Processing of the FMRFamide Precursor Protein in the Snail Lymnaea stagnalis: Characterization and Neuronal Localization of a Novel Peptide, 'SEEPLY'

In the pulmonate snail Lymnaea stagnalis , FMRFamide-like neuropeptides are encoded by a multi-exon genomic locus which is subject to regulation at the level of mRNA splicing. We aim to understand the post-translational processing of one resulting protein precursor encoding the tetrapeptide FMRFamide and a number of other putative peptides, and determine the distribution of the final peptide products in the central nervous system (CNS) and periphery of Lymnaea. We focused on two previously unknown peptide sequences predicted by molecular cloning to be encoded in the tetrapeptide protein precursor consecutively, separated by the tetrabasic cleavage site RKRR. Here we report the isolation and structural characterization of a novel non-FMRFamide-like peptide, the 22 amino acid peptide SEQPDVDDYLRDWLQSEEPLY. The novel peptide is colocalized with FMRFamide in the CNS in a number of identified neuronal systems and their peripheral motor targets, as determined by in situ hybridization and immunocytochemistry. Its detection in heart excitatory motoneurons and in nerve fibres of the heart indicated that the novel peptide may play a role, together with FMRFamide, in heart regulation in the snail. The second predicted peptide, STEAGGQSEEMTHRTA (16 amino acids), was at very low abundance in the CNS and was only occasionally detected. Our current findings, suggestive of a distinct pattern of post-translational processing, allowed the reassessment of a previously proposed hypothesis that the two equivalent sequences in the Aplysia FMRFamide gene constitute a molluscan homologue of vertebrate corticotrophin releasing factor-like peptides.     

A comparison of the effects of serotonin, substance P and thyrotropin-releasing hormone on excitability of rat spinal motoneurons in vivo

Lumbar spinal motoneurons of urethane-anesthetized rats were driven at stable low firing rates by automatically cycled iontophoretic applications of glutamate or aspartate. The effects of iontophoretically applied serotonin, substance P or thyrotropin-releasing hormone (TRH) on glutamate or aspartate-evoked activity were then tested. All 3 substances were found to enhance both glutamate- and aspartate-induced excitation of the motoneurons. This enhancement of excitability was usually preceded by a brief period of inhibition at current onset. Although the effects of serotonin and substance P were qualitatively remarkably similar, TRH differed in that TRH occasionally inhibited motoneuron excitability without subsequent facilitation, and tachyphylaxis developed for the facilitatory effects of TRH. After TRH desensitization, serotonin could still enhance spinal motoneuron excitability.     

Immunohistochemical evidence for the coexistence of calcitonin gene-related peptide- and choline acetyltransferase-like immunoreactivity in neurons of the rat hypoglossal, facial and ambiguus nuclei

The present immunocytochemical study demonstrates that calcitonin gene-related peptide-like immunoreactivity (CGRPI) coexists with acetylcholine in single cells of hypoglossal, facial and ambiguus nuclei. The experiments were done using alternate frozen sections from relevant regions of the rat brain. We further show that CGRPI is localized in the nerve terminals that form neuromuscular junctions in the tongue muscles.     

大鼠运动神经元对胶体金联结抗运动神经元单抗的摄取和转运

应用胶体金联结抗运动神经元(Mn)单抗(Au-McAb24BO)研究大鼠Mn对此单抗(IgG)的特异性摄取和转运。将Au-McAb24BO注射于成年Wistar大鼠的舌肌、面肌、四肢肌和胃壁内，舌肌内还注射胶体金联结人IgG和单纯胶体金(φ7nm)作对照，脑脊髓切片经银加强后显示舌下神经核、面神经核及脊髓颈、腰膨大的前角内有Au-McAb24BO阳性神经元，迷走神经背核及初级感觉神经元内束见阳性标记。实验证明Au-McAb24BO被躯体运动神经元特异性地摄取和逆行转运。神经元对免疫球蛋白的摄取和转运可能在某些自身免疫性运动神经元病发病中有重要意义。     

Motoneurons of the tail of young Xenopus tadpoles

The purpose of this study was to identify, characterize, and follow the morphogenesis of motoneurons innervating tail myotomes of Xenopus during early and middle larval stages (33-48). Application of horseradish peroxidase to a single myotome results in a column of labeled motor cells extending over four spinal segments. By midlarval stages two morphologically distinct cell types were recognizable in this column-primary and secondary motoneurons. Primary motoneurons occupied the more rostral and secondary motoneurons the more caudal portions of the column with some overlap of the two populations. Primary neurons developed earlier, were larger, and displayed thicker axons and proximal dendrites. Secondary neurons, in contrast, were smaller, more variable in configuration and dendritic distribution, and displayed thinner axons and dendrites. Development of the labeled motor column during the period observed involved the addition of more labeled neurons, principally secondary cells, and the enlargement of individual cell somata with the progressive elaboration of more and longer dendrites into the developing lateral fasciculus.     

雄性大鼠盆底横纹肌运动神经元的定位分布

目的：探讨研究正常雄性大鼠盆底横纹肌运动神经元的定位分布。方法：分别用辣根过氧化物酶(HRP)、荧光金(FG)注射于正常雄性SD大鼠的尿道外括约肌、坐骨海绵体肌、球海绵体肌、肌门外括约肌，行逆行神经追踪。结果：尿道外括约肌或坐骨海绵体肌注射HRP或FG后，标记神经元位于脊髓L5～S1段前角背外侧核；球海绵体肌或肛门外括约肌注射HRP或FG后，标记神经元位于脊髓L5～S1段前角背内侧核。结论：雄性大鼠支配盆底横纹肌的运动神经元位于两个区域：支配坐骨海绵体肌运动神经元聚集分布于脊髓L5～S1(以L6多见)段前角背外侧核；支配球海绵体肌和肛门外括约肌的运动神经元主要聚集分布于脊髓L5～S1(以L6多见)段前角背内侧核。     

Different classes of cat spinal neurons display differential sensitivity to sodium pentobarbital

The effect of graded doses of systemically injected sodium pentobarbital on several classes of spinal neurons was studied using spinal cats. Classes of spinal neurons included unidentified dorsal horn cells, ascending tract dorsal horn cells, and motoneurons. Single unit activity of spinal neurons was evoked by electrically stimulating a peripheral nerve with an intensity strong enough to excite both A and C fibers. The A- and C-fiber evoked activity was compared before and after intravenous injections of small incremental doses of sodium pentobarbital. The activity of different classes of spinal neurons showed different sensitivities to graded doses of systemically injected pentobarbital. The reflex activity of motoneurons elicited by stimulation of peripheral nerve was much more sensitive to pentobarbital than that of dorsal horn cells. In general, activity evoked by peripheral unmyelinated fibers was more susceptible to pentobarbital than was that evoked by myelinated fibers. However, intravenous injections of pentobarbital produced nondifferential suppression of dorsal horn cell activity evoked by noxious and innocuous mechanical stimuli applied to the peripheral receptive fields.     

Musculotopic organization of the facial motor nucleus in Macaca fascicularis: a morphometric and retrograde tracing study with cholera toxin B-HRP

Morphometric and retrograde tracing methods were used to determine the location and number of motoneurons innervating individual facial muscles in Macaca fascicularis. Intramuscular injections of the cholera toxin B subunit-horseradish peroxidase conjugate produced discrete labeling patterns in the ipsilateral facial motor nucleus with good definition of somata and their processes. The facial nucleus extended rostrocaudally in the pons for about 2 mm, varying in shape and cross-sectional area along this axis. Motoneurons were clustered in subnuclei, but their boundaries were not sharp and they were not segregated by fiber bundles. The length, number, and area of subnuclei varied with rostrocaudal location. Retrograde labeling patterns revealed that individual muscles were innervated by longitudinal columns of motoneurons with each muscle region represented at all rostrocaudal levels of its column. The columns began at different rostrocaudal levels and varied in length. Columns for closely related muscles, such as the orbicularis oris and mentalis of the lower lip, tended to overlap, whereas columns for disparate muscles, such as the perioral and orbital, did not overlap. The dendritic processes of most motoneurons branched extensively among several different columns or subnuclei. Some dendrites extended outside of the nucleus into the surrounding tegmentum. Mean soma diameter (10.4-42.2 microns) was distributed unimodally, reflecting the absence of gamma motoneurons and lack of muscle spindles in the facial muscles. Large and small motoneurons were found in all regions of the nucleus, but the largest ones were located caudally and innervated muscles of the upper and lower lip. The perioral muscles also had more neurons, longer columns, and a lower cell density than the other muscle groups examined. These features may reflect the functions of the perioral muscles in facial expression and vocalization.     

Evidence of a Monosynaptic Pathway Between Cells of the Ventromedial Medulla and the Motoneuron Pool of the Thoracic Spinal Cord in Rat: Electron Microscopic Analysis of Synaptic Contacts

Previous electrophysiological and anatomical data have suggested the existence of a descending pathway from the ventromedial medulla into the thoracic motoneuron pool. However, systematic light and electron microscopic analysis have not yet been done to reveal such a projection. In the present study, the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into several discrete regions of the medioventral medulla and descending PHA-L-labelled axons were investigated in the thoracic ventral horn using both light and electron microscopy. Light microscopic analysis of descending projections from 20 distinct areas of the medioventral medulla showed that neurons that project predominantly to the intermediate and ventral regions of the thoracic spinal grey matter are located caudal to the facial nucleus. Monosynaptic contacts were found between axons originating from five distinct regions of the medioventral medulla (containing raphé and/or gigantocellular reticular neurons) and cells in the thoracic motoneuron pool. PHA-L-labelled boutons formed synaptic contacts with large calibre dendrites and with somata. Seventy-two per cent of the investigated 32 boutons appeared to have symmetrical synaptic membrane specializations. The majority of the boutons contained only small, pleomorphic vesicles. Our findings show the existence of a direct monosynaptic pathway between the neurons of the ventromedial medulla and thoracic motor nuclei, providing anatomical support for previous physiological data.