Changing synaptic connections on cell bodies of growing identified spinal motoneurons of the eel, Anguilla.

As the target musculature they innervate grows throughout life, certain segmental motoneurons from the spinal cord of Anguilla, readily identified on the basis of their form and position, also increase in size. In doing so, they present a steadily increasing target to the spinal and supraspinal neurons that innervate them. How the afferent neurons respond was assessed by measuring features of their synaptic boutons contacting the motoneuronal perikarya, as seen with electron microscopy. About 60% of the perimeter of the perikaryal profile of each motoneuron was found to be covered with synaptic bouton profiles, a value that is independent of the size of the motoneuron. Furthermore, the distances between synaptic profiles, their contact sizes (measured as apposition length) and the number and size of the vesicles each profile contains were all found to be relatively constant and also independent of motoneuronal size. In contrast, the number of synaptic profiles contacting a motoneuron correlated well with its perikaryal size. Our findings indicate that the challenge of a growing neuronal target is met by a steady increase in the number of contacting boutons, the form and spacing of which are held relatively constant; this strategy will require continual synaptic realignment at the target.

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.

Corticosteroid actions on the expression of kainate receptor subunit mRNAs in rat hippocampus.

Previous studies have shown that corticosteroid hormones affect kainate-induced excitotoxic processes in the rat hippocampus. In the present study we employed in situ hybridization to examine the effect of adrenalectomy, and subsequent treatment with a low or a high lose of corticosterone on the mRNA levels for kainate receptor subunits in the hippocampus. We observed that adrenalectomy by itself does not affect the expression pattern for the GluR6, GluR7, KAR1 and KAR2 subunits. However, treatment of the adrenalectomized animals with a low dose of corticosterone (3 micrograms/100 g bodyweight) resulted in an enhanced expression of the KAR1, KAR2 and GluR6 subunit mRNAs, when compared to the expression levels in the untreated rats or the sham operated controls. Treatment with a high dose of corticosterone (1 mg/100 g bodyweight) yielded expression levels which were significantly lower than those observed in animals treated with a low corticosterone dose, for the KAR1, KAR2 and GluR7 subunit mRNAs; the levels did not differ from those in untreated rats or in the sham group. We conclude that changes in corticosteroid receptor occupancy, which may occur daily due to circadian or stress-induced variations in the circulating corticosterone level, potentially regulate high affinity kainate receptor activation and the processes in which these receptors are involved.

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.

Histochemistry of sarcoplasmic reticulum Ca-ATPase using dysprosium as capturing reagent.

This report describes the development of a histochemical method for the demonstration of sarcoplasmic reticulum Ca-ATPase activity in cross-sections of skeletal muscle. The demonstration of sarcoplasmic reticulum Ca-ATPase activity is complicated by the fact that capturing reagents for phosphate inhibit the enzyme. We present a minimal model for heavy-metal-phosphate precipitation reactions which gives a theoretical description of the effect of enzyme inhibition on the rate of phosphate precipitation in the section. The model indicates that the choice of capturing reagent is crucial: whether or not ATPase activity can be demonstrated depends mainly on the inhibition constant and the solubility product of the phosphate salt of the capturing reagent (but also on a fairly large number of other factors). All lanthanides tested can be used to demonstrate sarcoplasmic reticulum Ca-ATPase activity, but dysprosium results in the highest staining intensity. This suggests that dysprosium inhibits sarcoplasmic reticulum Ca-ATPase to a lesser degree than the other lanthanides and/or the solubility product of its phosphate salt is smaller. As an example, the method is used to investigate the effect of thyroid hormone on sarcoplasmic reticulum Ca-ATPase activity in individual fibres of the rat soleus muscle.

N-methyl-D-aspartate and kainate receptor gene expression in hippocampal pyramidal and granular neurons in the kindling model of epileptogenesis.

To investigate the changes underlying kindling epileptogenesis in the rat hippocampus, the levels of the messenger RNAs encoding for the subunits of the N-methyl-D-aspartate-receptor (1, 2A-D) and the kainate-receptor (1, -2, GluR-5, -6, -7) were determined in hippocampal principal neurons using in situ hybridization techniques and semi-quantitative analysis of the autoradiograms. Schaffer collateral-commissural pathway kindled rats were investigated at three different stages of kindling acquisition, always 24 h after the last stimulation. Furthermore, fully kindled rats were studied at long-term (28 days) after termination of kindling stimulations. NR1 messenger RNA levels were slightly decreased in CA1 area of fully kindled animals. In the fascia dentata region, a minor increase of NR2A and NR2B transcripts was found at all stages of kindling acquisition. KA-2 messenger RNA was enhanced in all hippocampal subfields during kindling development. However, none of these changes persisted at long-term after the last seizure and only the low-abundant GluR-7 expression was slightly depressed in the fascia dentata. From our observations we conclude that it is unlikely that alterations in N-methyl-Daspartate or kainate receptor gene expression play an important role in kindling acquisition or maintenance.

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.

Calibration of quantitative histochemical methods: estimation of glycogen content of muscle fibers using the PAS reaction.

A fairly simple method for calibrating microdensitometric histochemical assays is described. The method is based on paired biochemical and histochemical assays on single freeze-dried skeletal muscle fibers which differ widely in their properties. As an example, the method is applied to investigate the validity of the periodic acid-Schiff (PAS) reaction for the microdensitometric estimation of glycogen content. Some problems that may interfere with the calibration are discussed.

The use of texture analysis for the discrimination of Nissl substance in neurons.

Structural changes induced by cordotomy in the Nissl substance of identifiable spinal motoneurons innervating the white musculature of the European eel were quantified with the use of texture features calculated from digitized images. Data were evaluated and the motoneurons classified by using multivariate analysis. The study shows that there are differences in the structural organization of the Nissl substance of motoneurons taken from control and cordotomized fishes. Distinction could only be made by using texture features, as visual examination of the motoneurons did not reveal any alteration of the studied cellular substance. Reorganization of the Nissl substance might be the result of a changed protein metabolism or a changed neuronal activity pattern consequent upon cordotomy. The method employs quick and simple techniques and could be useful in several other neurobiological studies.

Further evaluation of quantitative nuclear image features for classification of lung carcinomas.

The usefulness of quantitative nuclear image features (QNI) for the histological classification of lung carcinomas was investigated. As no clear distinction could be established between the distributions of these features for the nuclei of squamous cell, adenocarcinoma, and large cell carcinoma, the attention was restricted to the discrimination between small cell lung carcinoma (SCLC) and non-small cell carcinoma (NSCLC). This discrimination is the crucial one in discussions about the choice of treatment. The differences between SCLC and NSCLC are statistically highly significant for various QNI features. The use of more than one QNI feature hardly raised the discriminatory performance with respect to the distinction between SCLC and NSCLC. Inferences were made about the probability and confidence interval of SCLC for a given QNI feature. It is concluded that in cases of uncertainty or disagreement, nuclear characteristics are useful for the discrimination between SCLC and NSCLC.

Resistance to fatigue of single muscle fibres from Xenopus related to succinate dehydrogenase and myofibrillar ATPase activities.

This report describes how the resistance to fatigue of a muscle fibre relates to the fibre's most important ATP-producing and ATP-consuming reactions. Twelve intact single muscle fibres were dissected from lumbrical muscles of Xenopus laevis. Their resistance to fatigue induced by repeated tetanic stimulation was determined, as well as their succinate dehydrogenase activity and calcium-stimulated myofibrillar ATPase activity. The enzyme activities were determined by means of quantitative histochemistry. It was found that resistance to fatigue correlates with succinate dehydrogenase activity (r = 0.83) and with myofibrillar ATPase activity (r = -0.74). The highest correlation was found between resistance to fatigue and the ratio of succinate dehydrogenase to myofibrillar ATPase activity (r = 0.93). It is concluded that muscular fatigue is closely related to cellular energetics.

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.

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.

Maximum rate of oxygen consumption and quantitative histochemistry of succinate dehydrogenase in single muscle fibres of Xenopus laevis.

Three different types of single living muscle fibre were dissected from the iliofibularis muscle of Xenopus laevis. The fibres were mounted in a glass chamber and their rate of oxygen consumption was determined as a function of twitch frequency at 20 degrees C. The rate of oxygen consumption increased with twitch frequency until it levelled off and reached a maximum. The maximum rate of oxygen consumption varied between fibres (0.019 to 0.161 nmol O2 s-1 mm-3) and was reached at different twitch frequencies (less than 0.2 to 5.7 stimuli s-1). After the determination of the maximum rate of oxygen consumption, the succinate dehydrogenase activity in cross sections of the fibre was determined by means of a quantitative histochemical method. A proportional relationship between the maximum rate of oxygen consumption and the succinate dehydrogenase activity was found. The maximum rate of oxygen consumption and the succinate dehydrogenase activity are also proportional to the volume density of mitochondria in the three fibre types reported by Smith and Ovalle (1973; J. Anat., Lond. 116, 1-24). It is concluded that quantitative histochemistry of succinate dehydrogenase reliably predicts the maximum rate of oxygen consumption of muscle fibres in Xenopus laevis and that the maximum rate of oxygen consumption of single muscle fibres is determined by the volume density of mitochondria.