Diversification of the myofibrillar M-band in rat skeletal muscle during postnatal development

Summary The fine structure of the M-band in soleus (SOL) and extensor digitorum longus (EDL) muscles in newborn and four-week-old rats was studied using electron-microscopic techniques. In newborn rats, all myotubes and fibres in both muscles had an identical myofibrillar appearance. A five-line M-band pattern was seen in longitudinal sections and distinct M-bridges in cross-sections. The Z-discs were of medium width. On the other hand, in four-week-old rats, different muscle fibre types were observed on the basis of their myofibrillar pattern. In SOL two fibre types were distinguished in longitudinal sections. One had a four-line M-band pattern and very broad Z-discs, whereas the other type had five lines in the M-band and broad Z-discs. In EDL, three different myofibrillar patterns were observed. The M-bands were composed of three, four or five lines. Fibres had either thin, broad or medium Z-disc widths, respectively. In cross-sections of the SOL muscle one group of fibres showed indistinct M-bridges, whereas distinct M-bridges were seen in the other fibres and in all observed EDL muscle fibres. We conclude that initially there seems to be a single intrinsic program for M-band genesis; this program becomes modified upon the induction of functionally differentiated fibres.     

Myofibrillar M-band proteins in rat skeletal muscles during development.

The distribution of three myofibrillar M-band proteins, myomesin, M-protein and the muscle isoform of creatine kinase, was investigated with immunocytochemical techniques in skeletal muscles of embryonic, fetal, newborn and four-week-old rats. Furthermore, muscles of newborn rats were denervated and examined at four weeks of age. In embryos, myomesin was present in all myotome muscle fibres of the somites, whereas M-protein was detected only in a small proportion of the myotome muscle fibres and muscle creatine kinase was not detected at all. In fetal and newborn muscles, all fibres contained all three M-band proteins. At four weeks of age, when fibre types (type 1 or slow twitch fibres and type 2 or fast twitch fibres) were clearly discernable, the pattern was changed. Myomesin and muscle creatine kinase were still observed in all fibres, whereas M-protein was present only in type 2 fibres. On the other hand, in muscle fibres denervated at birth all three M-band proteins were still detected. Our results suggest 1) that during the initial stages of myofibrillogenesis expression and incorporation of myomesin into the M-band precede that of M-protein and muscle creatine kinase; 2) that expression and incorporation of all three M-band proteins during fetal development is nerve independent and non coordinated to the expression of different forms of myosin heavy chains, and 3) that the suppression of M-protein synthesis during postnatal development is nerve dependent and reflects the maturation of slow twitch motor units.     

Myofibrillar M-band proteins in rat skeletal muscles during development

Summary The distribution of three myofibrillar M-band proteins, myomesin, M-protein and the muscle isoform of creatine kinase, was investigated with immunocytochemical techniques in skeletal muscles of embryonic, fetal, newborn and four-week-old rats. Furthermore, muscles of newborn rats were denervated and examined at four weeks of age. In embryos, myomesin was present in all myotome muscle fibres of the somites, whereas M-protein was detected only in a small proportion of the myotome muscle fibres and muscle creatine kinase was not detected at all. In fetal and newborn muscles, all fibres contained all three M-band proteins. At four weeks of age, when fibre types (type 1 or slow twitch fibres and type 2 or fast twitch fibres) were clearly discernable, the pattern was changed. Myomesin and muscle creatine kinase were still observed in all fibres, whereas M-protein was present only in type 2 fibres. On the other hand, in muscle fibres denervated at birth all three M-band proteins were still detected. Our results suggest 1) that during the initial stages of myofibrillogenesis expression and incorporation of myomesin into the M-band precede that of M-protein and muscle creatine kinase; 2) that expression and incorporation of all three M-band proteins during fetal development is nerve independent and non coordinated to the expression of different forms of myosin heavy chains, and 3) that the suppression of M-protein synthesis during postnatal development is nerve dependent and reflects the maturation of slow twitch motor units.     

Cellular patterns of post-mortem glycogenolysis in longissimus dorsi muscles of pigs at different live weights

Synopsis Serial frozen sections of longissimus dorsi muscles from twelve Canadian Yorkshire breed pigs at different live weights (13–86kg) were stained for glycogen by the periodic acid-Schiff (PAS) reaction and reacted for NAD tetrazolium reductase to determine the mitochondrial content of muscle fibres. Sections from muscles taken immediatelypost mortem and at 5 hrpost mortem were compared and the patterns of glycogenolysis in high, intermediate and low mitochondrial content fibres were assessed on the basis of the percentage of initially PAS-positive fibres which became PAS-negative 5 hrpost mortem. This form of assessment was necessary because not all fibres were PAS-positive immediatelypost mortem. In smaller pigs, only a few muscle fibres depleted their stainable glycogen by 5 hr and most of these fibres had a high mitochondrial content. In larger pigs, most or all initially PAS-positive fibres became PAS-negative by 5 hrpost mortem. Fibres with a low mitochondrial content accounted for most of the glycogenolysis detected histochemically in larger animals. The overall percentage of PAS-positive fibres was related to glycogen concentration (mg/g),r=0.52,P<0.025, when samples with all PAS-positive fibres were excluded.     

Low temperature activation of post mortem glycogenolysis in bovine skeletal muscle fibres.

Synopsis Trimmed strips of stermomandibularis muscles taken from freshlyslaughtered cattle were placed in an isotonic myograph and cooled to 1°C. Spontaneous activity due to neuromuscular irritability was minimized by keeping muscle surfaces moist and anaerobic and was monitored by electromyography. Muscle strips were removed and frozen for histochemical analysis after they had completed their initial phase of cold-induced shortening (several hours). Control strips maintained for an equal time at 24°C rarely depleted the stainable glycogen in any of their muscle fibres so as to become PAS-negative. In chilled muscle strips, however, glycogenolysis was activated in some muscle fibres and they became PAS-negative. In serial sections, most of the PAS-negative fibres exhibited strong ATPase and weak succinate dehydrogenase activity. Fibres with weak ATPase and strong succinate dehydrogenase activity rarely became PAS-negative. These results are in agreement with biochemical reports of a cold-induced (<5°C) activation of glycolysis in skeletal musclepost mortem. Investigations on untrimmed lengths of excised sternoman dibularis muscle indicated that longitudinal muscle damage caused in cutting muscle strips for the myograph and/or their more rapid rate of initial cooling had facilitated the depletion of stainable glycogen.     

Fine structure of single fibres of human skeletal muscle

Summary Individual muscle fibres were separated from freeze-dried needle biopsies and classed as type I or type II fibres according to their myofibrillar ATP-ase. Portions of the same fibres were processed for electron microscopy and their fine structure examined. Type I fibres were found to have thicker Z-bands and more mitochondria and lipid droplets than the type II fibres.     

Cytophotometry of post mortem glycogenolysis in different histochemical types of muscle fibres of the pig.

Synopsis Serial transverse sections of porcine longissimus dorsi muscle (18 pigs, 50 to 178 kg live weight) were reacted for NAD tetrazolium reductase and ATPase at pH9.4, and for glycogen with the periodic acid-Schiff (PAS) reaction. Three histochemical types of muscle fibre were identified; (1) strong ATPase and weak NADH oxidative activity; (2) strong ATPase and intermediate NADH oxidative activity; and (3) weak ATPase and strong NADH oxidative activity. Immediatepost mortem samples from one side of each animal were compared with a laterpost mortem sample from the other side by measuring the absorbance of PAS-stained glycogen at 570 nm with a microscope photometer. Laterpost mortem absorbance was expressed as a percentage of immediatepost mortem absorbance in each category of muscle fibre in order to compensate for distributional error and different starting levels of glycogen. Muscle fibres with weak ATPase and strong NADH oxidative activity showed a progressive decrease in absorbance of PAS-stained glycogenpost mortem. In some animals, fibres with strong ATPase and intermediate or weak NADH oxidative activity showed an initialpost mortem increase in absorbance of PAS-stained glycogen which was then followed by a progressive decrease. The maximum rates of decrease in absorbance in the three fibre types did not differ to any great extent.     

Stretch receptors in urodele limb muscles

Summary Non-encapsulated, fine beaded nerve endings were found histologically on some muscle fibres in a number of limb muscles in newts and axolotls. They were present in newt muscles that had been chronically de-efferented, and in which no efferent activity survived, and were therefore likely to be sensory. They were located only on muscle fibres on or near the outside surface of the muscle. These small-diameter muscle fibres were characterised histochemically by low lipid, SDH and phosphorylase content; ultrastructurally by low glycogen content, and relatively large myofilaments poorly delimited by a sparse SR. There were many of this type (Type 1) that did not support sensory endings. A few endings occurred on another larger-diameter type of fibre (Type 2) whose properties were opposite to those listed above for Type 1. There was virtually no specialization of muscle fibre structure beneath the sensory endings.Physiological experiments involving ramp-and-hold and sinusoidal stretch applied to the muscle whilst recording single-unit afferent responses in m.ext. dig. III of axolotls showed unit responses very similar to those known from muscle spindles, particularly those of the frog.We are grateful to the M.R.C. for an equipment grant (to R.M.A.P.R.), and to Mrs. Janis Taberner for her technical help. Part of this work was done during the tenure of a Nuffield-NRC Lectureship (Q.B.) which is gratefully acknowledged, as is financial support by Prof. J.R. Nussall during a visit to the University of Alberta at Edmonton.     

Cytophotometry of post mortem glycogenolysis in quiescent bovine muscle fibres in relation to temperature, succinate dehydrogenase activity and adenosine triphosphatase activity

Summary Immediatepost mortem samples of sternomandibularis muscles from six steers were maintained with a minimum of intrinsic activity at approximately 40°C or allowed to cool to approximately 22°C. Samples were frozen in liquid nitrogen at 0, 2, 4 and 6hpost mortem and serial transverse sections were stained by the periodic acid-Schiff (PAS) reaction for glycogen or reacted for adenosine triphosphatase (ATPase) or succinate dehydrogenase. Individual fibres were mapped and categorized from their ATPase and succinate dehydrogenase activity using a projecting microscope. The absorbance of PAS-stained glycogen in individual fibres was measured with a microscope photometer at 570 and 601 nm with a correction for distributional error. Overall, thepost mortem decline in absorbance was approximately twice as fast in body temperature samples relative to room temperature samples. Transientpost mortem increases in absorbance were detected in some situations, particularly in fibres with strong ATPase activity from room temperature samples. In fibres with strong ATPase activity, the rate of decline in absorbance increased progressivelypost mortem. Fibres with weak ATPase mostly had a lower initialpost mortem absorbance and were generally the first to become PAS-negative.     

The ultrastructure of the metathoracic femoral extensors of the cockroach,Periplaneta americana

The ultrastructure of the femoral extensors of the metathoracic leg of the cockroach, Periplaneta americana was studied to determine morphological correlations with the known patterns of innervation, physiological properties and biochemical properties. Three different types of muscle fibers were described. Type 1 consisted of short sarcomeres (mean 3.7 μm), few mitochondria and sparse glycogen-like material; Type 2, short sarcomeres (4.2 μm), numerous mitochondria, large amounts of glycogen; Type 3, long sarcomeres (7.5 μm), numerous mitochondria and large amounts of glycogen. A qualitative examination of the sarcoplasmic reticulum (SR) and transverse tubular system (TTS) revealed the density of SR and TTS to be greatest in Type 1 and least in Type 3. There were obvious correlations between the morphological features and the other known characteristics of these muscle fibers. The role of these different muscle fiber types in different locomotory behaviors was discussed. In summary, the three types of muscle fibers are used in three different behaviors: Type 1, rapid walking; Type 2, slow walking; Type 3, postural control.     

The Ultrastructure of the Indirect Flight Muscles of the Monarch Butterfly,Danaus plexippus (L.) with Implications for Fuel Utilization

The fine structure of the dorsal longitudinal flight muscle of the monarch butterfly, Danaus plexippus (L.), is described. The high actin: myosin filament ratio in this fast muscle is likely related to extensive actin-myosin filament interaction which must occur when tension is increased and maintained by repeated nervous stimulation as demonstrated by Kammer (1967) during the long downstroke of the wingbeat cycle. Glycogen particles are present in the granular fraction of the interfibrillar sacroplasm and among the myofilaments of the flight muscle of young butterflies. The distribution of the granules appears to be related to spatial forces within the muscle fibril for in resting butterflies the intermyofilament particles are generally located in rows parallel to the myofilaments, while they are in transverse bands in the H and I zones of animals fixed immediately after flight. Since the glycogen particles were not depleted during flight, this redistribution does not appear to be related to glycogen metabolism. Glycogen in the flight muscle was depleted during eight days of adulthood in butterflies fed on honey solutions, but lipid reserves increased in the same period.     

Mutants lacking glutelin subunits in rice: mapping and combination of mutated glutelin genes

Nine mutant lines lacking glutelin subunits were selected from M₂ seeds of about 10000 M₁ plants mutagenized with gamma rays or EMS and from 1400 mutant lines selected originally for morphological characters. There were three types of mutants, one line lacking the largest subunit among four minor bands of glutelin acidic subunits (Type 1), five lines lacking the second largest subunit band (Type 2), and three lines lacking the third largest subunit band (Type 3). Mutants lacking the smallest subunit band were not found. Type 1 lacked 2 of the 10 spots of glutelin acidic subunits separated by two-dimensional electrophoresis and 1 of the 11 spots of the 57-kDa glutelin precursor. Type 2 lacked 2 spots of acidic subunits and 1 spot of the 57-kDa glutelin precursor, and had low amounts of 1 of the 8 spots of glutelin basic subunits. Type 3 mutants lacked each of 1 spot of the acidic subunits and glutelin precursor and had low amount of 1 spot of the basic subunits. Genetic analysis of the mutated genes showed that these mutant characters were controlled by single recessive genes named glu-1, glu-2, and glu-3, respectively. Mutated genes of different lines of the same type were found to be at the same locus. RFLP analysis of F₂ plants between the mutant lines and cv `Kasalath' indicated that glu-1 is on chromosome 2, glu-2 on chromosome 10, and glu-3 on chromosome 1. These mutant genes were combined by crossing, and a line lacking the 3 minor bands of the glutelin acidic subunits was developed. However, the total glutelin content of this line was not remarkably reduced, showing a only 13% decrease. Received: 1 April 1996 / Accepted: 14 June 1996     

Dimerisation of myomesin: implications for the structure of the sarcomeric M-band

The sarcomeric M-band is thought to provide a link between the thick and the elastic filament systems. So far, relatively little is known about its structural components and their three-dimensional organisation. Myomesin seems to be an essential component of the M-band, since it is expressed in all types of vertebrate striated muscle fibres investigated and can be found in its mature localisation pattern as soon as the first myofibrils are assembled. Previous work has shown that the N-terminal and central part of myomesin harbour binding sites for myosin, titin and muscle creatine kinase. Intrigued by the highly conserved domain layout of the C-terminal half, we screened for new interaction partners by yeast two-hybrid analysis. This revealed a strong interaction of myomesin with itself. This finding was confirmed by several biochemical assays. Our data suggest that myomesin can form antiparallel dimers via a binding site residing in its C-terminal domain 13. We suggest that, similar to alpha-actinin in the Z-disc, the myomesin dimers cross-link the contractile filaments in the M-band. The new and the already previously identified myomesin interaction sites are integrated into the first three-dimensional model of the sarcomeric M-band on a molecular basis.     

Histoenzymatic characterization of sub-types of type I fibres in fast muscles of rats.

The histochemical activities of succinic dehydrogenase (SDH), myofibrillar Adenosine triphosphatase (ATPase) and alpha glycerophosphate dehydrogenase were studied in serial sections of rat vastus lateralis (red) (RVL), gastrocnemius and diaphragm muscles. Three main fibre-types were distinguished. The "Type I" fibres of RVL and gastrocnemius muscles fell into two distinct groups: one category--"Type IA" showed very low ATPase activity. The second category of "Type IB" fibres displayed moderate ATPase reaction. The "Type IA" fibres were divisible into two sub-groups when tested for SDH reaction. "Type IA1" fibres possessed a homogenous distribution of diformazan granules throughout the fibre: "Type IA2" fibres displayed characteristic "moth-eaten" pattern of diformazan localization. The diaphragm muscle did not show either "Type IB" or "Type IA2" varieties. The great majority of TypeI fibres were sub-type IA1 in the three fast muscles studied. It is also demonstrated here that an inherent heterogeneity exists between Type I filores of diaphragm and leg muscles in regard to alpha-GPD localization. This histochemical data emphasizes the fact that subdivision of TypeI striated muscle fibres of mammalian animals into two sub-types is only approximate and that a further subcategorization is possible.     

The claw closer muscle of two estuarine crab species,Cyrtograpsus angulatus and Neohelice granulata (Grapsoidea,Varunidae): histochemical fibre type composition

Longo, M.V. and Díaz, A.O. (2011). The claw closer muscle of two estuarine crab species, Cyrtograpsus angulatus and Neohelice granulata (Grapsoidea, Varunidae): histochemical fibre type composition. —Acta Zoologica (Stockholm) 00 : 1–7. This study permitted the characterization of four types of muscle fibres in the claw closer muscles of Cyrtograpsus angulatus and Neohelice granulata. Succinic dehydrogenase (SDH) for mitochondria, periodic acid Schiff (PAS) for glycogen, Sudan Black B for lipids and myosin‐adenosine triphosphatase (m‐ATPase) preincubated at alkaline and acid pHs were used for that purpose. The mean fibre diameters, the relative areas and frequencies of each muscle fibre type were calculated. Types I and IV would be considered ‘extreme’ groups with type I fibres large, weak and acid/alkaline‐labile m‐ATPase, weak SDH, PAS and Sudan, and type IV fibres small, very strong and acid/alkaline‐resistant m‐ATPase, strong SDH and PAS, and moderate Sudan. Types II and III would belong to a predominant ‘intermediate’ group. Type IV fibres were scarce in C. angulatus but represented 25% of the total fibre population in N. granulata. In C. angulatus, the relative area occupied by type I fibres was bigger than its relative proportion, whereas in N. granulata, types I and II had similar patterns. Concluding, variations in fibre type composition in the claw closer muscles of C. angulatus and N. granulata would be linked to different habitats and feeding behaviours.     

Fluorimetry of bovine myotendon junction by fibre-optics and microscopy of intact and sectioned tissues

Summary The autofluorescence of tendon, epimysium and endomysium at the myotendon junction of the deep digital flexor in the bovine forelimb was measured with a fluorescence microscope and with a bifurcated light guide composed of quartz optical fibres. Data were adjusted for spectral variation in the radiance of the halogen illuminator used to standardize the photometer. Samples of myotendon junction were examined intact, in slices several millimetres thick and after being frozen in liquid nitrogen and sectioned at 20 µm. Sections were examined with and without a mounting medium and with and without immersion oil objectives. Type I collagen fibres were identified by their scarcity of branching, relatively large size and yellow staining with silver. Type III collagen fibres were identified by their extensive branching, small size and black staining with silver. Purified Types I and III collagen were also examined. Type I collagen fibres had a strong fluorescence emission peak between 410 and 450 nm and a shoulder at 510 nm. For the strong peak, results obtained by fibre-optics were positively biased relative to those obtained by microscopy. Type III collagen reticular fibres lacked a strong emission peak at 410 to 450 nm. Although their overall fluorescence was weaker than that of Type I collagen fibres, Type III collagen fibres had similar or slightly stronger emissions around 510 nm. The Type I emission spectrum of collagen fibres was converted to a spectrum similar to the Type III spectrum by conditions that caused the fading of fluorescence (storage as dry or mounted sections and exposure of sections to UV light). It is suggested that, with fibre-optic fluorimetry of intact tissues, Type I collagen fibres may emit a pre-fading spectrum while Type III collagen fibres may emit a post-fading spectrum, and that the preservation of Type I and the fading of Type III collagen is a consequence of the surface to volume ratio of their fibres.     

In vitro cleavage of eIF4GI but not eIF4GII by HIV-1 protease and its effects on translation in the rabbit reticulocyte lysate system

The vertebrate muscle Z-band organizes and tethers antiparallel actin filaments in adjacent sarcomeres and hence propagates the tension generated by the actomyosin interaction during muscular contraction. The axial width of the Z-band varies with fibre and muscle type: fast twitch muscles have narrow (approximately 30-50 nm) Z-bands, while slow-twitch and cardiac muscles have wide (approximately 100-140 nm) Z-bands. In electron micrographs of longitudinal sections of fast fibres like those found in fish body white muscle, the Z-band appears as a characteristic zigzag layer of density connecting the mutually offset actin filament arrays in adjacent sarcomeres. Wide Z-bands in slow fibres such as the one studied here (bovine neck muscle) show a stack of three or four zigzag layers. The variable Z-band width incorporating variable numbers of zigzag layers presumably relates to the different mechanical properties of the respective muscles. Three-dimensional reconstructions of Z-bands reveal that individual zigzag layers are often composed of more than one set of protein bridges, called Z-links, probably alpha-actinin, between oppositely oriented actin filaments. Fast muscle Z-bands comprise two or three layers of Z-links. Here we have applied Fourier reconstruction methods to obtain clear three-dimensional density maps of the Z-bands in beef muscle. The bovine slow muscle investigated here reveals a Z-band comprising six sets of Z-links, which, due to their shape and the way their projected densities overlap, appear in longitudinal sections as either three or four zigzag layers, depending on the lattice view. There has been great interest recently in the suggestion that Z-band variability with fibre type may be due to differences in the repetitive region (tandem Z-repeats) in the Z-band part of titin (also called connectin). We discuss this in the context of our results and present a systematic classification of Z-band types according to the numbers of Z-links and titin Z-repeats.     

Muscle Z-band ultrastructure: titin Z-repeats and Z-band periodicities do not match

Vertebrate muscle Z-bands show zig-zag densities due to different sets of alpha-actinin cross-links between anti-parallel actin molecules. Their axial extent varies with muscle and fibre type: approximately 50 nm in fast and approximately 100 nm in cardiac and slow muscles, corresponding to the number of alpha-actinin cross-links present. Fish white (fast) muscle Z-bands have two sets of alpha-actinin links, mammalian slow muscle Z-bands have six. The modular structure of the approximately 3 MDa protein titin that spans from M-band to Z-band correlates with the axial structure of the sarcomere; it may form the template for myofibril assembly. The Z-band-located amino-terminal 80 kDa of titin includes 45 residue repeating modules (Z-repeats) that are expressed differentially; heart, slow and fast muscles have seven, four to six and two to four Z-repeats, respectively. Gautel et al. proposed a Z-band model in which each Z-repeat links to one level of alpha-actinin cross-links, requiring that the axial extent of a Z-repeat is the same as the axial separation of alpha-actinin layers, of which there are two in every actin crossover repeat. The span of a Z-repeat in vitro is estimated by Atkinson et al. to be 12 nm or less; much less than half the normal vertebrate muscle actin crossover length of 36 nm. Different actin-binding proteins can change this length; it is reduced markedly by cofilin binding, or can increase to 38.5 nm in the abnormally large nemaline myopathy Z-band. Here, we tested whether in normal vertebrate Z-bands there is a marked reduction in crossover repeat so that it matches twice the apparent Z-repeat length of 12 nm. We found that the measured periodicities in wide Z-bands in slow and cardiac muscles are all very similar, about 39 nm, just like the nemaline myopathy Z-bands. Hence, the 39 nm periodicity is an important conserved feature of Z-bands and either cannot be explained by titin Z-repeats as previously suggested or may correlate with two Z-repeats.     

Ultrastructure of the pericardium in chitons (Mollusca: Polyplacophora), in relation to filtration and contraction mechanisms

Summary The pericardium in Lepidopleurus asellus (Spengler), Tonicella marmorea (Fabricius), T. rubra L., Ischnochiton albus L., and Calleochiton laevis (Montagu), species taxonomically far apart, is described. It consists of a flat, simple epithelium facing the pericardial cavity, a basement membrane, a muscle layer with two types of muscle fibres, nerve processes, glio-interstitial cells, and fibrocytes, embedded in a loose collagen matrix. The epithelium in L. asellus and I. albus have convoluted lateral cell borders, and in L. asellus very long basal cell processes are seen. Type 1 muscle fibres resemble smooth molluscan muscle. Type 2 muscle fibres resemble cardiac muscle fibres in chitons. Nerve processes associated with glio-interstitial cells and cell processes, run free in the matrix. Synapses in type 1 fibres are covered with glio-interstitial cell processes, lacking in type 2 muscle fibres synapses.This work was supported by grants from the Norwegian Research Council for Science and the Humanities     

Immunoreactivity of Glutamic Acid Decarboxylase (GAD) Isoforms in the Central Nervous System of the Barn Spider,Araneus cavaticus

The γ‐aminobutyric acid (GABA) has long been considered as an inhibitory neurotransmitter in the central nervous system (CNS) of both vertebrates and arthropods. Since the glutamic acid decarboxylase (GAD) has a restricted tissue distribution and catalyzes the conversion of L‐glutamate to GABA, immunoreactivity of GAD isoforms can reveal distribution of GABAergic neurons in the CNS. In the CNS of the spider Araneus cavaticus, immunoreactivity of GAD isoforms can be detected in the optic lobes including neurons and neuropiles of the supraesophageal ganglia. Strong GAD‐like immunoreactive cell bodies are concentrated in two bilaterally symmetric cell clusters of the protocerebrum. Some intrinsic cell bodies near the central body also show strong immunoreactivity. However, the intrinsic nerve masses and some of the longitudinal and transverse tracts within the supraesophageal ganglion are only lightly labelled, and the fibers transverse the hemisphere and the central fibrous masses are not labelled. Among the three basic types of cell bodies surrounding the central body, several clusters of the Type‐C cells show strong GAD‐like immunoreactivity, however both of the Type‐A and Type‐B cells are not labelled at all.