Regeneration of supraspinal projection neurons in the adult goldfish

Regeneration of descending supraspinal projections were identified in adult goldfish following administration of HRP to different levels of the spinal cord. While in the untreated normal fish 17 nuclei were shown to project into the spinal cord, only 11 of them seem to have participated in the process of regeneration. The nuclei whose axons regenerated include the nucleus ventromedialis (NVMD), nucleus of the median longitudinal fasciculus (NMLF), nucleus reticularis superior (NRS), nucleus reticularis medialis (NRM), nucleus reticularis inferior (NRI), anterior octaval nucleus (AON), magnocellular octaval nucleus (MON), descending octaval nucleus (DON) and certain neurons of the facial lobe. The neurons of the magnocellular preoptic nucleus (NPO), raphe nucleus (NR), Mauthner cell (MC), posterior octaval nucleus (PON) and somata located adjacent to the descending trigeminal tract were not labeled. The nuclei that apparently participated in the regeneration process were significantly larger in size than the corresponding cell bodies in the untreated normal fish.     

Melatonin signal transduction in the goldfish, Carassius auratus

Generation and reception of melatonin signals in the goldfish, Carassius auratus, are reviewed. The photoreceptive pineal gland of the goldfish generates circulating melatonin rhythms according to a given photoperiod under light-dark cycles and in a circadian manner under continuous dark conditions. Melatonin is also produced in the retina in a similar fashion. Melatonin produced in the pineal gland and retina is considered to act as internal zeitgeber in the brain and retina, respectively, controlling various physiological events via specific melatonin binding sites that are coupled with G protein. The goldfish exhibit clear diurnal locomotor activity rhythms under light-dark cycles and free-running rhythms under constant conditions. However, the relationship between melatonin and locomotor activity rhythms in the goldfish remains unclear. Further studies should be required to demonstrate the roles of melatonin in the circadian system in this species.     

Melatonin signal transduction in the goldfish, Carassius auratus

Generation and reception of melatonin signals in the goldfish, Carassius auratus, are reviewed. The photoreceptive pineal gland of the goldfish generates circulating melatonin rhythms according to a given photoperiod under light-dark cycles and in a circadian manner under continuous dark conditions. Melatonin is also produced in the retina in a similar fashion. Melatonin produced in the pineal gland and retina is considered to act as internal zeitgeber in the brain and retina, respectively, controlling various physiological events via specific melatonin binding sites that are coupled with G protein. The goldfish exhibit clear diurnal locomotor activity rhythms under light-dark cycles and free-running rhythms under constant conditions. However, the relationship between melatonin and locomotor activity rhythms in the goldfish remains unclear. Further studies should be required to demonstrate the roles of melatonin in the circadian system in this species.     

Descending projection neurons to the spinal cord of the goldfish, Carassius auratus

The sources of descending spinal tracts in the goldfish, Carassius auratus, were visualized by retrograde transport of horseradish peroxidase (HRP) administered to the hemisected spinal cord. In the diencephalon, HRP-positive neurons were identified in the nucleus preopticus magnocellularis pars magnocellularis and ventromedial nucleus of the thalamus of the ipsilateral side. In the mesencephalic tegmentum, a few somata of the contralateral nucleus ruber and several ipsilateral neurons of the nucleus of the median longitudinal fasciculus were labeled. The reticular formation of the rhombencephalon was the major source of descending afferents to the spinal cord. A larger number of neurons were retrogradely labeled in the ipsilateral superior, middle, and inferior nuclei than in the contralateral nuclei. A few raphe neurons and the contralateral Mauthner neuron were also HRP-positive. The octaval area showed retrogradely labeled neurons in the anterior, magnocellular, descending, and posterior octaval nuclei of the ipsilateral side. A large number of neurons in the facial lobe and a few somata located adjacent to the descending trigeminal tract were labeled on the ipsilateral side. The pattern of descending spinal projections in goldfish is comparable to that of tetrapods and suggests that the spinal tracts have originated quite early in the course of vertebrate evolution.     

Stimulation of feeding behavior and food consumption in the goldfish, Carassius auratus, by orexin-A and orexin-B

The neuropeptides, orexin-A and orexin-B, have been demonstrated to have a physiological role in the regulation of food intake in mammals. The effects of human orexin-A and orexin-B intracerebroventricular (i.c.v.) injection on the feeding behavior of goldfish (Carassius auratus) were investigated. I.c.v. injection of orexin-A and orexin-B both caused a significant increase in appetite, as indicated by an increased number of feeding acts. Orexin-A and orexin-B both significantly stimulated food consumption, as indicated by increased total food intake during a 60-min observation period; the actions of orexin-A were dose dependent. Orexin-A was more potent than orexin-B in stimulation of both feeding behavior and food intake. These results indicate that orexin peptides are involved in the hypothalamic regulatory pathways of feeding behavior in goldfish.     

Axonal regeneration of descending and ascending spinal projection neurons in spinal cord-transected larval lamprey

The distributions of descending and ascending spinal projection neurons (i.e., spinal neurons with moderate to long axons) were compared in normal larval lamprey and in animals that had recovered for 8 weeks following a complete spinal cord transection at 50% body length (BL, normalized distance from the anterior head). In normal animals, application of HRP to the spinal cord at 60% BL (40% BL) labeled an average of 713.8 +/- 143.2 descending spinal projection neurons (718.4 +/- 108.0 ascending spinal projection neurons) along the rostral (caudal) spinal cord, most of which were unidentified neurons. Some of these neurons project for at least approximately 50-60 spinal cord segments (approximately 36-47 mm in animals with an average length of approximately 90 mm used in the present study). At 8 weeks posttransection, the numbers of HRP-labeled descending or ascending spinal neurons that extended their axons through the transection were about 40% of those in similar areas of the spinal cord in normal animals. Thus, in larval lamprey, axonal regeneration of descending and ascending spinal projection neurons is incomplete, similar to that found for descending brain neurons. The majority of restored projections were from unidentified spinal neurons that have not been documented previously. In contrast to results from several other lower vertebrates, in the lamprey ascending spinal neurons exhibited substantial axonal regeneration. Identified descending spinal neurons, such as lateral interneurons and crossed contralateral interneurons, and identified ascending spinal neurons, such as giant interneurons and edge cells, regenerated their axons at least 9 mm beyond the transection site in animals with an average length of approximately 90 mm, which is appreciably farther than previously reported. In contrast, most dorsal cells, which are centrally located sensory neurons, exhibited very little axonal regeneration.     

Immunohistochemical study of a dopaminergic system in the spinal cord of the ray, Raja radiata

Using a specific antibody we have demonstrated the presence of an intrinsic dopamine (DA)-containing system in the spinal cord of a cartilaginous fish. The DA neurons are distributed throughout the spinal cord in the subependymal layer that surrounds the central canal. These cells extend a thick process into the canal and thinner neurites throughout the gray matter; they resemble the liquor-contacting neurons reported for a range of vertebrates.     

Collateral branching in axonal projections to spinal cord from paramedian reticular nucleus neurons

Experiments were done in cats to identify neurons in the paramedian reticular nucleus (PRN) sending collateral axons to the region of the intermediolateral nucleus (IML) at different levels of the thoracic cord by using lectin-conjugated horseradish peroxidase (HRP) and double-labeling fluorochrome histochemistry to retrogradely label PRN neurons. Injections of Fast blue (FB) into the spinal cord at the T2 level centered in the region of the IML were coupled with injections of Nuclear yellow (NY) into the ipsilateral cord at either the T4 or T7 levels centered in the region of the IML. Neurons in the PRN retrogradely labeled after diffusion of HRP into the region of the IML at the T2 level were observed throughout the rostrocaudal extent of the ventral PRN. In addition, a few labeled neurons were noted in the ventral portion of the dorsal PRN. About 40% of the neurons in the PRN which were labeled with FB after an injection at the T2 level were also labeled with NY injected into the cord in further caudal segments. These data suggest that the PRN may exert its influence on the cardiovascular system partly through collateral axonal branches to widely separated populations of sympathetic preganglionic neurons in different spinal segmental levels.     

Distribution and time course of protein extravasation in the rat spinal cord after contusive injury

We have previously characterized a graded, spinal cord contusive injury in the rat. We have now used this reproducible model to examine vascular permeability to horseradish peroxidase (HRP) after injury. The relationship between severity of injury and distribution of protein extravasation was evaluated at 3 h after injury. After mild injury, tracer was primarily confined to central gray matter and the ventral part of the dorsal columns. After moderate injury, protein extravasation was similar to that observed after mild injury, with the exception that the central hemorrhage included pericentral while matter and occasionally extended to the pial surface. After severe injury, reaction product (RP) was more densely distributed within the central cord and peripheral white matter. The axial extent of tracer at sites proximal and distal to the impact site increased with severity of injury. At 2.0 cm from the injury, no leakage of tracer was noted after mild injury. In contrast, after moderate and severe injury limited microvascular leakage of HRP was noted. Furthermore, after severe injury, in addition to local sites of microvascular leakage, intense RP was present in the dorsal columns up to at least 2.0 cm from the injury. The time course for re-establishment of the blood-spinal cord barrier to protein was evaluated from 3 h to 14 days after moderate injury. At 3 h to 1 day, protein leakage was maximal and coincided with sites of extravasated blood components, although was consistently more extensive. By 7 days, despite resolution of the initial hemorrhage, there remained scattered evidence for protein extravasation at the injured site and at sites along the axis of the cord. The blood-spinal cord barrier to HRP was reestablished by 14 days after injury.     

甲基汞引发的大鼠脊髓前角大型运动神经元的脱落

目的研究有机汞中毒大鼠脊髓前角的病理学改变。方法使用每日１０ｍｇ／ｋｇ甲基汞，给成熟大鼠连续灌胃１０日，观察其脊髓、前根及肌肉的病理学所见。结果首次投药１４日起，脊髓前角大型运动神经元胞质内出现空胞及尼氏小体脱失；在１６日观察到噬节现象；到１８日，前角大型运动神经元高度脱落，而中、小型神经元则无明显减少。使用醋酸银金属自显影技术亦检测到脊髓前角大型运动神经元有汞的特异性沉积。结论以往报道的汞中毒性类肌萎缩侧索硬化综合征的主要病理学基础为脊髓前角运动神经元的变性、脱落，本实验动物模型可以作为研究运动神经元疾病病理生理学改变的有用模型。     

A new population of neurons with crossed axons in the lamprey spinal cord

Neurons with contralateral, rostrally and caudally projecting axons were studied in whole mounts of lamprey spinal cord using retrograde labelling techniques with fluorescent dextran-amines, cobalt-lysine or horseradish peroxidase. A previously unknown large population (180-300 cells per hemisegment) of small (less than 25 microns) cells with contralateral projecting axons is described. Their axons extend over less than 5 segments rostrally or caudally. The number of these cells per segment was relatively constant in the rostral half of the spinal cord, but increased significantly in the caudal half. In comparison, medium-sized cells with contralateral axons corresponding to previously identified premotor interneurons were far less numerous (14-21 per hemisegment) and their axons extended more than 5 segments. Contralaterally projecting edge cells (intraspinal stretch receptor neurons) with principal rostral or caudal axons plus short collaterals in the other direction were distributed throughout the length of the spinal cord, whereas large and giant cells with a varied morphology were found in the caudal half.     

Distribution of capsaicin-sensitive urinary bladder afferents in the rat spinal cord

The capsaicin-sensitive afferent innervation of the urinary bladder and the central nervous system distribution of urinary bladder afferents have been studied in the rat. Capsaicin-sensitive primary sensory neurones supplying the urinary bladder have been found in two groups of spinal ganglia located in the Th₁₃-L₂ and L₆-S₁ segments. Capsaicin-sensitive primary sensory afferents from the bladder terminate within Rexed's laminae I, V and X, and in the dorsal gray commissure of the lumbosacral spinal cord. In addition, the results point to a possible vagal sensory innervation of the urinary bladder.     

Demonstration of artifactual coupling between spinal neurons and glial cells during intracellular recording with micropipette electrodes

Intracellular recordings with electrophysiological properties that are characteristic of both neurons and glial cells were obtained in the isolated spinal dorsal horn. Intracellular staining supports an idea that these elements may become artifactually coupled via the recording electrode. This coupling can be a gradual process, occurring over periods up to 30 min, and may have implications for the interpretation of slow, afferent-evoked potentials in some dorsal horn neurons.     

Effect of transection on the blood-spinal cord barrier of the cat after isolation from descending sources

The disruption of descending pathways and subsequent release of vasoactive neurotransmitters may contribute to the abnormal vascular permeability observed after spinal cord injury. Therefore, the relationship between disruption of long descending fiber tracts in the rat spinal cord and the development of blood-spinal cord barrier breakdown to the protein horseradish peroxidase (HRP) was evaluated. This was accomplished by first transecting the cord in order to deplete transmitter stores in the distal (caudal) segments. One month after this isolation procedure, a second transection was made several segments distal to the first transection. The axial distribution of barrier permeability to HRP was evaluated at both the light and electron microscopic levels in this 'isolated cord' preparation. Camera lucida drawings, delineating the distribution of tracer leakage in the spinal cord, were used to quantify the extent of protein extravasation. Vascular leakage of the tracer was identified as early as 1 h postinjury but was restricted to segments adjacent to the second transection. By 1 day after injury, protein extravasation was more marked, as compared to the earlier time points, and axial spread of barrier breakdown occurred along more distal vascular sites. Abnormal permeability to HRP was confirmed at the ultrastructural level where the protein was present within vesicles in the endothelium and the surrounding smooth muscle layer and basal lamina. The tracer was also identified in the cytoplasmic compartment of neurons and glia and within the adjacent extracellular space.     

Regeneration of descending spinal axons after transection of the thoracic spinal cord during early development in the North American opossum, Didelphis virginiana

Opossums are born in an immature, fetal-like state, making it possible to lesion their spinal cord early in development without intrauterine surgery. When the thoracic spinal cord of the North American opossum, Didelphis virginiana, is transected on postnatal day 5, and injections of Fast Blue (FB) are made caudal to the lesion site 30–40 days or 6 months later, neurons are labeled in all of the spinal and supraspinal areas that are labeled after comparable injections in age-matched, unlesioned controls. Double-labeling studies document that regeneration of cut axons contributes to growth of axons through the lesion site and behavioral studies show that animals lesioned on postnatal day 5 use their hindlimbs in normal appearing locomotion as adults. The critical period for developmental plasticity of descending spinal axons extends to postnatal day 26, although axons which grow through the lesion site become fewer in number and more restricted as to origin with increasing age. Animals lesioned between postnatal day 12 and 26 use the hindlimbs better than animals lesioned as adults, but hindlimb function is markedly abnormal and uncoordinated with that of the forelimbs. We conclude that restoration of anatomical continuity occurs after transection of the spinal cord in developing opossums, that descending axons grow through the lesion site, that regeneration of cut axons contributes to such growth, and that animals lesioned early enough in development have relatively normal motor function as adults.     

Evidence for sequential degeneration of the neurons in the intermediate zone of the spinal cord in amyotrophic lateral sclerosis: a topographic and quantitative investigation

Summary To elucidate the degenerating mechanism of the neurons in the intermediate zone of the spinal cord in classical amyotrophic lateral sclerosis (ALS), the spinal neurons in a patient with ALS, whose muscular strength was fairly well preserved up to death, were examined quantitatively and topographically, and compared with the data of advanced ALS patients and age-matched control subjects reported previously. In advanced ALS patients, anterior horn cells completely disappeared and the medium-sized (nuclear area; 71–150 m²) and large (nuclear area; greater than 151 m²) neurons in the intermediate zone were severely reduced. In the present case, however, the loss of anterior horn cells was severe but the degree was not equal to that of advanced ALS patients, and the neurons in the intermediate zone were quite well preserved. The finding indicates that the primary degeneration may occur in the anterior horn cells and the neurons in the intermediae zone degenerate sequentially in the spinal gray matter in ALS.Supported in part by a Grant-in-Aid for Scientific Research (A) No. 60440046 from the Ministry of Education, Science and Culture, Japan     

c-fos Expression and NADPH-d reactivity in spinal neurons after fatiguing stimulation of hindlimb muscles in the rat

The distribution of Fos-immunoreactive (Fos-ir) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-reactive neurons in the rat lumbar spinal cord was examined following muscle fatigue caused by intermittent high-rate (100 s⁻¹) electrical stimulation of the triceps surae muscle or the ventral root L5 (VRL5) for 30 min. Following both types of stimulation, the fatigue-related c-fos gene expression was more extensive in the L2–L5 segments on the stimulated side, and the majority of Fos-ir neurons were concentrated in the dorsal horn. After direct muscle stimulation, the highest number of Fos-ir neurons were detected in two regions: layer 5, and superficial layers (1 and 2_o), although many labeled cells were also found in layers 3, 4, 6, and 7. In response to VRL5 stimulation, the maximal density of Fos-ir neurons was detected in the middle and lateral parts of layers 1 and 2_o, the zone of termination of high-threshold muscle afferents_. Statistically significant prevalence of Fos-ir cell number was also found in layers 5 and 7 on the stimulated side. A few Fos-ir neurons were detected in the ventral horn (layer 8 and area 10) on both sides. The lamellar distribution of NADPH-d-reactive neurons was similar over all experimental groups of animals. In the L3–L6 segments, such reactive cells were arranged in two distinct regions: dorsal horn (layers 2_i, 3, and 5) and area 10; in the L1 and L2 segments, an additional cluster of NADPH-d positive cells was found in the intermediolateral cell column (IML). Double-labeled cells were not detected. We suggest that c-fos expression in response to muscle fatigue reveals activity of functionally different types of spinal neurons which could operate together with NOS-containing cells in pre-motoneuronal networks to modulate the motoneuron output.     

Evidence for rapid fluid flow from the subarachnoid space into the spinal cord central canal in the rat

Horseradish peroxidase (HRP) was used as a CSF tracer in Sprague-Dawley rats. One group of rats received an injection of HRP in the cistema magna and a second group was injected in the thoracic spinal subarachnoid space. The animals were sacrificed 0, 10 or 30 min after HRP injection by rapid perfusion with paraformaldehyde and glutaraldehyde. In both groups, there was rapid HRP labeling of brain and spinal cord perivascular spaces. HRP was present in the central canal in a pattern that was not consistent with flow from the fourth ventricle: in both groups there were segments of unlabeled central canal between the fourth ventricle and central canal segments containing HRP. HRP-labeled perivascular spaces were seen in the central gray matter adjacent to the central canal. There was a distinctive pattern of interstitial HRP between perivascular spaces and the central canal. The results suggest that there is a normal flow of fluid from the subarachnoid space, into the perivascular spaces, across the interstitial space and into the central canal. The function of this flow may be to clear metabolites from the interstitial space. The existence of such a flow would add considerable support to the theory that non-communicating syringomyelia develops in segments of central canal isolated by occlusion or stenosis at each end.