NADPH-diaphorase activity and nitric oxide synthase-like immunoreactivity colocalize in the electromotor system of four species of gymnotiform fish.

The electric organ discharge (EOD) of gymnotiform electric fish is controlled by a well-characterized neural circuit in the brainstem and spinal cord. NADPH-diaphorase (NADPH-d) activity was previously found in phase-locking and/or rapidly firing neurons in the electromotor and electrosensory systems of Apteronotus leptorhynchus [Turner and Moroz, 1995]. These findings suggested that nitric oxide synthase (NOS) is expressed in these neurons and may regulate their precise, high frequency firing. We extended these results by examining the distribution of both NADPH-d activity and NOS-like immunoreactivity (NOS-lir) in the electromotor systems of four gymnotiform species that differ in the frequency and modulation of their EODs. NOS-lir colocalized with NADPH-d staining throughout the electromotor system, indicating that NADPH-d is a faithful indicator of NOS in this system. The distribution of NOS-lir and NADPH-d was similar in the electromotor systems of all four species in this study, with one exception: NOS and NADPH-d staining was consistently less intense in pacemaker and relay cells in Sternopygus macrurus, which produces low frequency EODs, than in the three other species that produce higher frequency EODs. This species difference in NOS expression in the pacemaker nucleus may be related to species differences either in EOD frequency or in modulations of the EOD (e.g., the jamming avoidance response). In Apteronotus species, NOS-lir and NADPH-d were concentrated in bands along the axons of their nerve-derived electric organs. These bands corresponded to regions surrounded by little or no staining with a Schwann cell-specific antibody, suggesting that the NOS-positive regions lie near nodes of Ranvier. In Sternopygus and Eigenmannia, the innervated, posterior membranes of muscle-derived electrocytes were more intensely labeled for NADPH-d and NOS than inexcitable portions of the membrane. Thus, in both muscle- and nerve-derived electric organs, NOS is concentrated near excitable membranes. These results indicate that NOS is well-positioned within the electromotor system to regulate the frequency, precision, amplitude, and waveform of EODs.

Distinct myosin heavy chain isoform transitions in developing slow and fast cat hindlimb muscles.

The expression of myosin heavy chain (MHC) isoforms leading to adult fiber phenotypes in the tibialis anterior (TA) and soleus muscles of the cat were investigated from embryonic day 35 to 1 year after birth. Electrophoresis and immunoblotting of myofibrils demonstrated the expression of 5 different MHC isoforms, i.e. I, IIa, IIx, embryonic, and neonatal, during development. Based on electrophoresis, the adult-like MHC composition of the soleus and TA were not observed until postnatal day 40 (P40) and 120 (P120), respectively. In contrast, immunohistochemical analyses revealed that the adult-like fiber phenotype composition was attained much later (P120) in the soleus. The existence of multiple MHC isoforms in individual fibers suggested that transitions occurred until P120 in both muscles. Adult type I fibers were first observed at P1. Adult IIA fibers were first observed at P30 in the TA and P40 in the soleus. IIX fibers were not identified until P40 in the TA. The transition to the predominantly slow phenotype of the soleus involved a gradual loss of embryonic and fast isoforms accompanied by an accumulation of slow MHC. In contrast, the expression of slow and fast MHC in the fast TA muscle was relatively unchanged throughout development. These results show that the establishment of a given MHC-based fiber phenotype varies significantly between slow and fast muscles in the kitten.

Development and regeneration of the electric organ.

The electric organ has evolved independently from muscle in at least six lineages of fish. How does a differentiated muscle cell change its fate to become an electrocyte? Is the process by which this occurs similar in different lineages? We have begun to answer these questions by studying the formation and maintenance of electrocytes in the genus Sternopygus, a weakly electric teleost. Electrocytes arise from the fusion of fully differentiated muscle fibers, mainly those expressing fast isoforms of myosin. Electrocytes briefly co-express sarcomeric proteins, such as myosin and tropomyosin, and keratin, a protein not found in mature muscle. The sarcomeric proteins are subsequently down-regulated, but keratin expression persists. We investigated whether the maintenance of the electrocyte phenotype depends on innervation. We found that, after spinal cord transection, which silences the electromotor neurons that innervate the electrocytes, or destruction of the spinal cord, which denervates the electrocytes, mature electrocytes re-express sarcomeric myosin and tropomyosin, although keratin expression persists. Ultrastructural examination of denervated electrocytes revealed nascent sarcomeres. Thus, the maintenance of the electrocyte phenotype depends on neural activity.

Reexpression of myogenic proteins in mature electric organ after removal of neural input.

The electric organ (EO) of the weakly electric fish Sternopygus macrurus derives from striated myofibers that fuse and suppress many muscle properties. Mature electrocytes are larger than muscle fibers, do not contain sarcomeres, or express myosin heavy chain (MHC) or tropomyosin. Furthermore, electrocytes express keratin, a protein not expressed in muscle. In S. macrurus the EO is driven continuously at frequencies higher than those of the intermittently active skeletal muscle. The extent to which differences in EO and muscle phenotype are accounted for by activity patterns, or innervation per se, was determined by assessing the expression of MHC, tropomyosin, and keratin 2 and 5 weeks after the elimination of (1) activity patterns by spinal transection or (2) all synaptic input by denervation. Immunohistochemical analyses showed no changes in muscle fiber phenotypes after either experimental treatment. In contrast, the keratin-positive electrocytes revealed an upregulation of MHC and tropomyosin. Nearly one-third of all electrocytes expressed MHC (35%) and tropomyosin (25%) 2 weeks after spinal transection, whereas approximately two-thirds (61%) expressed MHC 2 weeks after denervation. After 5 weeks of denervation or spinal transection, all electrocytes contained MHC and tropomyosin. Newly formed sarcomere clusters also were observed in denervated electrocytes. The MHC expressed in electrocytes corresponded to that present in a select population of muscle fibers, i.e., type II fibers. Thus, the elimination of electrical activity or all synaptic input resulted in a partial reversal of the electrocyte phenotype to an earlier developmental stage of its myogenic lineage.

Phenotypic conversion of distinct muscle fiber populations to electrocytes in a weakly electric fish.

In most groups of electric fish, the electric organ (EO) derives from striated muscle cells that suppress many muscle phenotypic properties. This phenotypic conversion is recapitulated during regeneration of the tail in the weakly electric fish Sternopygus macrurus. Mature electrocytes, the cells of the electric organ, are considerably larger than the muscle fibers from which they derive, and it is not known whether this is a result of muscle fiber hypertrophy and/or fiber fusion. In this study, electron micrographs revealed fusion of differentiated muscle fibers during the formation of electrocytes. There was no evidence of hypertrophy of muscle fibers during their phenotypic conversion. Furthermore, although fish possess distinct muscle phenotypes, the extent to which each fiber population contributes to the formation of the EO has not been determined. By using myosin ATPase histochemistry and anti-myosin heavy chain (MHC) monoclonal antibodies (mAbs), different fiber types were identified in fascicles of muscle in the adult tail. Mature electrocytes were not stained by the ATPase reaction, nor were they labeled by any of the anti-MHC mAbs. In contrast, mature muscle fibers exhibited four staining patterns. The four fiber types were spatially arranged in distinct compartments with little intermixing; peripherally were two populations of type I fibers with small cross-sectional areas, whereas more centrally were two populations of type II fibers with larger cross-sectional areas. In 2- and 3-week regenerating blastema, three fiber types were clearly discerned. Most (> 95%) early-forming electrocytes had an MHC phenotype similar to that of type II fibers. In contrast, fusion among type I fibers was rare. Together, ultrastructural and immunohistochemical analyses revealed that the fusion of muscle fibers gives rise to electrocytes and that this fusion occurs primarily among the population of type II fibers in regenerating blastema.

Limited fiber type grouping in self-reinnervation cat tibialis anterior muscles.

The percent and distribution patterns of three immunohistochemically identified fiber types within the anterior compartment of the cat tibialis anterior were determined 6 months after denervation and self-reinnervation. After self-reinnervation, mean frequencies of slow (9%) and fast (91%) fibers were similar to those in control (12% and 88%, respectively) muscles. However, a lower proportion of fast-1 (26%) and a higher proportion of fast-2 (65%) fibers were observed in self-reinnervated than control (32% and 56%) muscles. Quantitation of adjacencies between fibers of similar myosin heavy chain (MHC) phenotype, a measure of type grouping, revealed that the frequencies of two slow or two fast-1 fibers being adjacent in self-reinnervated muscles were similar to control. In contrast, the frequency of fast-2/fast-2 fiber adjacencies found in self-reinnervated muscles (45%) was significantly higher than in control muscles (37%). In both groups, the frequency of adjacencies between slow, fast-1, or fast-2 fibers was largely attributable to the number of each fiber type present. These data show that the incidence of grouping within each fiber type present was not altered after 6 months of self-reinnervation. Minimal changes in the spatial distribution of fiber types following self-reinnervation in adults suggests a limited degree of conversion of muscle fibers to a MHC phenotype matching the motoneuron characteristics.

Further evidence of incomplete neural control of muscle properties in cat tibialis anterior motor units.

To examine the influence of a motoneuron in maintaining the phenotype of the muscle fibers it innervates, myosin heavy chain (MHC) expression, succinate dehydrogenase (SDH) activity, and cross-sectional area (CSA) of a sample of fibers belonging to a motor unit were studied in the cat tibialis anterior 6 mo after the nerve branches innervating the anterior compartment were cut and sutured near the point of entry into the muscle. The mean, range, and coefficient of variation for the SDH activity and the CSA for both motor unit and non-motor unit fibers for each MHC profile and from each control and each self-reinnervated muscle studied was obtained. Eight motor units were isolated from self-reinnervated muscles using standard ventral root filament testing techniques, tested physiologically, and compared with four motor units from control muscles. Motor units from self-reinnervated muscles could be classified into the same physiological types as those found in control tibialis anterior muscles. The muscle fibers belonging to a unit were depleted of glycogen via repetitive stimulation and identified in periodic acid-Schiff-stained frozen sections. Whereas muscle fibers in control units expressed similar MHCs, each motor unit from self-reinnervated muscles contained a mixture of fiber types. In each motor unit, however, there was a predominance of fibers with the same MHC profile. The relative differences in the mean SDH activities found among fibers of different MHC profiles within a unit after self-reinnervation and those found among fibers in control muscles were similar, i.e., fast-2 < fast-1 < or = slow MHC fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of incomplete neural control of motor unit properties in cat tibialis anterior after self-reinnervation.

1. The mechanical, morphological and biochemical properties of single motor units from the anterior compartment of the tibialis anterior muscle in adult cats were studied six months after the nerve branches to that compartment were cut and resutured in close proximity to the muscle. 2. In these self-reinnervated muscles, the maximum tetanic tensions were lower in slow than fast units, a relationship similar to that observed among motor units from control adult muscles. The maximum tetanic tensions produced by the fast units were larger than those produced by the same motor unit types in control muscles. Direct counts of muscle fibres belonging to a motor unit showed that factors controlling the number of muscle fibres innervated by a motoneurone type persist during the reinnervation process in that fast motoneurones reinnervated more muscle fibres than slow motoneurones. Thus, the number of muscle fibres reinnervated by a motoneurone principally accounted for the difference in the maximum tension outputs among motor unit types, a relationship similar to that observed in control tibialis anterior muscles. 3. Monoclonal antibodies for specific myosin heavy chains were used to differentiate fibre types. By this criterion, motor units from control muscles were found to contain a homogeneous fibre type composition. In contrast, a heterogeneous, yet markedly biased, fibre type composition was observed in each unit analysed from self-reinnervated muscles. 4. Although not all of the muscle fibres of a motor unit developed the same type-associated parameters after reinnervation, the relationships among myosin heavy chain profile, succinate dehydrogenase activity and the fibre size were similar in fibres of control and self-reinnervated muscles. 5. The processes which dictate both motor unit size and the matching between motoneurone and muscle fibre type during the reinnervation process must be interdependent and result from a hierarchy of decisions which reflects their relative importance. The mechanisms responsible for these two processes may be a combination of: (1) selective innervation which may or may not incorporate a pruning process if multiple synaptic connections are initially formed and/or (2) conversion of enough fibres of a motor unit to form a predominant type.

Innervation patterns in the cat tibialis anterior six months after self-reinnervation.

The spatial distribution of fibers belonging to a single motor unit was analyzed in 10 motor units from the tibialis anterior of the cat 6 months after denervation and self-reinnervation of the anterior (superficial) compartment of the muscle. After self-reinnervation, the distribution patterns of the fibers in the fast motor units were significantly different than control, whereas the fiber distribution in the slow unit was similar to control. Reinnervated fast units had a significant increase in the number of adjacencies among motor unit fibers, and there were often distinct "clusters or groups" of fibers distributed within the motor unit territory. Clustering or grouping of fibers was evident within the motor unit, even though fiber type grouping was not evident within the muscle. The differences in distribution patterns between control and reinnervated units may be related to variations in the branching pattern of axons during reinnervation compared to the process that occurs during development.

Deficits in operant behaviour in monkeys treated with N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Six adult Macaca fascicularis monkeys were trained to perform an instrumentally conditioned, visually-guided forearm reaching task for fruit juice reinforcement. Once animals were overtrained on this task, they were given intravenous injections of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (0.15 to 0.33 mg/kg). Animals were tested daily for performance in the previously learned behavioural task and were assessed daily for abnormalities in motor functioning. Monkeys developed deficits in operant task performance characterized by termination of responses after an initial series of responses and long pauses between responses. Once an animal stopped responding to the task, responses could often be reinitiated if the experimenter guided the monkey through the task. This type of performance deficit was seen both before and without the appearance of distinct parkinsonian motor signs. Animals which developed motor signs had extensive ventral mesencephalic cell loss while an animal with performance deficits but without motor signs had cell loss restricted to the ventral substantia nigra pars compacta. The results demonstrate that operant performance deficits can be observed in MPTP-treated monkeys independent of the appearance of motor deficits.

Dopaminergic dorsal raphe neurons in cats and monkeys are sensitive to the toxic effects of MPTP.

Tyrosine hydroxylase immunohistochemical analysis of the dorsal raphe nucleus (DRN) of severely parkinsonian MPTP-treated cats and cynomolgus monkeys revealed a marked loss of catecholaminergic neurons in this region. Cell loss was more extensive in the ventral portion of the nucleus with a relative sparing of neurons in the dorsal-most portions of the DRN. These results demonstrate that catecholaminergic neurons other than those in the ventral mesencephalon and the locus ceruleus are affected by the toxic effects of MPTP.