Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

physiological property of the EOM that would allow continuous adaptation of single fiber functional properties in response to physiological needs. The existence of ongoing remodeling in normal adult EOM suggests that there may be ways to modulate this process in vivo to alter muscle size, force, or response to injury or disease. In particular, it suggests new hypotheses to explain the preferential sparing or involvement of the EOM in skeletal muscle disease.

Acknowledgments

This work was supported by EY15313 and EY11375 from the National Eye Institute, the Minnesota Medical Foundation, the Minnesota Lions and Lionesses, Research to Prevent Blindness (RPB) Lew Wasserman Mid-Career Development Award (LKM), and an unrestricted grant to the Department of Ophthalmology from RPB.

See also: Extraocular Muscles: Extraocular Muscle Metabolism; Extraocular Muscles: Functional Assessment in the Clinic; Eyelid Anatomy and the Pathophysiology of Blinking.

Further Reading

Asmussen, G., Punkt, K., Bartsch, B., and Soukup, T. (2008). Specific metabolic properties of rat oculorotatory extraocular muscles can be linked to their low force requirements. Investigative Ophthalmology and Visual Sciences 49: 4865–4871.

Caiozzo, V. J., Baker, M. J., Huang, K., et al. (2003). Single-fiber myosin heavy chain polymorphism: How many patterns and what proportions? American Journal of Physiology – Regulatory, Integrative and Comparative Physiology 285: R570–R580.

Harrison, A. R., Anderson, B. C., Thompson, L. V., and McLoon, L. K. (2007). Myofiber length and three-dimensional localization of

NMJs in normal and botulinum toxin-treated adult extraocular muscles. Investigative Ophthalmology and Visual Sciences 48: 3594–3601.

Jacoby, J., Chiarandini, D. J., and Stefani, E. (1989). Electrical properties of multiply innervated fibers in the orbital layer of rat extraocular muscles. Journal of Neurophysiology 61: 116–125.

Kallestad, K. M. and McLoon, L. K. (2008). Myogenic precursor cells in the extraocular muscles. In Low, W. C. and Verfaillie, C. M. (eds.)

Stem Cells and Regenerative Medicine. Hackensack, NJ: World Scientific.

Kaminski, H. J., Kusner, L. L., and Block, C. H. (1996). Expression of acetylcholine receptor isoforms at extraocular muscle endplates.

Investigative Ophthalmology and Visual Sciences 37: 345–351. Kjellgren, D., Thornell, L. E., Andersen, J., and Pedrosa-Domellof, F.

(2003). Myosin heavy chain isoforms in human extraocular muscles.

Investigative Ophthalmology and Visual Sciences 44: 1419–1425. Li, Z. B., Rossmanith, G. H., and Hoh, J. F. Y. (2000). Cross-bridge

kinetics of rabbit single extraocular and limb muscle fibers.

Investigative Ophthalmology and Visual Sciences 41: 3770–3774. Mayr, R. (1971). Structure and distribution of fiber types in the external

eye muscles of the rat. Tissue and Cell 3: 433–462.

McLoon, L. K., Rowe, J., Wirtschafter, J. D., and McCormick, K. M. (2004). Continuous myofiber remodeling in uninjured extraocular myofibers: Myonuclear turnover and evidence for apoptosis.

Muscle and Nerve 29: 707–715.

Shall, M. S., Dimitrova, D. M., and Goldberg, S. J. (2003). Extraocular motor unit and whole-muscle contractile properties in the squirrel monkey. Summation of forces and fiber morphology.

Experimental Brain Research 151: 338–345.

Stephenson, G. M. M. (2001). Hybrid skeletal muscle fibers: A rare or common phenomenon? Clinical and Experimental Pharmacology and Physiology 28: 692–702.

Glossary

Adenine nucleotide translocator – An inner mitochondrial protein that exchanges adenosine diphosphate (ADP) and adenosine triphosphate (ATP) between the mitochondrial matrix and the cytoplasm. It is also known as the ADP/ATP translocator.

Chronic progressive external ophthalmoplegia –

A syndrome characterized by progressive inability or difficulty to move the eyes and elevate the eyelids. It is a common manifestation of some mitochondrial diseases.

Electron transport chain – The set of mitochondrial protein complexes that couples the oxidation of electron donors (such as NADH) to the reduction of electron acceptors (such as oxygen) in order to produce ATP.

Gene expression profiling – The measurement of the activity (or expression) of large numbers of genes simultaneously.

Glycolysis – The metabolic pathway that converts glucose to pyruvate, with a net result of 2 ATP and 2 NADH.

M line – A dark band or line seen in the center of the sarcomeres, using electron microscopy.

NADH – Nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH are coenzymes involved in oxidation/reduction reactions as electron acceptors and donors.

Oxidative phosphorylation – The metabolic pathway that uses the energy released during the oxidation of nutrients to produce ATP.

The extraocular muscles exhibit the greatest diversity among mammalian skeletal muscles, a likely consequence of the varied functional requirements imposed by the ocular motor system. Extraocular muscle fibers differ from typical limb and respiratory skeletal muscles in mitochondrial content, innervation/contractile patterns, contractile protein isoforms, hormone receptors, cell surface markers, and a variety of other cell and molecular properties that may relate to their unique functions. The divergence from the skeletal muscle stereotype is further exemplified by the fact that extraocular muscles do not conform to traditional fiber type classifications, which are based primarily on myosin isoform expression. The most

accepted fiber-type classification scheme for extraocular muscles includes six fiber types based upon: (1) distribution into orbital and global layers, (2) innervation status, single versus multiple nerve contacts per fiber, and (3) mitochondrial/oxidative enzyme content. Figure 1 shows representative micrographs of extensor digitorum longus (EDL, which is a predominantly type IIB fiber – fast, fatigable – limb skeletal muscle), diaphragm (mixed fiber type, fatigue-resistant respiratory muscle), and the extraocular muscle from rats. EDL is recruited sporadically and diaphragm is constantly active as is the extraocular muscle. Despite the wide difference in activity, EDL and diaphragm sections are mostly indistinguishable. For sure, there are important biochemical differences between the two muscles that reflect their specific adaptations to their respective activation patterns. However, the divergent requirements of EDL and diaphragm motor systems are met with fairly stereotypical muscle fibers, as evident from the micrographs. In contrast, the extraocular muscle fibers are very different: small round fibers with prominent mitochondria, suggestive of atypical contractile and metabolic properties. This article outlines newly identified unique aspects of extraocular muscle metabolism and how they may correspond to contractile function.

Insights from Gene Expression Profiling

The fast and constant contractions of the extraocular muscles necessitate well-developed energy supply systems. It might be expected a priori that these muscles would upregulate all the main energy-supply metabolic pathways, from glycolysis to mitochondrial metabolism. Surprisingly, this may only apply to mitochondrial content, which is the highest reported in mammalian skeletal muscles. Studies comparing the gene expression profile of extraocular and limb muscles found that genes coding for key enzymes of glycogen synthesis and breakdown were repressed in the extraocular muscles. Glycogen content in the extraocular muscles is correspondingly reduced. These findings indicate that the extraocular muscles are seemingly less dependent on stored glycogen as a metabolic fuel than other skeletal muscles. They also suggest that the extraocular muscles rely, instead, on constant transport of blood-borne glucose and fatty acids through their extensive microvascular network. Interestingly, the expression of the lactate dehydrogenase (LDH) isoform that preferentially oxidizes lactate to pyruvate is increased

in the extraocular muscles, compared to typical skeletal muscles, and that may allow them to use lactate as a fuel for aerobic pathways. The importance of these alternative metabolic pathways is now being tested in extraocular muscles.

Lactate: An Oxidizable Substrate for the Extraocular Muscle

In limb skeletal muscles, glycogen breakdown drives glycolysis only during brief bursts of intense activity (Figure 2). In most muscles, metabolic demand during moderate activity is met by aerobic (mitochondrial) pathways. During periods of sustained peak activity, when mitochondrial capacity is exceeded in skeletal muscle, lactate is the end product of glycolysis in a reaction that reduces pyruvate at the expense of NADH and is catalyzed by LDH. For this and other reasons, increased production and accumulation of lactic acid during exercise has been associated with muscle fatigue. However, cells can also use the LDH reaction in the reverse direction from lactate oxidation to pyruvate, and lactate then goes on to become a substrate for aerobic metabolism. As mentioned above, the expression of the LDH isoform that preferentially oxidizes lactate to pyruvate is higher in the extraocular muscles. Combined with their high aerobic capacity, this reaction would allow the extraocular muscles to use lactate as a metabolic substrate. Cinnamate, a blocker of lactate transport, alone or in combination with exogenous lactate can be used to evaluate the role of lactate on fatigue resistance. Cinnamate accelerates fatigue in the extraocular muscles significantly: treated muscles lose their ability to generate force at a faster rate than untreated extraocular muscles. Conversely, cinnamate treatment does not affect the endurance or residual force of limb muscles. Replacing glucose with exogenous

lactate increases limb-muscle fatigability but has no effect on the extraocular muscles. However, the extraocular muscles fatigue faster when exposed to exogenous lactate combined with cinnamate treatment. These results indicate that LDH oxidation of lactate to pyruvate seems to be an important source of metabolic substrate for aerobic metabolism in the extraocular muscles. This conclusion is a significant deviation from the traditional view of lactate as a final waste product of glycolysis; increased lactate production and accumulation during vigorous contractile activity is typically associated with fatigue. Muscle fatigue is a complex phenomenon: substrate depletion, metabolite accumulation, and ionic imbalances are some of the factors that combine to reversibly impair contractile function. In the particular case of the extraocular muscles, lactate can be used via the LDH reaction as an additional substrate source for aerobic metabolism, a concept developed recently for other aerobic muscles and one that also applies to the nervous system.

Creatine Kinase, the Missing ATP Buffer

in the Extraocular Muscle

Skeletal muscles and other tissues with fluctuating metabolic needs rely on the creatine–phosphocreatine system to buffer intracellular ATP concentration: creatine kinase (CK) catalyzes the reversible transfer of the phosphoryl group from phosphocreatine to ADP in order to maintain constant ATP levels. Cellular CK activity is due to a family of oligomeric enzymes: two cytosolic, ubiquitous brain-type CK-B and muscle-type CK-M, and two mitochondrial isoforms, ubiquitous mitochondrial CK (uCK) and sarcomeric mitochondrial CK (sCK). In differentiated skeletal muscle, CK-MM and sCK are the predominant isoforms. In fast-twitch muscles, most CK activity is due to the CK-MM isoform, some of which is found

associated with the sarcomeric M line, the sarcoplasmic reticulum, and T-tubules. This arrangement couples the CK-dependent ATP-buffering system to the cellular sites with the highest ATPase activity, and is needed for normal contractile function. Given the predicted need for ATP buffering in the extraocular muscles, we proposed that (1) CK isoform expression and activity in rat extraocular muscles would be higher and (2) the resistance of these muscles to fatigue would depend on CK activity. Instead, we found that messenger ribonucleic acid (mRNA) and protein levels for all (cytosolic and mitochondrial) CK isoforms are lower in the extraocular muscles than in limb muscles. The muscle-enriched isoforms, CK-M and sCK, are less abundant in extraocular muscle, despite the fact that the extraocular muscles have a higher mitochondrial content than limb muscles. Total CK activity is also correspondingly decreased in the extraocular muscles. Moreover, cytoskeletal components of the sarcomeric M line, where a significant fraction of cytosolic CK activity is found, are downregulated in the extraocular muscles as was initially suggested by gene expression profiling. To explore the role of CK activity on muscle function, the CK inhibitor 2,4-dinitro-1-fluorobenzene (DNFB) was used during an in vitro fatigue protocol. Treatment with DFNB accelerates the development of fatigue in limb muscle, but has no detectable effect on the

extraocular muscles. These data support the conclusion that CK activity is not an important ATP buffer in the extraocular muscles. The myokinase reaction (2 ADP ! ATP þ AMP), catalyzed by adenylate kinase (AK), serves as an additional ATP-buffering system in skeletal muscle. While total AK activity is similar in extraocular and limb muscles, the mRNA content for two putative mitochondrial AK isoforms (AK3 and AK4) is over 13-fold more abundant in the extraocular muscles. This suggests that the relative lack of CK in the extraocular muscles may be compensated by upregulation of selected AK isoforms.

Mitochondrial Content in the Extraocular

Muscles

Aerobic capacity is typically measured by mitochondrial volume density (percentage of muscle fiber volume occupied by mitochondria). In general, mitochondrial volume density is well matched to the metabolic needs of skeletal muscle and it scales almost linearly with maximal oxygen uptake among muscles and across mammalian species. In other words, the consensus is that changes in the oxidative (aerobic) capacity of mammalian skeletal muscles are met by corresponding increases or decreases in mitochondrial volume density. Since the mitochondrial content and the

activity of respiratory complexes and enzymes of mitochondrial metabolic pathways change in parallel, enzymatic activities are used as indices of mitochondrial content and aerobic capacity. Highly aerobic muscle groups in mammals have abundant capillaries and elevated mitochondrial volume density. The extraocular muscles have, arguably, the highest mitochondrial content of all mammalian skeletal muscles. However, the mechanism responsible for maintaining mitochondrial abundance in the extraocular muscles remains unclear. We have identified a number of transcription factors that influence mitochondrial biogenesis and that are upregulated in the extraocular muscles. Surprisingly, these factors are different from the mitochondrial biogenesis program initiated in response to endurance training.

Mitochondria as Calcium Sinks in the

Extraocular Muscle

The fast-contracting extraocular muscles rely on tight regulation of free cytosolic calcium concentration ([Ca2+]i). In principle, the extraocular muscles have the profile of very efficient calcium handling capacity: extensive and welldeveloped sarcoplasmic reticulum and the expression of fast calcium ATPase isoforms. Moreover, the extraocular muscles contain parvalbumin, a low-weight calcium-bind- ing protein that serves as a temporary buffer to accelerate the removal of calcium off its binding sites on the myofilaments and facilitates muscle relaxation. Other investigators have already shown that the kinetics of calcium flux into mitochondria are fast enough to influence very rapid events such as neurotransmitter release from motor nerve terminals. The specific inhibition of mitochondrial calcium transport slows the relaxation of mitochondria-rich skeletal muscles. We recently reported that the magnitude and speed of calcium uptake by mitochondria are sufficient to influence contractile function. This property of the extraocular muscles appears to serve at least two complementary functions. First, it couples metabolic supply to demand because higher mitochondrial calcium stimulates enzymes that control substrate oxidation: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, isocitrate dehydrogenase, and glycerol 3-phosphate dehydrogenase. The combined activity of these enzymes sustains a high NADH/NAD+ level and maximizes oxidative phosphorylation and ATP production. Second, by limiting the [Ca2+]i increase during contractions in response to submaximal stimulation frequencies, mitochondria widen the dynamic range of the extraocular muscles. In other words, the capacity of the extraocular muscles to produce force is spread over a wider stimulation frequency range, increasing the fine control of the effector arm of the ocular motor system.

Are Extraocular Muscle Mitochondria

Different?

The adenine nucleotide transporter 1 (Ant1) gene encodes an inner mitochondrial membrane protein that transports ADP into mitochondria and ATP from mitochondria to the cytosol. Mutations within Ant1 have been shown to produce a syndrome of chronic progressive external ophthalmoplegia (CPEO) in humans. Ant1 knockout (Ant1–/–) mice develop cardiomyopathy and severe exercise intolerance. Despite this dramatic phenotype, the extraocular muscles are mostly unaffected. Histologically, the extraocular muscles from Ant1–/– mice present a relatively mild mitochondrial myopathy. There are no measurable ocular motor abnormalities in Ant1–/– mice, and their peak eye velocities overlap with those measured in control mice. Moreover, their extraocular muscles do not show evidence of increased fatigability. In addition, the extraocular muscles have higher levels of Ant2 mRNA compared to the limb muscles. Ant2 is a nonskeletal muscle isoform previously described in the heart. Its presence in the extraocular muscles may explain the lack of effects of Ant1 loss, and it was the first documented difference between extraocular muscle and limb muscle mitochondria.

The ability of muscles to perform aerobic work depends on their mitochondrial volume density, with the assumption that the composition of these organelles is fairly constant across muscle types and mammalian species. One of these components is the electron transport chain, a series of multimeric complexes (complexes I–IV, plus the ATP synthase which is sometimes called complex V) in the inner mitochondrial membrane responsible for most of the aerobic ATP generation (Figure 3). Recently, we found that the extraocular muscle mitochondria have lower content or lower activity of some enzyme complexes of the electron transport system, causing them to respire at slower rates. This is puzzling given that the extraocular muscles are constantly active and aerobic capacity was predicted to be elevated, given their high mitochondrial content. These findings are not explained by differences in the ultrastructure of extraocular muscle mitochondria: the surface area of their inner membrane is comparable to values reported for other skeletal muscle. Furthermore, the differences are not generalized or systematic: complex II content and activity, and complex III content are similar in mitochondria from triceps surae (a limb skeletal muscle) and extraocular muscle. Complexes I and IV give a more puzzling result: their activities are lower, but their content is higher in the extraocular muscle mitochondria. These are multimeric protein complexes, and differential expression of isoforms of some subunits has been described in skeletal muscle and other tissues.

Therefore, the content of some electron transport chain complexes (I, IV, and V) and the subunit composition of some others (I and IV) may not be the same in the extraocular muscles compared to limb muscles. This demonstrates that the metabolic divergence between extraocular and limb muscles includes major differences in the composition and basic function of their respective mitochondrial populations. Intrinsic differences in mitochondrial structure and function may explain the susceptibility of the extraocular muscles to some hereditary and acquired mitochondrial myopathies such as CPEO and related syndromes. For example, the extraocular muscles present the most severe age-dependent loss of mitochondrial respiratory complex activity among muscles. There is a significant increase in the number of fibers with cytochrome c oxidase defects in the extraocular muscles of humans and other primates, even when compared to other highly aerobic muscles such as the diaphragm and heart. This can be at least partially explained by mitochondrial DNA mutations, presumably due to reactive oxygen species generated during mitochondrial respiration or present as part of a more generalized cellular oxidative stress.

Matching Mitochondrial Capacity to Contractile Function

The primary role of mitochondria is to generate ATP. Recent studies lead to an obvious question: How do extraocular muscles sustain their contractile function with mitochondria that respire half as fast as mitochondria from other muscles? The content of respiratory complexes is one parameter behind tissue variations in mitochondrial respiration, although some argue that it is not particularly relevant for metabolic control. Under experimental conditions, mitochondrial respiration in the skeletal muscle and heart is regulated at the level of the respiratory chain, while in the liver, kidney, and brain it is controlled mainly at the phosphorylation level by ATP synthase (complex V) and phosphate carrier. That may not be the case in vivo, where different parameters such as cellular steady state, the energy demand, and the energy supply of the tissue may also regulate mitochondrial respiration. In the case of the extraocular muscles, allosteric regulation of respiratory complexes may combine with changing metabolite concentrations to maintain mitochondrial respiration closer to its theoretical maximum.

For example, a mechanism to enhance energy production in the extraocular muscle is mitochondrial calcium influx during contractile activity in order to activate enzyme systems that exert strong control on substrate oxidation, as mentioned above.

Matching Energy Supply to Demand

Initially inspired by morphological characteristics and gene-expression-profiling results, a more global perspective of extraocular muscle metabolism is beginning to emerge. First, glycogen content is low and the glycogenolysis pathway seems to be downregulated in the extraocular muscles. Second, CK activity and content, including the mitochondrial isoform, are lower in the extraocular muscles, indicating that phosphocreatine may be a less important temporal and spatial ATP buffer in these muscles. In other words, mitochondrial ATP production may be sufficiently high and close to cellular sinks as to obviate the need for an energy buffer. Third, the extraocular muscles can use lactate as an oxidizable substrate due to the presence of a LDH isoform that catalyzes the conversion of lactate to pyruvate that then goes to the Krebs cycle. Finally, the mitochondrial population in extraocular muscles appears to respond to a different biogenesis program, and exhibits atypical functional characteristics that may influence the contractile activity of these muscles significantly.

Acknowledgements

The author’s work in this field is supported by the National Eye Institute (grant R01 EY012998).

See also: Extraocular Muscles: Extraocular Muscle Anatomy.

Further Reading

Andrade, F. H. and McMullen, C. A. (2006). Lactate is a metabolic substrate that sustains extraocular muscle function. Pflu¨gers Archiv-European Journal of Physiology 452: 102–108.

Andrade, F. H., McMullen, C. A., and Rumbaut, R. E. (2005). Mitochondria are fast Ca2+ sinks in rat extraocular muscle: A novel regulatory influence on contractile function and

metabolism. Investigative Ophthalmology and Visual Science 46: 4541–4547.

McMullen, C. A., Hayeß, K., and Andrade, F. H. (2005). Fatigue resistance of rat extraocular muscles does not depend on creatine kinase activity. BMC Physiology 5: 12.

Porter, J. D., Khanna, S., Kaminski, H. J., et al. (2001). Extraocular muscle is defined by a fundamentally distinct gene expression profile.

Proceedings of the National Academy of Sciences of the Unites States of America 98: 12062–12067.

Spencer, R. F. and Porter, J. D. (2006). Biological organization of the extraocular muscles. Progress in Brain Research 151: 43–80.

Yin, H., Stahl, J. S., Andrade, F. H., et al. (2005). Eliminating the Ant1 isoform produces a mouse with CPEO pathology but normal ocular motility. Investigative Ophthalmology and Visual Science 46: 4555–4562.

Glossary

Choline acetyltransferase – The enzyme responsible for the synthesis of the neurotransmitter acetylcholine, causing the transfer of acetate to choline.

Choline transporter – These recapture choline from the synaptic cleft after acetylcholine release and degradation. This process is critical for new acetylcholine synthesis at the synapse.

Golgi tendon organ – A proprioceptive organ that provides information to the brain about changes in muscle tension. In contrast to muscle spindles, these are in series with muscle fibers, interwoven with the collagen in the muscle tendon. Tension on the tendon caused by muscle contraction activates these proprioceptors.

Muscle spindles – Proprioceptive organs that provide the brain with information about changes in muscle length and are organized in parallel with the skeletal muscle fibers. They contain modified muscle fibers called intrafusal fibers, in contrast with the muscle fibers themselves, which are called extrafusal fibers. They have a complex structure; they are surrounded by a connective tissue capsule, and contain several types of modified myofibers within them. They are innervated by sensory afferents.

Myotendinous cylinders or palisade endings –

Proprioceptive organs found at the myotendinous junction consisting of dense axonal branching which invests the tips of single muscle fibers. These are unique to the extraocular muscles. The nerves establish synaptic contacts with both collagen fibrils and muscles fibers at the myotendinous junction. The function of these structures is unknown.

Proprioception – The sense that provides information about the location of various parts of the body in relation to each other and in relation to the space.

Proprioceptors – Sensory receptors which are found in muscles and tendons that bring sense of body position to the brain.

Vesicular acetylcholine transporter –

A membrane protein which is necessary for the uptake of acetylcholine into synaptic vesicles.

Proprioception

Proprioception refers to a sense that provides information about the location of various parts of the body in relation to each other and in relation to space. It is of practical importance for activities in everyday life and allows a person to use the foot pedal of a car properly while driving or to learn to walk in darkness. Moreover, sportsmen use specific training devices to sharpen their proprioceptive sense. Proprioceptive signals come from specialized sensory nerve endings called proprioceptors that occur throughout skeletal muscle. Typical proprioceptors in skeletal muscle are muscle spindles and Golgi tendon organs which constantly transmit information to the brain. In this way the brain knows, at any given time, the spatial position of our body parts.

The eyes are the most mobile organs of the body, and vision is useful only if the brain knows the position of the eyes in the orbit. By knowing where the eyes are pointing, the brain is aware of the position of objects in the surrounding space: if objects are leftwards, straight ahead, or rightwards. Several studies indicate that the brain has access to proprioceptive information from the extraocular muscles (EOMs). Specifically, neuronal tracing experiments have demonstrated projections from the EOM to various peripheral and central nervous system structures, including the trigeminal ganglion, the mesencephalic trigeminal nucleus, the superior colliculus, the vestibular nuclei, and the cerebellum. A recent physiology experiment showed that the primary somatosensory cortex, which receives proprioceptive input from all other skeletal muscles, also receives signals from EOM. This new finding completes the somatotopic representation of the body in the primary somatosensory cortex which, thus far, had lacked a map of the eye muscles.

Indication that there is proprioceptive input from the EOM has also come from psychophysical investigations. Patients suffering from strabismus were tested after surgery, and it was detected that they had deficits in spatial perception. These results were interpreted to mean that the surgical intervention has damaged the proprioceptors at the myotendinous junction resulting in a loss of eye position signals.

Despite this evidence for EOM proprioception, there are also counterarguments. Specifically, no stretch reflex has been observed in the EOM of monkey. By cutting the ophthalmic nerve, which is supposed to carry the afferent fibers from EOM, deficits in eye movements would be

expected. However, findings indicate that deafferentation does not affect ocular alignment or eye movements, including saccades and smooth pursuit. Such observations led scientists to doubt whether EOM proprioception really exists. Instead, it has been hypothesized that the motor command that is sent to the EOMs is copied, called efference copy, and this copy provides the necessary information for the brain to be aware of the eye’s position.

If there is sensory feedback from EOM, the eye muscles should have proprioceptors. In the last century, the EOMs of several mammalian species and man have been screened for muscle spindles and Golgi tendon organs. Interestingly, the endowment with classical proprioceptors varies widely among the species, and there are even some species that do not have proprioceptors at all. In view of these interspecies variations, it is not clear where the source of EOM proprioception lies. By searching for alternative sensory organs in the EOMs, palisade endings (also called myotendinous cylinders) have been detected, and so far, palisade endings have been observed in each species investigated. In the following section, we discuss EOM proprioceptors, including muscle spindles, Golgi tendon organs, and palisade endings. We give an overview about the occurrence, distribution, number, and structure of these organs and speculate about their putative function. Recent studies have focused on the molecular characteristics of palisade endings, and we also refer to these findings.

Muscle Spindles

Occurrence, Distribution, and Number of

Muscle Spindles

Muscle spindles are regularly observed in the EOMs of even-toed ungulates (sheep, cow, camel, goat, and pig) and in the EOMs of primates (monkey and man). In other animal species, including fellidae (cat), rodents (rat and guinea pig), odd-toed ungulates (horse), and lagomorphs (rabbit), muscle spindles have not been found (Table 1).

In the EOMs of even-toed ungulates, muscle spindles are uniformly distributed throughout the entire muscle length. The number of muscle spindles is remarkably high, and counts per muscle yield between 146 and 333 muscle spindles in pig; between 100 and 181 muscle spindles in camel; and more than 200 muscle spindles in cow. In man the distribution of muscle spindles exhibits differences when compared with that in even-toed ungulates. Specifically, muscle spindles are located predominantly in the proximal and distal parts of the EOM, and each muscle has a spindle-free zone approximately in the middle. The number of human EOM spindles varies between 13 and 42. Only in the inferior oblique muscle has a lower number of muscle spindles been counted (3–7). The density of human EOM spindles is comparable

Table 1 Occurrence of muscle spindles, Golgi tendon organs, and palisade endings in the extraocular muscles of man and mammals

aSo far, palisade endings have only been demonstrated in sheep. bSo far not analyzed for palisade endings.

to that of muscle spindles in finely controlled skeletal muscle such as the hand lumbrical and deep dorsal neck muscles. In monkey (rhesus monkey and cynomolgus monkey), very few muscle spindles (2–6) have been observed in some EOMs, and none in the others.

Structure of Muscle Spindles

Muscle spindles in the EOMs of even-toed ungulates conform in their structure with those in other skeletal muscles. The muscle spindles have a fusiform shape with a wide central region (equatorial region) and two narrow polar regions. Muscle spindles are ensheathed by a capsule consisting of several layers of perineural cells. The capsule space is filled with a viscous fluid containing acidic mucopolysaccharides. Inside the capsule two types of intrafusal muscle fibers (nuclear chain fibers and nuclear bag fibers) can be distinguished, which both exhibit modifications concerning their myonuclei in the spindle’s equatorial region. Nuclear chain fibers have a single row of centrally arranged nuclei, whereas nuclear bag fibers show an accumulation of nuclei. In the muscle spindle’s equatorial region, a large tissue-free space (periaxial space) separates the intrafusal muscle fibers from the capsule.

Muscle spindles in the EOMs of even-toed ungulates receive a double innervation from sensory and motor nerve fibers. In the equatorial region, both types of intrafusal muscle fibers are endowed with sensory nerve endings (annulospiral sensory endings) which are wrapped spirally around the muscle fibers. Whether a second type of sensory nerve ending (flower-spray ending) that is common in other mammalian skeletal muscle spindles is also present in ungulate EOM spindles is unclear. Fine structural analyses have shown that sensory nerve terminals contain mitochondria and a few clear vesicles. The synaptic cleft separating the nerve terminal from the muscle fiber surface is free from a basal lamina. At the muscle spindle’s pole, intrafusal muscle fibers receive motor terminals. Motor terminals contain mitochondria

and dense aggregations of clear vesicles, and the synaptic cleft is filled with a basal lamina.

Muscle spindles in EOM of primates exhibit structural differences when compared with those in even-toed ungulates. Specifically, in most muscle spindles of monkey and man the periaxial space exhibits little or no expansion. Only in human infants have some muscle spindles with a wide periaxial space been observed. Thorough analyses of the intrafusal fiber composition have been done in human EOM spindles. The findings indicate that human EOM spindles contain nuclear chain fibers but most of them lack nuclear bag fibers. Only 2% of the spindles contain nuclear bag fibers and, when present, the bag region is poorly developed with only two nuclei lying side by side. In addition to nuclear chain fibers, anomalous muscle fibers are also regularly observed in human EOM spindles. Anomalous muscle fibers exhibit no nuclear modification in the spindle’s equatorial region and are indistinguishable from muscle fibers outside the spindle. The unique morphology of human EOM spindles was initially described in aged persons (67–83 years old) and later was confirmed in infants (Figure 1(a)).

The innervation pattern of primate EOM spindles has only been analyzed in humans. In human EOM spindles, sensory nerve endings have been observed on nuclear chain and, when present, on nuclear bag fibers, but only 7% of the anomalous fibers are endowed with sensory nerve terminals. In their fine structure, sensory nerve terminals in human EOM spindles do not differ from sensory nerve terminals in EOM spindles of even-toed ungulates. At the muscle spindle’s pole, intrafusal muscle fibers are equipped with motor terminals. Motor terminals in human EOM spindles are identical in their structure with those in EOM spindles of even-toed ungulates (Figure 1(b)).

Function of Muscle Spindles

Muscle spindles in mammalian skeletal muscle are stretch receptors which register changes in muscle length. Indications that muscle spindles in the EOMs of even-toed ungulates are capable of monitoring muscle length have come from electrophysiological investigations. Specifically, in goat and sheep the EOMs were stretched and afferent signals were recorded in the sensory trigeminal ganglion. Recorded signals exhibited characteristics that are the same as muscle spindles in other skeletal muscles.

There is controversy whether muscle spindles in human EOMs are functional. Due to their unusual morphology, some authors suppose that human EOM muscle spindles are not functional. On the other hand, muscle spindles in human EOMs are numerous, and their nerve terminals exhibit a normal morphology. This is why other authors suggest that human EOM spindles are functional, and their unusual morphology might indicate special functional properties. In particular, as most human EOM muscle spindles lack nuclear bag fibers, muscle spindles might have a predominantly static function and monitor the degree of muscle stretch rather than the contraction velocity of muscle fibers.

Golgi Tendon Organs

Occurrence, Distribution, and Number of Golgi

Tendon Organs

Golgi tendon organs are exclusively found in the EOMs of even-toed ungulates (pig, sheep, camel, and cow). They have not been found in other mammals and man. In even-toed ungulates, Golgi tendon organs are distributed throughout the proximal and distal EOM tendons, their number always being higher in the distal tendons (Table 1). The number of Golgi tendon organs per muscle has been counted to be