ß-1,4-Galactosyltransferase III suppresses extravillous trophoblast invasion through modifying ß1-integrin glycosylation.

INTRODUCTION: Glycosylation controls diverse protein functions and regulates various cellular phenotypes. Trophoblast invasion is essential for normal placental development. However, the role of glycosylation in human placenta throughout pregnancy is still unclear. The ß-1,4-galactosyltransferase III (B4GALT3) has been found to regulate cancer cell invasion. We therefore investigated the expression of B4GALT3 in placenta and its roles in trophoblast. METHODS: B4GALT3 protein expression was examined by quantitative Western blotting analysis in human placentas. For identification of B4GALT3-positive cells in normal human placenta, immunohistochemistry and immunofluorescence methods were used. To investigate effects of B4GALT3 on extravillous trophoblast (EVT)-like cell and primary EVT cells, we analyzed cell growth, adhesion, migration, and invasion in mock and B4GALT3-transfected cell. RESULTS: B4GALT3 expression significantly increased in third trimester human placenta. Immunostaining revealed that B4GALT3 expressed in placental villous cytotrophoblast, syncytiotrophoblast, and a subpopulation of EVT cells throughout pregnancy. Interestingly, we found increases in the expression level and percentage of B4GALT3-positive cells in third trimester EVT, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. Overexpression of B4GALT3 in HTR8/SVneo cells and primary trophoblast cells significantly suppressed cell migration. In addition, B4GALT3 suppressed cell invasion, and enhanced cell adhesion to laminin in HTR8/SVneo cells. Notably, we found that B4GALT3 modified glycans on ß1-integrin, suppressed focal adhesion kinase (FAK) signaling, and enhanced ß1-integrin degradation. DISCUSSION: We propose that B4GALT3-mediated glycosylation change not only enhances ß1-integrin binding to laminin, but also attenuates ß1-integrin stability. Our findings suggest that B4GALT3 is a critical regulator for suppressing EVT invasion in the late stages of pregnancy.

Expression of GALNT2 in human extravillous trophoblasts and its suppressive role in trophoblast invasion.

Extravillus trophoblast (EVT) invasion plays a critical role in placental development. Integrins bind to extracellular matrix (ECM) proteins to mediate EVT cell adhesion, migration, and invasion. Changes in O-glycans on ß1-integrin have been found to regulate cancer cell behavior. We hypothesize that O-glycosyltransferases can regulate EVT invasion through modulating the glycosylation and function of ß1-integrin. Here, we found that the GALNT1 and GALNT2 mRNA were highly expressed in HTR8/SVneo and first trimester EVT cells. Immunohistochemstry and immunofluorescence staining showed that GALNT2 was expressed in subpopulations of EVT cells in deciduas, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. The percentage of GALNT2-positive EVT cells increased with gestational ages. Overexpression of GALNT2 in HTR8/SVneo cells significantly enhanced cell-collagen IV adhesion, but suppressed cell migration and invasion. Notably, we found that GALNT2 increased the expression of Tn antigen (GalNAc-Ser/Thr) on ß1-integrin as revealed by Vicia Villosa agglutinin (VVA) binding. Furthermore, GALNT2 suppressed the phosphorylation of focal adhesion kinase (FAK), a crucial downstream signaling molecule of ß1-integrin. Our findings suggest that GALNT2 is a critical initiating enzyme of O-glycosylation for regulating EVT invasion.

Influence of sleep deprivation coupled with administration of melatonin on the ultrastructure of rat pineal gland.

The effects of sleep deprivation with or without melatonin treatment on the pineal morphology in rats were studied. Five days after sleep deprivation and using electron microscopy, many of the pinealocytes exhibited structural alterations including dilation of the cisternae of the rough/smooth endoplasmic reticulum, Golgi saccules and mitochondria, and an increase in the numbers of lipid droplets, vacuoles and dense-core vesicles. These features were considered as morphological evidence of increased synthesis or secretion by the pineal gland. In addition, numerous membranous profiles, considered to be degraded cellular organelles, were observed in some pinealocytes and sympathetic nerve terminals. It is suggested that the occurrence of degenerating organelles had resulted from the deleterious effect of sleep deprivation. This may be attributed to an overload of secretory activity of the pineal gland during stress elicited by the long-term sleep deprivation, leading to functional exhaustion and irreversible damage of the oxidation-related organelles. In sleep-deprived rats receiving a single injection of melatonin (10 mg/kg) for 5 consecutive days, the above features indicative of pinealocytic activation were attenuated. In fact, all signs of degeneration of cellular organelles were rarely found. These results suggest that the pineal gland is itself a target for exogenously administered melatonin. Thus, melatonin when administered systemically may be used as a potential neuroprotective drug against neuronal damage induced by sleep deprivation.

Sensory restoration of the skin graft on a free muscle flap: experimental rabbit study.

Transplantation of a muscle flap with free skin graft for wound coverage is a common procedure in reconstructive microsurgery. However, the grafted skin has little or no sensation. Restoration of the sensibility of the grafted skin on the transferred muscle is critically important, especially in palmar hand, plantar foot, heel, and oral cavity reconstruction. The purpose of this study was to investigate the possibility of sensory restoration of the grafted skin on a trimmed muscle surface that has been sensory neurotized after sensory nerve-to-motor nerve transfer, using the rabbit gracilis muscle as an animal model. The ipsilateral saphenous nerve (sensory) was transferred to the motor nerve of the gracilis muscle for sensory neurotization. A 4 x 4-cm2 area of skin island over the midportion of the gracilis muscle was harvested as a full-thickness skin graft. The upper half of the gracilis muscle was then excised, becoming a rough surface. The harvested skin was reapplied on the trimmed rough surface of the muscle. After 6 months, retrograde and antegrade horseradish peroxidase labeling studies were performed through skin and muscle injection. The group with a free skin graft was compared with the group with an intact surface of the gracilis muscle. This study clearly shows that sensory nerves can regenerate and penetrate into the trimmed muscle surface and grow into the overlying grafted skin. However, if the muscle surface is intact as with the compared group, sensory reinnervation of the grafted skin is not possible.

Different astroglial reaction between the vagal dorsal motor nucleus and nucleus ambiguus following vagal-hypoglossal nerve anastomosis in cats.

The dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA) were both traced with horseradish peroxidase (HRP) retrograde labelling technique after vagal-hypoglossal nerve anastomosis (VHA). By light microscopy, reinnervation of the new target, viz. tongue skeletal musculature, by DMV and NA was established at 22 days postoperation (dpo) as shown by the neuronal labelling with HRP. Ultrastructurally, signs of retrograde degeneration occurred in some DMV and NA neurons between 3 and 25 days after VHA. The incidence of darkened dendrites, an early sign of dendritic loss, was more common in the DMV compared to the NA. Accompanying the neuronal alteration were drastic astrocytic reactions in the DMV, but not in the NA. Between 3 and 7 dpo, the astrocytes in the DMV showed extensively hypertrophied processes and by 22 dpo, the somata and dendrites of HRP-labelled DMV neurons, but not NA's, appeared to be delineated by the increased lamellar astrocytic processes. Such a feature was sustained throughout the remaining postoperative intervals up to 500 dpo. It is concluded that the DMV motoneurons being autonomic in nature are probably not conducive to the newly acquired target organ. Hence, the insulation of the regenerating DMV motoneurons by the astroglial ensheathment would be vital in the neuronal remodelling and reconstruction of the vagal-hypoglossal pathway.

Target-dependent axonal sprouting following vagal-hypoglossal nerve anastomosis in cats.

PURPOSE: To investigate the relationships between the axonal sprouting and target neurotization by central neurons after nerve heteroconnection. METHODS: Unilateral (right) vagal-hypoglossal nerve anastomosis (VHA) was performed in adult cats. Following 3-315 days postoperation (dpo), quantitative analyses and ultrastructural changes in the proximal portion of the vagal-hypoglossal heteroconnected nerve as well as the time course of neuronal regeneration were studied. Along with this, horseradish peroxidase (HRP) retrograde tracing technique was used to label the neurons of dorsal motor vagal nucleus (DMV) and nucleus ambiguus (NA) to ascertain if target neurotization was established. RESULTS: The contralateral (left) intact vagus nerve proximal to the level of ansa cervicalis showed an average of 33 ± 1 myelinated and 74 ± 4 unmyelinated axons in 727 µm2 sectional area of the nerve. In the heteroconnected nerve at the corresponding level just proximal to the anastomosis site, there was a marked increase in the number of small axons sprouting from the unmyelinated nerve fibers between 18 and 25 dpo. The number of these axonal sprouts appeared to decline at 32 dpo but its increase of 131 % was sustained until the late regeneration stage at 315 dpo when compared with the contralateral nerve serving as a control. The mean number of myelinated axons per area unit (727 µm2) was reduced to 18 at 3 dpo but was immediately restored to the normal range at 7 dpo. The retrograde labelling of neurons in both the DMV and NA was first detected at 22 dpo and was progressively increased peaking by about 67 dpo. CONCLUSIONS: We conclude that compared with the unmyelinated axons, the myelinated axons may acquire a superior interaction with the new target. Furthermore, the postoperative neurotization of tongue muscles may initiate and facilitate the retraction of the redundant axonal sprouts.

Evidence of a direct projection from the cardiovascular-reactive dorsal medulla to the intermediolateral cell column of the spinal cord in cats as revealed by light and electron microscopy.

To ascertain whether the dorsal medulla, a well-established vasopressor structure, would project directly to the sympathetic intermediolateral cell column of the spinal cord, we have carried out both anterograde and retrograde tracing studies in cats. For anterograde tracing, biotin-dextran was iontophoretically delivered into the cardiovascular-reactive dorsal medulla following its functional identification by electrical stimulation. The anterogradely transported biotin-dextran was then visualized using the avidin-biotin-horseradish peroxidase complex method. By light microscopy, dextran-labelled varicose axons were observed bilaterally in the intermediolateral nucleus extending from segments T1 to L3, but concentrated in segments T1-T5, notably at levels T2-T4. Electron microscopic examination revealed the localization of biotin-dextran reaction product in some small myelinated axons and axon terminals in the intermediolateral cell column. The majority of tracer-labelled axonal boutons contained spherical synaptic vesicles and made asymmetric synaptic contacts primarily with small dendrites. A few boutons contained polymorphic synaptic vesicles and tended to form symmetric axodendritic synapses. Spinally projecting neurons of the dorsal medulla were identified using the retrograde transport of horseradish peroxidase injected into the electrically cardiovascular-reactive intermediolateral nucleus. The labelled neurons were localized in the medullary dorsomedial reticular formation ventromedial to the nucleus of the solitary tract, approximately 0.5-5 mm rostral to the obex. The projection was bilateral, but was relatively denser in the rostral portion of the contralateral dorsal medulla. The present findings support the hypothesis that the dorsal medulla, through its direct pathway innervating the intermediolateral nucleus, may serve as a sympathetic premotor structure for regulation of arterial pressure.

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.

Ultrastructural study of phenylethanolamine-N-methyltransferase, corticotropin-releasing factor and neurotensin immunoreactive neurons in the external cuneate nucleus of the gerbil.

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.

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.

Ultrastructural identification of cholinergic neurons in the external cuneate nucleus of the gerbil: acetylcholinesterase histochemistry and choline acetyltransferase immunocytochemistry.

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.

A comparative ultrastructural study of primary afferents from the brachial and cervical plexuses to the external cuneate nucleus of gerbils.

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.

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.

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.

Multiple origins of cerebellar cholinergic afferents from the lower brainstem in the gerbil.

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.

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.

Cells of origin, thalamic relay and termination of the external cuneothalamocortical tract in the gerbil.

The present study is concerned with the connections of the external cuneate nucleus (ECN) in the gerbil following an injection of horseradish peroxidase (HRP) into the ventralis posterior pars oralis (VPLo) or adjacent nuclei of the thalamus. The number, soma size and distribution of the retrograde-labelled ECN neurons were studied and quantified. The application of two retrograde fluorescent tracers was also used to determine whether the ECN neurons would project to the thalamus as well as to the cerebellum through their collaterals. The HRP-positive ECN neurons projecting to the thalamic VPLo were confined to the contralateral caudal half of the ECN, primarily within the intermediate portion represent the forearm and arm territories with a small part of the thoracic and shoulder areas. Labelled neurons were classified into small and medium-sized cells. The majority (96%) of the external cuneothalamic neurons were of the small variety. No double-labelled cells were detected in the ECN following injections of Rhodamine-labelled latex microspheres and Fast blue into the cerebellum and thalamus respectively, suggesting that the ECN neurons projecting to the thalamus form a separate cell group different from those projecting to the cerebellum. The injected HRP into the VPLo was also transported in an anterograde direction by the thalamocortical fibers. The HRP-labelled axonal terminals were distributed within motor area 4 and the dysgranular zones (DZs) of the primary somatosensory cortex (SmI), reaching the deep layers IV and VI as well as superficial layer I. The external cuneothalamocortical pathway shown in the present study may be related to the proprioceptive feedback control of the coordinating motor activity, especially during forelimb muscle movement.

Anatomical studies on the cuneocerebellar neurons in the gerbil by using HRP method.

The projection from the external cuneate nucleus (ECN) to the cerebellum was studied in the gerbil following the retrograde transport of minute injections (0.05-0.1 microliter, 30% solution) or implantations of horseradish peroxidase (HRP) in various folia of the cerebellar cortex and deep nuclei. The morphology of the labelled neurons, as well as their quantitative distribution, was also examined. The projections were in general bilateral but predominantly ipsilateral. A remarkable finding, however, was the predominant contralateral projection from the medial cell column of ECN representing the paw region. Topographically, the nuclear areas receiving the thoracic, shoulder and neck muscle afferents projected bilaterally to vermis lobules I-V and VIII-IX, lobulus simplex, crura Ia and Ib, paraflocculus and flocculus, and contralaterally to crus II and nucleus interpositus; the nuclear regions receiving the forelimb muscle afferents sent fibers bilaterally to vermis lobules V-VIII, paramedian lobule and copula pyramidis, and ipsilaterally to crus II and nucleus interpositus. Based on their somal size, three classes of neurons were distinguished: small, medium and large cells. The small and medium cells constituted 79 and 20% of the population of the labelled cells respectively, whereas the large cells were only occasionally identified. In the cell columns of the caudal ECN representing the forelimb (Lan et al. 1994), most labelled neurons were medium cells which made up 65% of the total labelled medium cells. In contrast, the majority (90%) of the labelled cells in the rostral ECN were of the small-sized variety. The functional significance of the segregation, both in the projection patterns and sizes of somata of the cuneocerebellar neurons is discussed.

Musculotopic organization of muscle afferents in the external cuneate nucleus of the gerbil as revealed by transganglionic transport of horseradish peroxidase.

The projections of muscle afferents from six regions (hand, forearm, arm, thorax, shoulder and neck) to the external cuneate nucleus (ECN) of gerbils were investigated using transganglionic transport of horseradish peroxidase (HRP). These were chiefly ipsilateral projections which terminated in six discrete longitudinal columns in the ECN. A medial cell column confined to the caudal ECN received projections from the hand muscles, while the ventromedial and ventrolateral cell columns in the posterior half of the ECN received projections from the forearm and arm muscles respectively. In the caudal ECN, the thoracic and shoulder muscles were represented by the dorsomedial and dorsolateral cell columns respectively. The last mentioned two columns extended anteriorly into the rostral ECN occupying a dorsal and intermediate portion leaving the ventral portion which represented the neck muscles. The latter also extended posteriorly into the middle 1/3 of ECN occupying a lateral portion. Serial sections and reconstruction studies showed that the ECN was located in the dorsal region of the upper medulla, approximately 0.3-1.8 mm rostral to the obex. The rostrocaudal extent of the six cell columns from hand proximally to neck were measured to be 0.5-0.8, 0.3-1.1, 0.3-1.3, 0.4-1.8, 0.4-1.8 and 0.8-1.8 mm rostral to the obex.