Cross-reinnervation changes the expression patterns of the monocarboxylate transporters 1 and 4: An experimental study in slow and fast rat skeletal muscle.

The monocarboxylate transporters 1 and 4 are expressed in brain as well as in skeletal muscle and play important roles in the energy metabolism of both tissues. In brain, monocarboxylate transporter 1 occurs in astrocytes, ependymocytes, and endothelial cells while monocarboxylate transporter 4 appears to be restricted to astrocytes. In muscle, monocarboxylate transporter 1 is enriched in oxidative muscle fibers whereas monocarboxylate transporter 4 is expressed in all fibers, with the lowest levels in oxidative fiber types. The mechanisms regulating monocarboxylate transporter 1 and monocarboxylate transporter 4 expression are not known. We hypothesized that the expression of these transporters would be sensitive to long term changes in metabolic activity level. This hypothesis can be tested in rat skeletal muscle, where permanent changes in activity level can be induced by cross-reinnervation. We transplanted motor axons originally innervating the fast-twitch extensor digitorum longus muscle to the slow-twitch soleus muscle and vice versa. Four months later, microscopic analysis revealed transformation of muscle fiber types in the cross-reinnervated muscles. Western blot analysis showed that monocarboxylate transporter 1 was increased by 140% in extensor digitorum longus muscle and decreased by 30% in soleus muscle after cross-reinnervation. In contrast, cross-reinnervation induced a 62% decrease of monocarboxylate transporter 4 in extensor digitorum longus muscle and a 1300% increase in soleus muscle. Our findings show that cross-reinnervation causes pronounced changes in the expression levels of monocarboxylate transporter 1 and monocarboxylate transporter 4, probably as a direct consequence of the new pattern of nerve impulses. The data indicate that the mode of innervation dictates the expression of monocarboxylate transporter proteins in the target cells and that the change in monocarboxylate transporter isoform profile is an integral part of the muscle fiber transformation that occurs after cross-reinnervation. Our findings support the hypothesis that the expression of monocarboxylate transporter 1 and monocarboxylate transporter 4 in excitable tissues is regulated by activity.

A novel postsynaptic density protein: the monocarboxylate transporter MCT2 is co-localized with delta-glutamate receptors in postsynaptic densities of parallel fiber-Purkinje cell synapses.

Confocal immunofluorescence microscopy showed strong monocarboxylate transporter 2 (MCT2) labeling of Purkinje cell bodies and punctate labeling in the molecular layer. By immunogold cytochemistry, it could be demonstrated that the MCT2 immunosignal was concentrated at postsynaptic densities of parallel fiber-Purkinje cell synapses. The distribution of MCT2 transporters within the individual postsynaptic densities mimicked that of the delta2 glutamate receptor, as shown by use of two different gold-particle sizes. The MCT2 distribution was also compared with the distributions of other monocarboxylate transporters (MCT1 and MCT4). The MCT1 immunolabeling was localized in the endothelial cells, while MCT4 immunogold particles were associated with glial profiles, including those abutting the synaptic cleft of the parallel fiber-spine synapses. The postsynaptic density (PSD) molecules identified so far can be divided into five classes: receptors, their anchoring molecules, molecules involved in signal transduction, ion channels, and attachment proteins. Here, we provide evidence that this list of molecules must now be extended to comprise an organic molecule transporter: the monocarboxylate transporter MCT2. The present data suggest that MCT2 has specific transport functions related to the synaptic cleft and that this transporter may allow an influx of lactate derived from perisynaptic glial processes. The expression of MCT2 in synaptic membranes may allow energy supply to be tuned to the excitatory drive.

Effect of cross-reinnervation on the expression of GLUT-4 and GLUT-1 in slow and fast rat muscles.

We determined whether the twitch-velocity phenotype or the metabolic phenotype of a muscle influences the content of GLUT-4 and GLUT-1 proteins. The soleus (Sol) and extensor digitorum longus (EDL) muscles were cross-reinnervated (X-Sol, X-EDL). After 3 mo the X-EDL had become enriched in slow-twitch oxidative (SO) fibers (70.5% SO) compared with its control (3.8% SO), whereas the X-Sol became enriched in fast-twitch oxidative-glycolytic (FOG) fibers (78.6% FOG) compared with its control (10% FOG). Thus the twitch phenotype of X-Sol shifted to fast-twitch muscle, whereas X-EDL shifted to a slow-twitch muscle. In the X-EDL, the oxidative nature of the X-EDL was increased to 97% oxidative fibers compared with 43% oxidative fibers in the normal EDL. In the Sol the oxidative nature of the X-Sol was retained at 100%. GLUT-4 content was increased 1.6-fold in the X-EDL (P < 0.05) but was not changed in the X-Sol (P > 0.05). GLUT-1 content was increased fourfold in X-EDL but was not altered in the X-Sol. We conclude that GLUT-4 and GLUT-1 content in muscle is related to the oxidative phenotype of the muscle rather than the twitch-velocity phenotype.

Factors causing different properties at neuromuscular junctions in fast and slow rat skeletal muscles.

Neuromuscular junctions on fast and slow skeletal muscle fibers have different properties. Possible reasons for these differences were examined in adult rat soleus (SOL) muscle fibers reinnervated at new ectopic or old denervated sites by fast fibular (FIB) or slow SOL motoneurons. FIB motoneurons formed large ectopic junctions with a high density of nerve terminal varicosities (fast appearance), whereas SOL motoneurons formed small ectopic junctions with a low density of varicosities (slow appearance). Both FIB and SOL motoneurons formed small junctions with a slow appearance when reinnervating old SOL endplates. FIB nerves innervating ectopic sites and SOL nerves reinnervating old sites had the same appearance whether they contacted the SOL fibers alone (single innervation) or together (dual innervation). Continuous stimulation of the FIB nerve at 10 Hz for 3-4 months reduced the size of ectopic FIB and intact extensor digitorum longus (EDL) junctions and caused a modest reduction in density of terminal varicosities in EDL. Junction size and muscle fiber diameter were positively correlated, but the slope describing this relation was steeper for FIB junctions than for SOL junctions. It is concluded that in the present system (1) motoneuron type and not muscle fiber type determines the fast or slow character of the neuromuscular junction. (2) denervated endplates of one type place stable and severe constraints on the termination pattern of reinnervating axons of another type, (3) the appearance of fast EDL junctions undergoes a modest fast to slow transformation when exposed to long-term slow pattern stimulation, and (4) not only the size of the muscle fibers, but also the type and firing pattern of the motoneurons and the spatial constraints at preformed endplates influence the relation between junction size and muscle fiber diameter.

Demonstration of glutamate-like immunoreactivity at rat neuromuscular junctions by quantitative electron microscopic immunocytochemistry.

Motor nerve terminals and adjacent structures in the extensor digitorum longus and soleus muscles of young adult rats were examined for their content of glutamate by means of quantitative, electron microscopic immunocytochemistry employing colloidal gold particles as markers. The level of glutamate immunoreactivity was stronger in the extensor digitorum longus terminals than in the soleus terminals. In both muscles the glutamate immunolabelling was stronger in the nerve terminals than in the synaptic clefts and the postsynaptic tissue separating the secondary clefts, but the differences were larger in the extensor digitorum longus than in the soleus muscle. The myofibrils of the soleus muscle were more densely labelled than those in the extensor digitorum longus muscle. The level of immunoreactivity was high in the Schwann cells of both muscles. By comparing the labelling intensity of motor nerve terminals with that of muscle fibres and hippocampal mossy fibres (compartments that have been analysed previously with respect to their glutamate content), the mean concentration of fixed glutamate in the extensor digitorum terminals was estimated to be in the range of 10-20 mmol/l. An association of glutamate immunoreactivity with synaptic vesicles was demonstrated in the most strongly labelled terminals. Whether these epitopes were localized in the interior of the vesicles or at their external surface could not be resolved with the present technique. These data indicate that motor nerve terminals contain glutamate, and that the enrichment of this amino acid is more pronounced in the terminals of the extensor digitorum longus muscle (a fast muscle) than in those of the soleus muscle (a slow muscle). A possible modulatory or trophic role of glutamate in the mammalian neuromuscular junction should be considered.

Different effects of physical training on the morphology of motor nerve terminals in the rat extensor digitorum longus and soleus muscles.

The morphology of nerve terminals in the rat extensor digitorum longus and soleus muscles was studied with light microscopy in 13-week-old male animals after 6 weeks of treadmill running and compared with data from untrained controls. The terminals were stained with methylene blue. Physical training tended to increase the area and length of the nerve terminals in relation to the corresponding muscle fiber diameter, and to reduce the density of nerve terminal varicosities, but significant differences between the trained group and the control group were obtained only in the extensor digitorum longus muscle. The different degrees of effect on the nerve terminals in the two muscles may be due to different abilities to respond to the training, but may also be due to differences in work load caused by the training. The effect of training on extensor digitorum longus junctions may reflect some transformation from fast to slow morphological characteristics.

Postnatal development of rat motor nerve terminals.

The nerve terminals of neuromuscular junctions in the rat diaphragm, extensor digitorum longus muscle and soleus muscle have been studied in animals between 3 weeks and 2.5 years of age using methylene blue stain and light microscopy. Dimensions, structure and organization of the nerve terminals were shown to change during life at various rates in different muscles and postnatal periods. The area and length of the terminals increase in all three muscles until young adult age. Later these dimensions continue to increase in the extensor digitorum longus and soleus muscles. In the diaphragm only the length increases, and this occurs late in adult life. The area also increases in relation to the diameter of the corresponding muscle fiber. Adult soleus terminals are more elongated than terminals in the diaphragm and extensor digitorum longus muscle. During adult life the extension of nerve terminals in relation to muscle fiber length increases in the extensor digitorum longus and soleus muscles, but is almost unchanged in the diaphragm. The nerve terminal branches are mainly coarse and irregular in young animals, but possess varying numbers of varicosities in adult animals. The number of varicosities is high in the extensor digitorum longus muscle and low in the diaphragm. In old animals the number of varicosities tends to be reduced. With increasing age the nerve terminal branches become organized in distinct groups with increasing distance between the groups. This is prominent in the soleus.

Species specific morphology of mammalian motor nerve terminals.

Motor nerve terminals in the diaphragm, extensor digitorum longus and soleus muscles of young and adult rat, hamster and guinea pig were studied with the light microscope after staining with methylene blue. In adult animals the nerve terminals are smaller in the diaphragm than in the two other muscles. In the rat and hamster the extensor digitorum longus and soleus terminals are of similar area, but the terminals in extensor digitorum longus are shorter. In the guinea pig the terminals are smaller and shorter in soleus than in extensor digitorum longus. The density of nerve terminal varicosities is lowest in the diaphragm in all three species. In the rat and hamster the density is higher in extensor digitorum longus than in soleus. In the guinea pig the converse is found. In all three muscles the density of varicosities is higher in the rat than in the hamster and guinea pig. The nerve terminal branches in the diaphragm are mostly organized in one group. In the rat and hamster the soleus terminal branches are more separated in groups than the extensor digitorum longus terminal branches. In the guinea pig the number of groups is almost the same in the two muscles. These muscle and species-specific differences appear already in very young animals.

Motor endplates in fast and slow muscles of the rat: what determines their difference?

Motor endplates in fast and slow skeletal muscles have different functional and morphological characteristics, and for brevity, are termed fast and slow respectively. We have examined the terminal arborization patterns of fast fibular and slow soleus axons 3-4 and 6 months after they reinnervated old preformed endplates or formed new ectopic endplates with denervated rat soleus muscles. Ectopic endplates formed by transplanted fibular and soleus nerves were fast and slow in appearance respectively. Both the fibular and the soleus nerves formed endplates of slow appearance when they reinnervated the original endplates. The fast appearance of ectopic fibular nerve endplates was unaffected by reinnervation of the original endplates by the slow soleus nerve. Dually innervated fibres had intermediate contraction speed compared to the fast fibres reinnervated only by the fibular nerve and the slow fibres reinnervated only by the soleus nerve. Continuous stimulation of the transplanted fibular nerve at 10 Hz for 3-4 months, starting just before the onset of ectopic endplate formation, prevented the increase in contraction speed seen without stimulation. The ectopic endplates of the stimulated axons were much smaller than usual and showed some signs of fast to slow transformation, but the transformation was incomplete and varied in degree between preparations. Transplanted soleus axons were less prone to growing along foreign pathways and to forming ectopic endplates than fibular axons.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of regular physical activity on the nervous system.

There are only a very small number of research reports on the effect of regular physical activity on the nervous system. Some of these are based on inadequately defined physical activity, and some of the conclusions are poor. There is little reliable information about which particular forms of regular physical activity affect particular structures and functions in the nervous system. The available results provide only tenuous evidence for assessment of the significance of physical activity for health.