Identification of molecular motors in the Woods Hole squid, Loligo pealei: an expressed sequence tag approach.

The squid giant axon and synapse are unique systems for studying neuronal function. While a few nucleotide and amino acid sequences have been obtained from squid, large scale genetic and proteomic information is lacking. We have been particularly interested in motors present in axons and their roles in transport processes. Here, to obtain genetic data and to identify motors expressed in squid, we initiated an expressed sequence tag project by single-pass sequencing mRNAs isolated from the stellate ganglia of the Woods Hole Squid, Loligo pealei. A total of 22,689 high quality expressed sequence tag (EST) sequences were obtained and subjected to basic local alignment search tool analysis. Seventy six percent of these sequences matched genes in the National Center for Bioinformatics databases. By CAP3 analysis this library contained 2459 contigs and 7568 singletons. Mining for motors successfully identified six kinesins, six myosins, a single dynein heavy chain, as well as components of the dynactin complex, and motor light chains and accessory proteins. This initiative demonstrates that EST projects represent an effective approach to obtain sequences of interest.

Prions in the peripheral nerves of bovine spongiform encephalopathy-affected cattle.

With the use of increasingly sensitive methods for detection of the abnormal isoform of prion protein (PrP(Sc)) and infectivity in prion diseases, it has recently been shown that parts of the peripheral nervous system (PNS) of bovine spongiform encephalopathy (BSE)-affected cattle may become infected. It has been reported that prions spread to the central nervous system (CNS) via the PNS in sheep scrapie, but the pathogenesis of BSE in cattle is less well understood. To determine whether parts of the PNS other than those implicated directly in the hypothetical pathogenetic spread of agent from the intestine to the CNS become involved before or after the CNS is affected, PrP(Sc) distribution was investigated by a highly sensitive Western blotting technique in dorsal root ganglia, stellate ganglion, phrenic, radial and sciatic nerves, adrenal gland and CNS of cattle that were inoculated orally with BSE-affected brain and culled sequentially. In experimentally BSE-affected cattle, PrP(Sc) was first detected in the CNS and dorsal root ganglia; subsequently, PrP(Sc) accumulation was detected in the peripheral nerve trunks. PrP(Sc) was also detected in the adrenal glands of cattle that showed clinical signs. No PrP(Sc) was detected in the PNS of BSE-negative cattle. This study shows that, with respect to dorsal root ganglia, a paravertebral sympathetic ganglion and the somatic nerves examined, PrP(Sc) is detected in the PNS during the disease course at the same time as, or after, it accumulates in the CNS.

Diabetes-induced biochemical changes in central and peripheral catecholaminergic systems.

A great variety of alterations have been described in the nervous system of diabetic animals. They are named as diabetic neuropathy and affect the brain, spinal cord and peripheral nerves. In diabetic animals, plasma and tissue catecholamine levels have been reported to be increased, decreased or unchanged, and these disparities have been explained by differences in the tissues selected, severity or duration of diabetes. Dopamine, norepinephrine and epinephrine from different tissues were extracted by absorption onto alumina, and measured by high performance liquid chromatography with electrochemical detection. We found that diabetes alters catecholaminergic systems in a highly specific manner. The dopamine content is reduced in the dopaminergic nigrostriatal system only. Norepinephrine is differently altered in several areas of the sympathetic nervous system. It is increased in cardiac ventricles, and decreased in stellate ganglia and the blood serum. However, it is not altered in the central nervous system. Finally, epinephrine is only altered in the adrenal gland where it is increased, and in the serum where it is reduced. Our results suggest that diabetes reduces the activity of the nigrostriatal dopaminergic system. Changes found at the sympathoadrenal level could be explained by reduced norepinephrine and epinephrine synthesis, with increased storage due to a reduced release from synaptic vesicles.

The perikaryal surface of spinal ganglion neurons: differences between domains in contact with satellite cells and in contact with the extracellular matrix.

The perikaryal surface of spinal ganglion neurons undergoes dynamic changes throughout life. In particular, numerous slender projections develop and retract continuously from this surface. We showed previously that the outgrowth of these projections, while an intrinsic property of spinal ganglion neurons, is also influenced by the surrounding microenvironment. Since the latter consists of satellite cells and the extracellular matrix, we sought to determine the relative contributions of each of these components to the outgrowth of perikaryal projections. To this end, we took advantage of a little known characteristic of the satellite cell sheaths: in the rabbit, these sheaths can exhibit gaps that leave the nerve cell body surface directly exposed to the extracellular matrix. We compared the surface domains covered by satellite cells with those in direct contact with the extracellular matrix. We found that the perikaryal projections are abundant in the former domains but are absent in the latter. We also found that the perineuronal extracellular matrix of rabbit spinal ganglia contains laminin and fibronectin, two glycoproteins that have been reported to promote the growth of axonal processes from sensory ganglion neurons. Laminin and fibronectin were also present at the level of the gaps in the satellite cell sheath. These results: (1) provide additional evidence that environmental factors influence the outgrowth of perikaryal projections from spinal ganglion neurons; (2) suggest that satellite cells permit the outgrowth of these projections; (3) suggest that in the spinal ganglia of adult rabbits the perineuronal extracellular matrix is not in itself able to promote the outgrowth of these projections. This study provides a further example of the influence that supporting neuroglial cells have on sensory ganglion neurons.

Topographic relationship of neurotensin-containing axon terminals with cardiac and noncardiac principal ganglion cells in the stellate ganglia of the cat.

The pattern of association between neurotensin (NT)-immunoreactive (NTIR) preganglionic nerve terminals and cardiac and noncardiac neurons in the stellate ganglion of the cat is analyzed, based on the finding of an excitatory modulation effect of exogenous NT on cardiac functions. For this purpose, NT-containing terminals were labeled by immunohistochemistry, and ganglion cells were detected by retrograde labeling of cardiac and vertebral nerves to identify cardiac and noncardiac neurons. To determine a possible regional localization of NTIR terminals and ganglion cells, the ganglia were divided into four areas: caudal, dorsomedial, cranial, and ventromedial, related to the two major afferent nerves (thoracic white rami 3 [T3WR] and 2 [T2WR]) and the two efferent nerves (vertebral and cardiac). NTIR terminals were widespread in the complete ganglion tissue; they covered practically all the regions explored, although two clusters of high concentration of NTIR terminals were detected in the cranial and caudal areas. By retrograde labelling it was found that cardiac cells were arranged around the exit of the cardiac nerve and that the vertebral neurons were extended from the exit of the vertebral nerve to the entrance of T3WR. The finding of association of NTIR terminals with cardiac neurons may account for the cardioregulatory effect of NT; however, since the presence of NTIR terminals close to the noncardiac neurons is notorious, other regulatory functions of NT must be considered.

Noradrenergic hyperinnervation in the heart of stroke-prone spontaneously hypertensive rats (SHRSP).

Noradrenergic (NA) nerve fiber density was investigated in the subepicardium and myocardium of ventricles in stroke-prone spontaneously hypertensive rats (SHRSP) and was compared with that of normotensive Wistar Kyoto (WKY) rats. Fluorescent NA nerve fibers in the subepicardium of the right and left ventricles of both strains at the ages of 10, 30, 60, 90, and 180 days were examined by the glyoxylic acid method. NA nerve fibers in the myocardium of the right and left ventricles and the ventricular septum of both strains at the ages of 30, 90, and 180 days were also examined in a similar manner. The density of NA nerve fibers was measured by quantitative image analysis. The distribution pattern of NA nerve fibers in the entire subepicardium of ventricles of both strains showed a meshwork pattern throughout the examination period. In sections of the myocardium, NA nerve fibers were distributed between heart muscle cells and around blood vessels in both strains at all ages examined. The densities of NA nerve fibers in the subepicardium of the ventricles of SHRSP were significantly higher than those of WKY rats at all ages examined except for the subepicardium of the left ventricle at 90 days of age. The densities in the myocardium of the right ventricle in 30- and 90-day-old SHRSP were significantly higher than those in WKY rats. The ratios of NA nerve fiber density of SHRSP to that of WKY rats were greater in the subepicardium of the right and left ventricles, except at 90 days of age, and in the myocardium of the right ventricle of younger animals as compared with older ones. NA hyperinnervation in the subepicardium and myocardium of the ventricles of SHRSP may be a primary change of the heart before the onset of hypertension and may be caused by hyperfunction of the stellate ganglia.

Localization of choline acetyltransferase in rat peripheral sympathetic neurons and its coexistence with nitric oxide synthase and neuropeptides.

Indirect immunofluorescence methods using a mouse monoclonal antibody raised to rat choline acetyltransferase (ChAT) revealed dense networks of ChAT-immunoreactive fibers in the superior cervical ganglion, the stellate ganglion, and the celiac superior mesenteric ganglion of the rat. Numerous and single ChAT-immunoreactive cell bodies were observed in the stellate and superior cervical ganglia, respectively. The majority of ChAT-immunoreactive fibers in the stellate and superior cervical ganglia were nitric oxide synthase (NOS) positive. Some ChAT-immunoreactive fibers contained enkephalin-like immunoreactivity. Virtually all ChAT-positive cell bodies in the stellate ganglion were vasoactive intestinal polypeptide (VIP)-positive, and some were calcitonin gene-related peptide (CGRP)-positive. After transection of the cervical sympathetic trunk almost all ChAT- and NOS-positive fibers and most enkephalin- and CGRP-positive fibers disappeared in the superior cervical ganglion. The results suggest that most preganglionic fibers are cholinergic and that the majority of these in addition can release nitric oxide, some enkephalin, and a few CGRP. Acetylcholine, VIP, and CGRP are coexisting messenger molecules in some postganglionic sympathetic neurons.

Autologous transplantation of the cervical sympathetic ganglion into the parkinsonian brain: case report.

The authors have reported a clinical trial of an autologous cervical sympathetic ganglion transplanted into the brain of a parkinsonian patient. A 45-year-old woman presented with bradykinesia and a gait disturbance for 8 years under L-dopa treatment. The patient underwent stereotactic transplantation of the right stellate ganglion into the right putamen. She showed marked amelioration of bradykinesia and gait disturbance 1 month after the operation, and she was able to conduct her activities of daily living without requiring L-dopa administration. The patient continued to improve gradually until 3 months after the operation. Two years after surgery, the patient functions independently as a housewife. The right hand tremor, however, became slightly worse after the operation, but it was transient. The patient developed a permanent right-sided Horner's syndrome after resection of the cervical sympathetic ganglion. Taken together with our previous data obtained from animal experiments, this case suggests that the autologous cervical sympathetic ganglion can be donor tissue for neural transplantation in Parkinson's disease.

Ultrastructural localization of neurotensin immunoreactivity in the stellate ganglion of the cat.

The morphological features and cellular relationships of neurotensin-containing axon terminals were studied at light and electron microscopic levels in the cat stellate ganglion using peroxidase and immunogold immunocytochemistry. By light microscopy, neurotensin immunoreactivity was detected within thin varicose fibres distributed throughout the ganglion. Immunoreactive fibres were no longer apparent following chronic deafferentation of the ganglion indicating that they were of extrinsic origin. Ultrastructural analysis of peroxidase immunostained material confirmed the presence of neurotensin immunoreactivity within a subpopulation of axonal varicosities which made synaptic contacts with the dendrites of ganglion cells. Within labelled varicosities neurotensin immunoreactivity was found by both immunoperoxidase and immunogold methods to be concentrated within large dense core vesicles 80-120 nm in diameter. These large dense core vesicles were characteristically distant from the active zone, in keeping with a possible extrasynaptic release of the peptide.

Immunohistochemical evidence for different pathways immunoreactive to substance P and calcitonin gene-related peptide (CGRP) in the guinea-pig stellate ganglion.

The colocalization of immunoreactivities to substance P and calcitonin gene-related peptide (CGRP) in nervous structures and their correlation with other peptidergic structures were studied in the stellate ganglion of the guinea pig by the application of double-labelling immunofluorescence. Three types of fibre were distinguished. (1) Substance P+/CGRP+ fibres, which sometimes displayed additional immunoreactivity for enkephalin, constituted a small fibre population of sensory origin, as deduced from retrograde labelling of substance P+/CGRP+ dorsal root ganglion cells. (2) Substance P+/CGRP- fibres were more frequent; some formed baskets around non-catecholaminergic perikarya that were immunoreactive to vasoactive intestinal polypeptide (VIP). (3) CGRP+/substance P- fibres were most frequent and were mainly distributed among tyrosine hydroxylase (TH)-immunoreactive cell bodies. The peptide content of fibre populations (2) and (3) did not correspond to that of sensory ganglion cells retrogradely labelled by tracer injection into the stellate ganglion. Therefore, these fibres are thought to arise from retrogradely labelled preganglionic sympathetic neurons of the spinal cord, in which transmitter levels may have been too low for immunohistochemical detection of substance P or CGRP. CGRP-immunoreactivity but no substance P-immunolabelling was observed in VIP-immunoreactive postganglionic neurons. Such cell bodies were TH-negative and were spared by substance P-immunolabelled fibre baskets. Retrograde tracing with Fast Blue indicated that the sweat glands in the glabrous skin of the forepaw were the targets of these neurons. The streptavidin-biotin-peroxidase method at the electron-microscope level demonstrated that immunoreactivity to substance P and CGRP was present in dense-cored vesicles of 50-130 nm diameter in varicosities of non-myelinated nerve fibres in the stellate ganglion. No statistically significant difference in size was observed between vesicles immunolabelled for substance P and CGRP. Immunoreactive varicosities formed axodendritic and axosomatic synaptic contacts, and unspecialized appositions to non-reactive neuronal dendrites, somata, and axon terminals. Many varicosities were partly exposed to the interstitial space. The findings provide evidence for different pathways utilizing substance P and/or CGRP in the guinea-pig stellate ganglion.