neurons were small cells (mean area = 198+/-1.9 microm2, n = 2862) with ovoid or spindle-shaped somata. Statistical analysis showed that the size of the glycine-IR neurons in the rostral level was significantly smaller than that in the middle and caudal levels. Immunogold labelled glycine-IR terminals which contained predominantly pleomorphic synaptic vesicles were mostly small (mean area = 1.24+/-0.03 microm2, n = 286) and they constituted 24.7% (286/1158) of the total terminals surveyed. They formed axodendritic, axosomatic and axoaxonic synapses with unlabelled elements. It is suggested from this study that glycine is one of the major neurotransmitters involved in the depression of synaptic transmission in the cuneate nucleus.

The present study examined the expression of different antigens in amoeboid microglial cells (AMC) in fetal rat brain extending from 12 to 20 d postconception (E12-E20) using a panel of monoclonal antibodies which recognised the major histocompatibility complex (MHC) class I (OX-18) and class II (OX-6) antigens, leucocyte common antigen (OX-1), CD4 receptor (OX-35), complement type 3 receptor (OX-42) or macrophage antigens of unknown function (ED1 and ED2). Of the above-mentioned antigens, ED1 and ED2-labelled AMC were observed in the neuroepithelia as early as embryonic day 12 (E12); other antigens were not detected at this stage. At E14, except for MHC class I antigen, all other antigens were expressed by AMC distributed predominantly in the developing white matter. At E16, AMC in the intermediate zone lateral to the striatum were endowed with all the above-mentioned antigens including MHC class I. At E18, the immunoreactivities of AMC stained with OX-6, OX-18, OX-35 and OX-42 antigens were noticeably reduced when compared with those cells at E16. At E20, amoeboid microglial cells exhibited full complement of antigen expression similar to those cells at E16; some of the labelled cells emitted a variable number of cytoplasmic processes. It is suggested that the successive and differential expression of various macrophage related antigens on AMC in fetal brain is related to the specific requirement of local environment in different stages of development.

The cranial components and central terminations of the sensory nerves supplying the concave surface of the puppy's pinna, namely, the rostral, middle and caudal internal auricular nerves (RIAN, MIAN and CIAN) were investigated using horseradish peroxidase retrograde and transganglionic labelling techniques. All the 3 internal auricular nerves received contributions from the vagus. The RIAN received additional fibres from the trigeminal nerve while the MIAN and CIAN contained fibres derived from the facial nerves. In the brainstem, collaterals from the descending fibres of the afferents were given off at all levels to the medially located spinal trigeminal nucleus (SpV) which extended rostocaudally from the principal nucleus of the trigeminal nerve, subnuclei oralis, interpolaris and caudalis of the SpV to C1 or C2 cervical segment. The greatest density of central projections was observed in the subnucleus caudalis and C1. In the latter, the terminal field in the dorsal horn was roughly wedge-shaped, tapering off medially from lamina I towards lamina V. A somatotopic organisation was observed in the spinal trigeminal tract (SpVtr) and spinal trigeminal nucleus (SpV) in which the projection fibres and terminal fields of the RIAN were located lateral to those of the MIAN and CIAN. Some nontrigeminal nuclei, e.g. paratrigeminal and cuneate nuclei, nucleus X and the nucleus of the solitary tract were also labelled following HRP application to the internal auricular nerves. The localisation of the central projections of the internal auricular nerves as well as the 2nd order neurons in some specific nuclei to which the afferent fibres project is consistent with the concept of a brainstem somatovisceral link.

The synaptic organisation of the primary afferents from the brachial and cervical plexuses to the external cuneate nucleus of gerbils was compared following an intraneural injection of horseradish peroxidase into the musculocutaneous, median, ulnar and radial nerves of the brachial plexus or the main branches of the cervical plexus; 407 labelled primary afferent terminals from the brachial and 459 from the cervical plexus were studied. These boutons made synaptic contacts with 586 and 633 dendritic profiles, respectively. 99.0% of the primary afferent boutons from the brachial plexus contained clear round synaptic vesicles (R boutons); the remaining 1% of boutons contained pleomorphic synaptic vesicles (P boutons). For boutons from the cervical plexus, 95% were R boutons and 5% were P boutons. The labelled R bouton profiles had a wide range of cross-sectional area from 0.4 to 13.1 microns 2, while the P boutonal profiles were of a small variety (range, 0.4-2.3 microns 2; mean, 1.5; S.D., 0.6 micron 2). The R boutons from the brachial plexus (mean, 3.9 microns; S.D., 2.1 microns 2) were generally larger than those from the cervical plexus (mean, 3.3 microns 2; S.D., 1.9 microns 2). On close analysis, 72.4% of R boutons from the brachial plexus were found to synapse on distal dendrites, 15.9% on secondary dendrites, 9.5% on dendritic spines and 2.2% on proximal dendrites. For R boutons from the cervical plexus, 81.1% synapsed on distal dendrites, 12.1% on dendritic spines and 6.8% on secondary dendrites; none was observed on proximal dendrites. Such a different synaptic organisation between the two nerve plexuses may be related to their different perceptuomotor executions.

The possible origins of cerebellar cholinergic afferents from the lower brainstem of the gerbil were examined using immunohistochemistry combined with retrograde neuronal labelling techniques. Choline acetyltransferase (ChAT) monoclonal antibody was used in conjunction with a retrogradely transported tracer, horseradish peroxidase (HRP). The use of this technique allowed an unequivocal localisation of cholinergic neurons in different parts of the lower brainstem projecting to the cerebellum. In addition, single labelling of acetylcholinesterase (AChE), ChAT and HRP was carried out to elucidate the efferent projection from the lower brainstem to the cerebellum as well as the cholinergic distribution in these two areas. Our results showed the presence of HRP/ChAT double-labelled neurons in (1) the midline medulla: the periventricular gray beneath the 4th ventricle, C3 adrenergic area, raphe obscurus nucleus and medial longitudinal fasciculus, (2) the reticular formation: the medullary, lateral, intermediate, gigantocellular, lateral paragigantocellular and dorsal paragigantocellular reticular nuclei and gigantocellular reticular nucleus ventralis, and (3) sensory nuclei: the gracile nucleus, cuneate nucleus, external cuneate nucleus, spinal trigeminal nucleus interpolaris, prepositus hypoglossal nucleus and medial vestibular nucleus. In the cerebellum, AChE-positive mossy fibres were chiefly localised in the vermian lobules VIb,c, VII and X, paramedian lobule, crura I and II, paraflocculus and flocculus, and they were distributed in the white matter and granular layer of the cortex. The 3 above-mentioned cerebellar cholinergic afferent systems associated with the unique AChE distribution pattern in the cerebellum may be of important functional significance.

The distribution of various morphological forms of microglia stained with lectin in different regions of postnatal rat brain was examined in 2 planes of section, coronal and sagittal. In the coronal sections (mediolateral plane) taken at the level of the optic chiasma, the lectin-labelled cells were examined in the gustatory (GU), lateral somatosensory (LSS), medial somatosensory (MSS), motor (MO) and cingulate (CI) cortex. In the sagittal sections (rostrocaudal plane), the lectin-labelled cells in the medial orbital (MOA), motor (MO) and primary visual area (PVA) were studied. The cells in the motor area in coronal plane were further analysed with reference to their distribution in each of the laminae (layers). Based on the variation of their external morphology which represents different degrees of differentiation, all lectin-labelled microglial cells of the above-mentioned regions in newborn (P0) and rats aged 4 d (P4) and 8 d (P8) postnatum were classified and quantified. In the mediolateral plane of any given age group, the most differentiated ramified cells were located in GU except in P8 rats where the cells were also concentrated in CI. Of the 3 regions in the rostrocaudal plane the majority of the more differentiated ramified cells at P0 were found in the PVA but were the major cell type in MO in P4 and P8 rats. For the distribution of cells in MO, the most differentiated cells were located in the intermediate zones. It was concluded from this study that microglial cells in the developing cerebrum showed a gradient of differentiation in relation to different regions of the cerebral cortex but this appeared to vary with age. Thus in the mediolateral plane in P0 and P4 rats, the gradient extended from GU to CI in a lateromedial fashion but in P8, in the direction towards MSS from GU and CI. In the sagittal section, the gradient was directed caudorostrally in P0 rats. In P4 and P8 rats, however, the gradient was from MO to both poles (MOA, PVA). In the motor cortex, the gradient was from the intermediate zone towards the superficial and deep laminae. The gradient of differentiation of microglia may be related to the growth of the respective regions in the cerebral hemisphere but inherent genetic factors were also considered.

The morphometric and quantitative changes associated with the differentiation of amoeboid microglia into ramified microglial cells in the corpus callosum of rats between 21 d postconception (E21) and 15 d postnatally are described. Using lectin labelling, 5 morphological types of labelled cells (R, SP, KLP, TLP, AP) based on cell body shape, the configuration of their cytoplasmic processes and their staining intensity, were recognised. Round cells (R) and cells with stout processes (SP) were aggregated in the central part of the developing corpus callosum whereas the highly branched labelled cells were distributed at its periphery. When the morphometric data and labelling intensities of labelled cells were analysed with the aid of an image analysis system, the values for cell length, area and perimeter increased as the complexity of branching increased, whereas the lectin-labelling intensity became reduced. Quantitative study showed that the proportion of the different morphological types of lectin-labelled cells peaked at different ages. The sequential peaking of R, SP and highly branched cells with advancing age suggests a similar chronological order of differentiation of R into branched cells. The quantitative study also showed a rapid increase in the density of lectin-labelled cells in the postnatal period between P4 and P8, attributed primarily to the active proliferation of the cell type. The consequent reduction of cell density (after P13) was probably due to cell death, a feature which appeared to increase with development.

Immunogold labelling was used to identify GABA-immunoreactive (GABA-IR) terminals in the cuneate nucleus of the rat. About 30% of the terminals surveyed were GABA-IR. They were mostly small, although a few of medium size were encountered. The terminals contained either polymorphic or round synaptic vesicles; these occurred in an approximately 5:1 ratio. Most of these terminals formed symmetric synapses with dendrites of various sizes. The GABA-IR terminals also made synaptic contacts with somata of cuneate neurons and axon terminals of unknown origin. In addition, a few GABA-IR terminals forming asymmetric synapses were found in the cuneate neuropil. The possible functional significance of the synaptic organisation is discussed.

Virtually all the ganglion cells in the nodose ganglion in hamsters underwent rapid degeneration following an intraneural injection of RCA-60 into the vagus nerve in the cervical region. The earliest signs of neuronal degeneration were evident in animals which survived 5 days after the ricin application. A remarkable feature was the appearance of a variable number of granular dense bodies measuring 1-4 microns in diameter in the cytoplasm. They were composed of closely stacked cisternae which were continuous at the periphery with those of the rough endoplasmic reticulum. Associated with the membranous cisternae were large accumulations of glycogen. With longer survival time, these glycogen-membrane complexes appeared to disintegrate. Numerous vacuoles and neurofilaments accumulated in their vicinity. Satellite cells were activated between the 7th and 10th postoperative days. These penetrated deeply into the degenerating neurons dividing them into numerous fragments by their extensive cytoplasmic prolongations. The cytoplasmic fragments of the RCA-poisoned neurons eventually became necrotic and disintegrated in the satellite cells, suggesting a rapid mode of neuronophagia. The biosynthesis of acetylcholinesterase was inhibited by the ricin injected as shown by the drastic reduction of the enzyme activity in the rough endoplasmic reticulum and nuclear envelope. Some isolated ganglion cells apparently survived the RCA injection as shown by their occurrence in long surviving animals (30-90 days). A few of them displayed an enhanced density of their cytoplasm and neurites. It is postulated that this was induced by the RCA released from the RCA-poisoned neurons.

The present study has shown the selective destruction of large ventral horn neurons in the lumbosacral cord segments following a single injection of RCA-60 into the sciatic nerve. The neurons appeared to undergo structural alteration beginning 3 days after the RCA application. In the postoperative period extending from 1 to 60 days, degeneration of neurons was progressive and irreversible and this elicited a rapid increase in the number of microglial cells. They were most numerous in the 7 days postoperative animals. The massive microglial cells penetrated the neuropil and appeared to strip off the axon terminals from the postsynaptic somata. Occasional axon terminals were phagocytosed by microglia. The numerous microglial cells often formed a multilayered 'barrier' encircling the somata of the RCA-poisoned neurons which eventually became totally disorganised. It is postulated that in the course of neuronal degeneration induced by RCA, microglial cells serve to prevent the leakage or diffusion of the toxic lectin from the neuronal somata into the neighbouring neuropil. They also function as scavenger cells in the removal of degenerating myelinated axons in the longer surviving rats. Oligodendrocytes do not appear to react actively to the degeneration process. However, astrocytes showed a significant increase in the 7 and 15 day postoperative rats and this coincided with the presence of mitotic astrocytes in the same period.

The dorsal motor nucleus of the vagus nerve of the hamster was studied electron microscopically. Two types of neuron distinguished by their ultrastructural features were identified. Type I cells, which had an average dimension of 20 X 12 microns, were rich in cytoplasmic organelles. The smaller Type II cells (15 X 9 microns) had scanty cytoplasm and a deeply indented nucleus. Type I cells were labelled by horseradish peroxidase (HRP) following its injection into the vagus nerve, whereas Type II cells were not. Seven days after cervical vagotomy, Type I neurons underwent typical retrograde degeneration. By the fourteenth postoperative day, the density of the cells was greatly enhanced, an early sign of cell death. Type II cells were not affected by vagotomy. With the electron microscope, three types of axon terminals were identified in the neuropil of the DMN: (1) boutons containing round agranular vesicles, (2) boutons containing round and elongated dense-cored vesicles, and (3) boutons containing pleomorphic agranular vesicles, a few of which were dense-cored. All of them showed synaptic contacts with HRP-labelled dendrites. Occasional boutons containing pleomorphic vesicles showed degenerative changes after vagotomy. Moreover, a few of them were seen to contain HRP granules. The observations made in the present study strongly suggest that the Type I neurons send efferent fibres to the vagus nerve. The Type II cells which are not labelled by HRP nor affected by vagotomy, probably serve as interneurons, or they may be cells projecting to other areas of the brain.

Following an injection of horseradish peroxidase solution into the cervical or lumbosacral segments of the spinal cords, or into the parietal cortices, of postnatal (1-10 days), developing (15-20 days) and mature (2 months and older) rats, only postnatal rats demonstrated peroxidase-positive amoeboid microglial cells in the supraventricular part of the corpus callosum. The amoeboid microglial cells were bilaterally labelled following a unilateral injection into the parietal cortex or the spinal cord. They were, however, not labelled after 15 minutes, but were definitely labelled 1 hour after the injection of the solution into the spinal cord. Also, they were not labelled when the solution was injected into the central canal or the subarachnoid space. The results suggest an ascending diffusion of the injected horseradish peroxidase in the spinal cord via the wide interstitial spaces to reach the cerebrum where it is engulfed by the amoeboid microglial cells. An enhanced activity of endogenous peroxidase was also indicated by the observation of peroxidase product in the lysosomal granules of the amoeboid microglial cells.

After injection of horseradish peroxidase (HRP) into the lumbar cord segments of adult albino rats, the rubrospinal (RS-LS) neurons projecting to lumbar cord segments are located predominantly in the ventral and ventrolateral region of the contralateral red nucleus, with a small contingent located ipsilaterally, as reported previously (Shieh et al. 1983). The same pattern of labelling occurs in animals subjected to right cervical and thoracic cord hemisections in rats aged 3-10 days, 20 days and 2-3 months. Nor are there statistically significant differences in the numbers of contralateral (P less than 0.3) and ipsilateral (P less than 0.3) RS-LS neurons between the control and experimental animals. In the experimental group, there are also no significant differences between the numbers of contralaterally (P less than 0.2) versus ipsilaterally (P less than 0.5) labelled RS-LS neurons in animals hemisected 3-5 days and 20-90 days postnatally. The lack of sprouting to the lumbar cord from the RS-LS neurons on either side might be correlated with the existence of the rubrospinal projection at birth. In at least two 3 days, two 10 days, two weanling and two adult experimental animals, an increase in the total area of contralaterally labelled RS-LS neurons could be demonstrated. An increase also occurs in the proportion of medium sized neurons in the lateral horn region of the contralaterally labelled RS-LS neurons in at least three neonatal and three adult experimental animals.