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.

Changes in enzyme histochemical profiles of identified spinal motoneurons of the European eel, Anguilla anguilla, following cordotomy.

The enzyme histochemical profiles of glucose-6-phosphate dehydrogenase (a marker of synthetic performance), succinate dehydrogenase (an indicator of oxidative metabolism), and NADH tetrazolium reductase (a marker of overall neuronal activity) were determined for identified white muscle motoneurons in six control and six cordotomized cels. Images were digitized and mean integrated absorbances obtained using appropriate hardware and software. For motoneurons caudal to the transection site there was a significant decrease in the mean absorbance value for NADH tetrazolium reductases which declines from 0.28 a.u. (arbitrary units) in control animals to 0.23 a.u. in cordotomized animals. However, no significant changes were detected in the activities of glucose-6-phosphate and succinate dehydrogenases. The cross-sectional area of the motoneuronal cell body was not affected by cordotomy. The decrease by around 20% in overall neuronal activity, as expressed by NADH tetrazolium reductase activity, might be expected from the decline in body motility that follows cordotomy. Changes in SDH and G6PDH activities would also be expected to follow this surgery, but none were seen, perhaps because they are compensated for by changes in neuronal metabolism that result from deafferentation.

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.

Energy metabolism in spinal motoneurons of the zebrafish.

Enzyme histochemical profiles of the energy metabolism in spinal motoneurons of fish, ranging in length from 18 mm to 53 mm, were investigated. Energy is used for ion pumps to maintain the resting membrane potential and for other processes of which transport and biosynthesis may be the most important. In this paper particular attention is paid to the impact of biosynthesis on the energy metabolism in motoneurons. As the innervation ratio of fish motoneurons increases dramatically during growth, we suppose that the formation and maintenance of functional connections with the target muscle accounts for the high level of the energy metabolism in motoneurons.

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.

Ultrastructural characteristics of zebrafish spinal motoneurons innervating glycolytic white, and oxidative red and intermediate muscle fibers.

Spinal motoneurons in the zebrafish were classified using morphological criteria. Dorsomedial white motoneurons which innervate the fast, glycolytic white muscle fiber compartment were distinguished from ventrolateral red and intermediate motoneurons which innervate the slow, oxidative, red and intermediate muscle fiber compartments. Synapses on cell somata and cell organelles were studied in detail. The motoneurons which innervate white muscle fibers (W motoneurons) are considerably larger than those which innervate red and intermediate muscle fibers (RI motoneurons; W > RI). Significant differences were also found in the size of the nucleus (W > RI) and in the ratio size nucleus/size soma (W < RI); small differences were found regarding endoplasmic reticulum (W > RI) and mitochondria (W < RI). There were no differences in synaptic apposition length or percentage of terminals with flat vesicles. Small differences were discerned with regard to covering percentages (W < RI) and percentage of terminals with round vesicles (W > RI). Terminals with dense cored vesicles appeared on W motoneuron somata only. Within the motoneuron population, there was a positive correlation between the coverage of terminals containing flat vesicles and the perimeter of the cell soma. In RI motoneurons, there was a positive correlation between the perimeter of the cell and the amount of endoplasmic reticulum. A negative correlation was found between the RI cell perimeter and mitochondria, which is in line with a high succinate dehydrogenase activity in small cells.

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.

Histochemical profiles of motoneurons innervating muscle fibres with different activity patterns in the zebrafish, Brachydanio rerio.

Enzyme histochemical profiles of spinal motoneurons in the zebrafish were determined. Five enzymes of glucose metabolism were chosen: glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), phosphofructokinase (PFK), succinate dehydrogenase (SDH) and NADH tetrazolium reductase (NADH-TR). Motoneurons were traced with Fluorogold and classified as those that innervate white muscle fibres (W-MNs) and those that innervate red and intermediate muscle fibres (R/I-MNs). The average enzyme activities per volume of tissue in the somata of both populations differed at most by 25%. Both the average soma volume and the average number of muscle fibres innervated are three times larger for the W-MNs than for the R/I-MNs. This suggests that the total amount of enzyme activity within a neuron soma matches target size. In the R/I-MNs, the activities of SDH and NADH-TR were closely correlated (correlation coefficient, r = 0.99; p less than 0.05) and HK activity correlated well with G6PDH activity (r = 0.94; p less than 0.05), but not with PFK (r = 0.64; p greater than 0.05). In the W-MNs, there was no correlation between SDH and NADH-TR (r = -0.59; p greater than 0.05) or between HK and G6PDH (r = 0.50; p greater than 0.05) and the correlation coefficient between HK and PFK activity was close to zero (r = 0.04; p greater than 0.05). It was concluded that in the R/I-MNs, which are continuously active, firing activity is fuelled by oxidative metabolism. We suggest that in the W-MNs glucose is stored in the form of glycogen and that, despite high levels of NADH-TR present, the energy for intermittent firing activity is provided by glycolysis.

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.

Identification of motoneurons in the spinal cord of the zebrafish (Brachydanio rerio), with special reference to motoneurons that innervate intermediate muscle fibers.

We investigated the location and size distribution of motoneurons that innervate red, intermediate and white muscle fibers in the axial musculature of the zebrafish (Brachydanio rerio). Motoneurons were identified by retrograde labeling from the respective myotomal compartments with horseradish peroxidase applied either in small polyacrylamide gel fragments or as pure crystals. We found a spatial relationship between the three myotomal muscle fiber types and the corresponding motoneurons. The white motoneurons are grouped in the dorsal part of the motorcolumn, near the central canal. Motoneurons of the red and intermediate muscle are clustered in the ventral part of the motorcolumn. The average position of the red motoneurons is ventral to that of the intermediate motoneurons. Some sizes are distributed over wide overlapping ranges, spanning from 41 to 352 microns 2 for red and intermediate and from 56 to 894 microns 2 for white motoneurons. These data are discussed in relation to the recruitment order of myotomal muscle fibers of different types as revealed by electromyographic recordings in fish, and the so called "size principle" for motoneuron recruitment.

Enzyme reaction rate studies in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus.

A histochemical analysis of reaction rates of a series of enzymes was performed in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus. These neurons were selected because of their functional homogeneity. The high metabolic activity of these cells as well as their large size facilitate cytophotometric analysis in cryostat sections. Sections were incubated for the activity of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH dehydrogenase, NADPH ferrihaemoprotein reductase and beta-hydroxybutyrate dehydrogenase. All media contained polyvinyl alcohol as tissue stabilizer and Nitro BT as final electron acceptor. Measurements were performed with a Vickers M85a cytophotometer. Linear relationships between the specific formation of formazan (test minus control reaction) and incubation time were obtained for all enzymes although some reactions showed an initial lag phase or an intercept with the ordinate. The relatively high activities of hexokinase, succinate dehydrogenase and the extremely low activity of hydroxybutyrate dehydrogenase indicate that energy is mainly supplied by glycolysis. Glucose-6-phosphate dehydrogenase showed a high activity whereas NADPH reductase and dehydrogenase activity were low in electromotor neurons, indicating that the NADPH generated is largely used for biosynthesis. Despite their synchronous firing pattern activity, electromotor neurons showed a considerable heterogeneity with respect to their metabolic activity.

Synaptonemal complex antigen location and conservation.

The axial cores of chromosomes in the meiotic prophase nuclei of most sexually reproducing organisms play a pivotal role in the arrangement of chromatin, in the synapsis of homologous chromosomes, in the process of genetic recombination, and in the disjunction of chromosomes. We report an immunogold analysis of the axial cores and the synaptonemal complexes (SC) using two mouse monoclonal antibodies raised against isolated rat SCs. In Western blots of purified SCs, antibody II52F10 recognizes a 30- and a 33-kD peptide (Heyting, C., P. B. Moens, W. van Raamsdonk, A. J. J. Dietrich, A. C. G. Vink, and E. J. W. Redeker, 1987, Eur. J. Cell Biol., 43: 148-154). In spreads of rat spermatocyte nuclei it produces gold grains over the cores of autosomal and sex chromosomes. The cores label lightly during the chromosome pairing stage (zygotene) of early meiotic prophase and they become more intensely labeled when they are parallel aligned as the lateral elements of the SC during pachytene (55 grains/micron SC). Statistical analysis of electronically recorded gold grain positions shows that the two means of the bimodal gold grain distribution coincide with the centers of the lateral elements. At diplotene, when the cores separate, the antigen is still detected along the length of the core and the enlarged ends are heavily labeled. Shadow-cast SC preparations show that recombination nodules are not labeled. The continued presence suggests that the antigens serve a continuing function in the cores, such as chromatin binding, and/or structural integrity. Antibody III15B8, which does not recognize the 30- and 33-kD peptides, produces gold grains predominantly between the lateral elements. The grain distribution is bimodal with the mean of each peak just inside the pairing face of the lateral element. The antigen is present where and while the cores of the homologous chromosomes are paired. From the location and the timing, it is assumed that the antigen recognized by III15B8 functions in chromosome pairing at meiotic prophase. The two anti-rat SC antibodies label rat and mouse SCs but not rabbit or dog SCs. A positive control using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) anti-centromere serum gives equivalent labeling of SC centromeres in the rat, mouse, rabbit, and dog. It is concluded that the SC antigens recognized by II52F10 and III15B8 are not widely conserved. The two antibodies do not bind to cellular or nuclear components of somatic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of two major components of the lateral elements of synaptonemal complexes of the rat.

This paper describes the identification of two major components of the lateral elements of synaptonemal complexes of the rat by immunocytochemical techniques. We prepared monoclonal antibodies against synaptonemal complexes (SCs) by immunization of mice with purified SCs. One of these antibodies, II52F10, reacts with a 30 and a 33 kDa polypeptide, which are major components of purified SCs. Using this antibody, we studied the localization of its antigens light microscopically, by means of the indirect immunoperoxidase technique, as well as ultrastructurally, by means of the immunogold labeling technique. The immunolocalization was carried out on whole-mount preparations of lysed spermatocytes. The antibody reacts with paired as well as unpaired segments of zygotene, pachytene and diplotene SCs. In light microscopic preparations, the attachment plaques, particularly those of late pachytene and diplotene SCs, also appear to react strongly. In electron micrographs the lateral elements in paired as well as unpaired segments could be seen to react. No reaction was observed in the attachment plaques; however, in late pachytene and diplotene SCs the swollen terminal segments of the lateral elements did react with the antibody. Thus, we conclude that a 30 and a 33 kDa polypeptide make part of the lateral elements of synaptonemal complexes of the rat.

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.