Long term effects of spinal cord transection in zebrafish: swimming performances, and metabolic properties of the neuromuscular system.

This study concerns functional recovery of zebrafish following spinal cord transection. Spinal cords were transected at the level of the 14th vertebra, just rostral to the dorsal fin. Recovery was tested at one month after transection when descending fibers start to regrow across the transection site and at three months after transection when fish perform kick and glide swimming. To estimate the rate of regrowth across the lesion site we analysed the tyrosine hydroxylase (TH) and dorsal 5-hydroxytryptamine (5-HT) systems in distal parts of lesioned cords. Both systems have cell bodies in the brainstem and in control fish TH- and dorsal 5-HT-containing fibers descend to all spinal segments. Swimming performance was studied by subjecting lesioned fish to endurance tests in a swimming tunnel with water flowing at a constant rate of 2 or 4.5 body lengths per second (BL/s). At 2 BL/s slow myotomal muscles are active whereas at 4.5 BL/s fast myotomal muscles are recruited. Control fish endured sustained swimming at both speeds for at least 3 hours. As a measure for the condition of the neuromuscular system in trunk and tail, we analysed aerobic metabolic capacities, assessed by NADH-tetrazolium reductase (NADH-TR) histochemistry of myotomal muscle fibers and spinal lateral neuropil. We found that TH- and dorsal 5-HT-immunoreactive fibers were absent in the entire distal part of lesioned cords at one month but at two months after transection they were present at approximately 6000 microns caudally to the site of the lesion. Thus the rate of outgrowth of these fibers is at least 200 microns per day. Sustained swimming at the slow speed (2 BL/s) could be endured for about 14.4 min at one month and for 23.5 min at two months after transection; there was no further improvement in the period that followed. In contrast, in the 10 weeks following transection, fast swimming (4.5 BL/s) could be endured for about 5 to 6 minutes. A significant improvement was gained in the period of 10 to 12 weeks after transection when fish could endure the high speed for almost 15 min. The aerobic capacity of muscle fibers in distal parts of the body was not strongly affected by the lesion. The only important change in aerobic capacity was observed in the neuropil of distal parts of the cords where, at three months after transection, NADH-TR activity was increased to approximately 150% of control values. On the basis of our findings, we assume that it is not the condition of the neuromuscular system, but rather a deficient co-ordination between proximal and distal body parts of lesioned fish that accounts for the relatively poor performances in endurance tests. Furthermore, differences in timing of improvements in swimming at 2 and 4.5 BL/s indicate that the spinal circuitries serving the slow parts of the neuromuscular system recover at an earlier stage than those serving the fast parts.

Changes in the synaptology of spinal motoneurons in zebrafish following spinal cord transection.

Effects of spinal cord transection on the synaptology of zebrafish spinal motoneurons were studied. The transection was made at the level of the 14th vertebra and the synaptology of motoneuron somata and dendrites was analysed at the level of the 21st to the 23rd vertebrae at one month and three months after transection. Horseradish peroxidase, applied to the myotomal muscle, was used to label motoneuron somata and dendritic branches in central and in lateral areas of the neuropil (referred to as central and lateral dendritic profiles). Boutons impinging on motoneurons were classified according to the morphology of the vesicles. We discerned R-boutons with spherical vesicles, F-boutons with flat vesicles and DC-boutons with at least one dense core vesicle. The apposition lengths of R-, F- and DC-boutons and the circumference of labelled profiles were determined to assess the proportional covering of boutons on somata and dendrites. Ratio's of covering with R- and F-boutons (R/F ratio) for somata, central and lateral dendritic profiles were 1.1, 2.1, and 2.1 in control fish and 0.5, 0.5 and 0.9 in lesioned fish at one month after transection, respectively. The total covering of motoneurons in lesioned fish was decreased by 20% on somata and by 30% on lateral dendritic profiles, whereas central dendritic profiles did not change significantly. At three months after transection the R/F ratio's for somata, central and lateral dendritic profiles were 0.5, 0.7 and 0.6, respectively. The total covering on somata and central and lateral dendritic profiles was at control levels. The anatomical aspects of the changes in synaptology indicate that in control fish 50 to 60% of the R-boutons on the motoneuron surface originate from descending axons. In contrast, almost all F-boutons seem to be from local origin.

Quantitative enzyme- and immunohistochemistry of hexokinase and cytochrome c oxidase of spinal neurons in the zebrafish.

A combined quantitative enzyme- and immunohistochemical procedure to demonstrate hexokinase (HK) was developed and tested on sections of spinal cord tissue of the zebrafish. In both procedures, the amount of final reaction product was linearly related with section thickness. When applied to serial sections of fish spinal neurons, the enzyme- and immunohistochemical activities appeared to correlate significantly (r = 0.61; p < 0.001). As HK and cytochrome c oxidase (COX) histochemistry have been used regularly to screen the average level of chronic activity of neurons, we subsequently analysed the relationship between HK and COX in fish spinal neurons, using previously published methods of quantitative enzyme- and immunohistochemistry for COX. The enzyme- as well as the immunohistochemical localisation patterns of HK showed a weak correlation with the enzyme- and immunohistochemical COX localisation respectively. Therefore, it is concluded that both enzyme- and immunohistochemical localisation of COX provide a poor estimate for the relative level of glucose utilisation in fish spinal neurons.

Organisation of the zebrafish spinal cord: distribution of motoneuron dendrites and 5-HT containing cells.

In order to find arguments for a selective innervation and modulation of the fast and slow spinal motoneurons in the zebrafish, we determined: the territories occupied by the dendritic trees of the large spinal motoneurons innervating the fast white muscle (WMNs) and those of the smaller motoneurons (RIMNs) innervating the slower red and intermediate fibers; the distribution of 5-hydroxytryptamine (5-HT) immunoreactive cells, which constitute one of the systems for the modulation of motoneuron activity. Motoneurons were either retrogradely labelled with horseradish peroxidase or iontophoretically filled with Lucifer yellow. The 5-HT cells were identified immunohistochemically. We found that the dendritic territories of the WMNs and those of the RIMNs partly overlap but they also occupy unique areas. The unique area of the WMN dendrites is dorsal to the lateral neuropil, where axon collaterals of the bulbospinal tract invade the motor column; the unique area of the RIMN dendrites lies in the ventral part of the cord, near the ventral commissural tract. 5-HT immunoreactive cells were observed in the ventromedial part of the spinal cord and among the motoneuron somata. The large overlap in dendrite territories between the WMNs and RIMNs argues for a motoneuron recruitment pattern as may be expected from the size principle; while the distinct areas occupied by dendrites of either WMNs or RIMNs hints at a selective innervation. The presence of the 5-HT cells close to the motoneuron somata and in the ventral area, where dendrites of the RIMNs ramify, suggests that the activities of the motoneuron types can be selectively modulated.

Combined quantitative immuno- and enzyme cytochemistry of cytochrome c oxidase in sections of neural tissue and cultured cells.

Immuno- and enzyme cytochemical procedures were tested for their applicability to determine quantitatively the concentration and the activity of cytochrome c oxidase (COX) in tissue sections of fish spinal cord and sections of cultured human Molt-4 cells. Sections of gelatin gels containing known amounts of human skeletal muscle COX served as standard sections. The selected procedures fulfilled an important criterion for valid quantitative cytochemistry: the amount of final reaction product was linearly related with section thickness. When the immuno- and enzyme cytochemical methods were applied to sections of fish spinal neurons or cultured human cells, the COX concentration appeared to correlate significantly with COX activity. These cytochemical methods might therefore be valuable to detect differences in molecular COX activities between individual cell types as may occur in tissue sections of biopsies taken from patients with mitochondrial dysfunctions.

Metabolic profiles of white and red-intermediate spinal motoneurons in the zebrafish.

To study the interactions between the citrate cycle and amino acid metabolism in zebrafish spinal motoneurons, we composed enzyme histochemical profiles from the activities of NAD-linked isocitrate dehydrogenase (NAD-ICDH), glutamate dehydrogenase (GDH), succinate dehydrogenase (SDH) and glucose 6-phosphate dehydrogenase (G6PDH). The enzyme assays were performed on serially-sectioned motoneuron somata. The motoneurons were identified by retrograde tracing from the trunk muscle and classified, on the basis of their location in the motor column, as those innervating the white, fast glycolytic fibers (WMNs) or those innervating the red and intermediate slow oxidative fibers (RIMNs). We found the following relationships between enzyme activities in WMNs: GDH correlates with G6PDH activity (r = 0.31; p = 0.02) and NAD-ICDH correlates with GDH activity (r = 0.37; p < 0.01); correlations between NAD-ICDH and SDH and between SDH and GDH are not significant. In RIMNs we found correlations between NAD-ICDH and SDH (r = 0.34; p = 0.03), between NAD-ICDH and GDH (r = 0.41; p < 0.01) and between GDH and SDH (r = 0.50; p < 0.01); the correlation between GDH and G6PDH is not significant. The differences in metabolic profiles between WMNs and RIMNs can be explained in the following way: in WMNs, alpha-ketoglutarate is drawn off from the citrate cycle and is used in amino acid metabolism whereas in RIMNs the removal of alpha-ketoglutarate from the cycle is balanced by formation of alpha-ketoglutarate. The data suggest that the functional role of the citrate cycle differs in the two motoneuron populations: in RIMNs energy generation predominates but in WMNs a role in biosyntheses seems most important.

The impact of target size on the level of the energy metabolism of spinal motoneurons. An enzyme histochemical and morphological study.

We investigated the relationship between the enzyme histochemical properties of motoneurons and the size of their peripheral target. The study was carried out on zebrafish (Brachydanio rerio) of 18 to 53 mm body length. Motoneurons of the white, fast glycolytic muscle (WMN) and of the slower red and intermediate oxidative muscle (RIMN) were investigated. We determined the average soma size of the WMNs and the RMNs and the following histochemical characteristics: Glucose 6-phosphate dehydrogenase (G6PDH), a marker for reductive biosynthesis; Phosphofructokinase (PFK), a marker for glycolytic activity; Succinate dehydrogenase (SDH) enzyme of the citric acid cycle and NADH tetrazolium reductase (NADH-TR) a marker for oxidative activity. The product of enzyme activity and the soma volume was denoted the enzyme capacity of a soma. As a measure for the average target of the motoneurons, we took the number of endplate regions within the white and the red-intermediate muscle. The age dependent increase in G6PDH capacity of motoneuron somata correlates significantly with the increase in the number of endplate regions. On the basis of changes in the soma size, enzyme capacity and target size, we estimate that in 18 mm fish about 45% and in 53 mm fish about 60% of the oxidative capacity is used to maintain connections with the target. This holds for WMNs as well as for RIMNs. Similar calculations, on basis of data known from the literature, indicate that in motoneurons of the cat, less than 10% of the oxidative capacity is used for connections with the target. The PFK capacity increased unproportionally with age in WMNs but not in RIMNs. This indicates that the energy metabolism in WMNs of large fish tends to be anaerobic glycolytic. The study shows that enzyme histochemical characteristics of motoneurons should be interpreted in a morphological context, regarding motoneuron soma size and target size.

Spinal neurons in the zebrafish labeled with fluoro-gold and wheat-germ agglutinin.

This study concerns the identification and location of interneurons in the spinal cord of the zebrafish by way of retrograde tracing from the body musculature. To distinguish motoneurons from interneurons; two tracers were applied: the fluorochrome Fluoro-Gold to label motoneurons, and the trans-synaptically transported compound wheat-germ agglutinin to label motoneurons and the associated interneurons that are considered to be premotoneurons. Because the tracer accumulated mainly in cell bodies (not in neurites), premotoneurons labeled directly from motoneurons could not be distinguished from those labeled via interjacent cells. Both tracers were unilaterally injected into the myotomal muscle at the anal level of the animal. The number of labeled cell bodies in the spinal cord was examined 6, 10 and 16 days after injection. Several technique-oriented experiments were performed in order to map all pathways along with the tracers were incorporated in the neurons. The following observations were made. (1) All Fluoro-Gold-positive cells contained wheat-germ agglutinin, but yet more cells contained only wheat-germ agglutinin; the number of wheat-germ agglutinin-labeled cells was about tenfold higher than the number of Fluoro-Gold-labeled cells. (2) Fluoro-Gold labeling was restricted to cells within one to two spinal cord segments corresponding to the injection site, whereas wheat-germ agglutinin labeling was more diffuse. (3) The position and size of Fluoro-Gold-labeled cells corresponded to those of motoneurons described in previous horse-radish peroxidase experiments. (4) Statistical analysis of the group of wheat-germ agglutinin-labeled cells showed two subpopulations, one with a mean cell size and position corresponding to motoneurons and one with a smaller mean cell size, also positioned within the motor column. The smaller cells were considered to be premotoneurons. The ratio motoneuron:premotoneuron was lowest in the ventrolateral area of the motor column.

Enzyme histochemical profiles of fish spinal motoneurons after cordotomy and axotomy of motor nerves.

Histochemical profiles were made of identified spinal motoneurons from normal adult zebrafish and from animals subjected to cordotomy or unilateral axotomy of the motor nerves. The lesions caused an increase of the myotomal area with oxidative muscle fibers. We studied the question: do changes in the myotomal muscle configuration concur with changes in the enzyme histochemical profiles of innervating motoneurons? Based on the location and size of cell somata, two categories of motoneurons are distinguished: large white (W) motoneurons that innervate the deep fast, glycolytic muscle fibers, and smaller red and intermediate (RI) motoneurons that innervate the superficial slow oxidative and intermediate muscle fibers. In normal animals, glucose-6-phosphate dehydrogenase activity is high in the large W motoneurons and relatively low in the small RI motoneurons. The reverse holds for succinate dehydrogenase activity is high in the large W motoneurons and relatively low in the small RI motoneurons. The reverse holds for succinate dehydrogenase activity. W and RI motoneurons show similar nicotinamide adenine dinucleotide diaphorase activity. Short- (2 weeks) and long- (8 weeks) term effects of lesions were studied. The results show that: (1) the 3 types of lesions lead to prolonged changes in the enzyme histochemical profiles of spinal motoneurons. The type of change depends on the type of lesion and on the type of motoneuron; (2) unilateral axotomy of the motor nerves affects the histochemical characteristics of spinal motoneurons and the myotomal muscle fiber type configuration on the ipsi- and contralateral side. The contralateral effects are conceived as adaptations to maintain a left-right symmetry in the motor output.

Quantitative cytochemical analysis of cytochrome oxidase and succinate dehydrogenase activity in spinal neurons.

This paper describes cytophotometric determinations of cytochrome oxidase (COX) and succinate dehydrogenase (SDH) activities in neurons in 3 areas of the spinal motor column of the teleost fish Brachydanio rerio (the Zebrafish). Purpose of this investigation was to analyse the correlation between the oxidative metabolic capacity of motoneurons with their activity patterns. The spatial organization of the spinal cord of the zebrafish allows such an analysis, because the motoneurons which innervate different muscle fiber types (slow tonic red and fast phasic white, respectively) occupy separate areas of the motor column (Van Raamsdonk et al. 1983). We analysed the COX and SDH activities on serially sectioned neurons, We found large variations in the ratio of COX/SDH activity: the ratio was high for large neurons (in the "white" area) and low for small neurons (in the "red" area). These findings were contrary our expectations, because COX as well as SDH activity have been proposed as indicators for neuronal activity if both activities are reliable indicators, then their ratio should be constant. In addition, COX and SDH activities were analysed on serially sectioned anterior horn neurons of the cat spinal cord. In contrast to the situation in fish, we observed a statistically significant proportionality between COX and SDH activities. We conclude that the histochemical reactions for COX or SDH activity have no general validity as markers for the same type of neuronal activity.

Differentiation of neurons and radial glia in the spinal cord of the teleost Brachydanio rerio (the zebrafish): An immunocytochemical study.

The differentiation of neurons and glial cells in the spinal cord of the zebrafish was studied by means of immunohistochemistry, using antisera against the 68 kD subunit of neurofilament (anti-NFP68) and against glial fibrillary acidic protein (anti-GFAP), both isolated from the bovine brain. Anti-NFP68 and anti-GFAP reactivity appear in the spinal cord at about 60 h after fertilization. At that time the anti-NFP68 reactivity is detectable in the dorsal Rohon-Beard neurons. About 12 h later, NFP68 positive neurons appear in the prospective motor column. In this respect the differentiation of the primary sensory system precedes that of the spinal motor system. During development the configuration of the glial cell processes changes from a horizontal arrangement in embryos to a radial frame work in larvae and in adults. From these observations together with data on the organization of the adult spinal motor column(28) we conclude that the motoneurons of the white and those of the red myotomal muscle fibers may have different origins in the neuroepithelial germinal layer. The anti-NFP68 serum recognizes a 120 and a 94 kD component of fish neurofilaments. Thus the subunit composition of neurofilament in fishes differs from that in mammals.

The development of the spinal motor column in relation to the myotomal muscle fibers in the zebrafish (Brachydanio rerio). I. Posthatching development.

The neuromuscular system in the trunk of larval and adult zebrafishes was studied by means of light and electronmicroscopical methods. Spinal motoneurons were identified with the horseradish peroxidase retrograde transport method. We correlated the differentiation and growth of the myotomal muscle with the number of motoneurons per spinal cord segment and the size of the motoneuron somata. The adult number of motoneurons is reached in an early larval stage, before the muscle fiber type differentiation in the myotomes is completed. The mean motoneuron size does not bear a clear correlation with the size of the myotomal muscle. In adult zebrafishes we could distinguish the motoneurons which innervate the superficial slow red and the deep fast white muscle fibers on the basis of soma size and position in the motor column. The motoneurons of the red muscle part are small; they are located in the ventrolateral part of the motor column. The motoneurons of the deep fast white fibers are large; they lie near the central canal.