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Reflexes as tools to study human neuromuscular system
Clinical Neurophysiology 121 (2010) 1599–1601
Contents lists available at ScienceDirect
Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Editorial
Reflexes as tools to study human neuromuscular system Knowledge of the synaptic connections between neurons is a key prerequisite to understanding of the operation of the nervous system. While the anatomy of these connections (‘wirings’) can be obtained by histochemical methods, their functional connections can only be determined using electrophysiological recordings. The functional connection of selected afferent inputs or corticospinal fibres to motoneurons can be studied directly in animal preparations. Compared with the studies on human subjects, experiments on animals have advantages as precise stimulation of selected nerve fibres/neurons and intracellular recordings from accurate sites are possible. Therefore, interpretations of the connections of the stimulated fibres and neurons are straight forward and easy in animal experiments. However, there are also limitations in studies on experimental animals. To prepare animals for experiments, they have to be reduced (anaesthetized, decerebrated, sliced, etc.). It is well recognized that such reductions have severe effects on synaptic potentials (Nicoll, 1972; Wali, 1985). Furthermore, one needs also to remember that active involvement of the supraspinal pathways to discharges of motoneurons is either completely suppressed or altered significantly in these reduced animal preparations. Therefore, although experiments on animals deliver the influence of a stimulated fibre or neuron to another fibre or neuron directly, one needs to accept the results of such experiments with the experimental settings in mind. In particular, describing findings on reduced animal preparations as ‘functional connections of neurons’ should be done with reservations. Although such direct experiments cannot be performed in humans, any experimental findings on conscious human subjects are likely to be more functional since they are not influenced by anesthetics or any other reduction processes and since the activity of supraspinal centres are maintained. To study functional connection of neurons in human subjects it has been customary to use stimulus-evoked changes in the discharge probability and rate of one or more motor units in response to stimulation of a set of peripheral afferents or corticospinal fibers. These are the most common ways to investigate the workings of peripheral and central pathways in human subjects. Although these are indirect methods of studying human nervous system, they are nevertheless extremely useful as there is no other method available yet to record synaptic properties directly in human subjects. The following 10 articles are based on invited lectures at the International Workshop and Conference on Human Reflexes: _ Wiring and Firing of Motoneurons (11–15 May 2009, Izmir, Turkey). The first five articles deal with the issue of estimating the properties of motoneurons using surface EMG and/or single motor unit activity. The last five articles use the surface EMG and single unit techniques to estimate neuronal connections both in healthy individuals and patients with spinal or cerebral injuries.
Although studies on surface EMG can provide much useful information regarding the excitability of the motoneurons especially when it is averaged around the timing of a stimulus, action potentials of individual motor units are more useful as they represent the activation of their motoneurons in a one-to-one basis. That is why researchers have been using intramuscular electrodes along with the surface electrodes to study the characteristics of motoneurons. Intramuscular electrodes however are painful to insert and unpleasant to keep and cannot be used in children and pain patients for ethical reasons. Therefore, there has been a growing interest in obtaining individual motor unit action potentials from the surface EMG records without in the same time inserting intramuscular electrodes. The first two articles deal with this very issue of decomposing the surface EMG into its constitutive motor unit action potentials. Nawab et al.’s article on this subject entitled: ‘‘High-yield decomposition of surface EMG signal” is an eye opener as it shows not only the difficulty of the subject in hand but also how this could be overcome with persistence. This article introduces the development of a five pin active electrode that can record single unit potentials with a certain level of confidence up to very high contraction levels. The article by Farina et al. entitled: ‘‘Decoding the neural drive to muscles from the surface electromyogram” also attempts to decompose surface EMG into its constitutive motor unit action potentials. Due to nonlinear representation of motor unit action potentials to the surface EMG and due to signal cancellation problems (Keenan et al., 2005; Tucker and Türker, 2005), these authors agree with the view that single motor unit discharge rate but not the whole muscle surface EMG represents the drive to a motoneuron pool (see also Fig. 6 in Türker, 1993). To study the discharge rate of single motor units and also without using invasive methods, these authors utilized a special grid electrode system where individual motor unit action potentials are recorded simultaneously in several surface electrodes located at different parts of the muscle of interest. Distinguishing action potentials of different motor units is facilitated by recording the surface EMG from several locations over the muscle using this ‘‘high density surface EMG” system which comprises several closely spaced surface electrodes. The authors have also successfully compared the timing of identified single unit action potentials as recorded on the surface electrode arrays with that of the ‘gold standard’ intramuscular electrodes. They agree however that the surface decomposition approach is limited to isometric contractions, localized portions of active units, and to low forces, in contrast to the more widespread applications of surface EMG recordings. Despite these limitations, surface EMG decomposition allows the accurate detection of relative changes in neural activation.
1388-2457/$36.00 Ó 2010 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2010.04.019
1600
Editorial / Clinical Neurophysiology 121 (2010) 1599–1601
Schmied and Descarreaux’s article entitled: ‘‘Influence of contraction strength on single motor unit synchronous activity” uses an intramuscular technique to obtain data from simultaneously active single motor units so that they can study the changes in the level of common input to pairs of motoneurons at various contraction levels. Using pairs of motor unit discharge data, they compare various synchronization indices to identify the index that is minimally affected by the discharge rate of motor units. They found that the index k’, that is the ratio of the total counts in the peak region of the cross-correlogram to the chance counts in that region (Ellaway and Murthy, 1985), is the index least affected by the contraction strength. In agreement with data obtained in rat motoneurons (Türker and Powers, 2002), the authors also showed that the index SIP (or E) (Datta and Stephens, 1990) was the index least affected by the motor unit firing rates. In their modeling work, Dick Stegeman and colleagues studied the question of translation of cortical oscillations into the EMG signal (‘‘The a-motoneuron pool as transmitter of rhythmicities in cortical motor drive”). They used a model to obtain insight into the corticomuscular signal transmission at various frequencies. Using coherence analysis between simulated motoneuron spike trains and surface EMG signals they have illustrated the importance of common input and found that the coherence between these variables augmented substantially with the increased level of common input. The strength of this coherence encouraged them to suggest that common inputs are needed to make corticomuscular coupling visible and that the coupling strength hardly depended on the frequency in a range from 1 to 100 Hz. They have concluded that the rhythmicities are translated into alpha motoneuron activity without strong non-linearities. The article by Powers and Türker entitled: ‘‘Deciphering the contribution of intrinsic and synaptic currents to the effects of transient synaptic inputs on human motor unit discharge” reviews the current methods of estimating synaptic potentials in human motoneurons. This article compares the methods of quantifying synaptic inputs to human motoneurons and comments on their accuracy based on recording synaptic potentials and their effects on motoneuron discharge in reduced animal preparations. Using the effects of synaptic noise, AHP summation, variations in spike threshold and subthreshold intrinsic currents on the responses of motoneurons, this review also discusses the contributions of the synaptic inputs and intrinsic motoneuron properties to altered motoneuron responses following CNS injury. The article by Maria Knikou entitled: ‘‘Neural control of locomotion and training-induced plasticity after spinal and cerebral lesions” reviews expression and regulation of spinal reflexes at rest and their modulations during locomotion. While summarizing extension and flexion reflexes, Maria reviews the mechanisms that underlie phase-dependent modulation of these reflexes in both healthy subjects and patients with spinal or cerebral lesions. She ties the recovery of walking in patients after locomotor training to the capabilities of these adjustable interneuronal and cortical circuits. She concludes her review with a suggestion that a multidisciplinary approach including rehabilitation and electrical stimulation is needed for maximizing sensorimotor recovery. ElBasiouny et al.’s article on: ‘‘Persistent inward currents in spinal motoneurons: important for normal function but potentially harmful after spinal cord injury and in amyotrophic lateral sclerosis” is an authoritative review on the findings of plateau potential in motoneurons. The authors note that the activation of motoneurons depends on interaction between synaptic inputs to motoneurons and their intrinsic properties. One of the intrinsic properties of motoneurons is the ability of the motoneuronal membrane to generate persistent inward currents (PICs). The authors review the
current findings to illustrate how these PICs can significantly change the motoneuron output to a given input and hence contribute substantially to the activation of motoneurons. In this review it is also indicated how these normally useful currents can turn out to be harmful after spinal cord injury and in ALS. The authors indicate that changes in PICs after injury or disease lead to exaggerated long-lasting reflexes and muscle spasms that contribute eventual neuronal degeneration. Shemmell et al.’s article on ‘‘Stretch sensitive reflexes as an adaptive mechanism for maintaining limb stability” examines the short and long latency stretch reflex in human subjects. They note that short latency stretch reflex’s role in regulation of stiffness in humans is well described and that this reflex generally strengthens the capabilities of a muscle to oppose external perturbations thereby increasing stability of the musculoskeletal system. The role of longer latency stretch reflexes is however less clear. According to these authors, this reflex increases its sensitivity during interactions with compliant environment relative to the sensitivity during interactions with rigid environment. This differential adaptation suggests that the longer latency stretch reflex sensitivity increases to boost limb stability when that steadiness is not provided by the environment. Deriu et al.’s article on ‘‘Reflex responses of masseter muscles to sound” reviews reflexes in the masseter muscle EMG in response to acoustic stimuli. Although previous studies have shown that these reflexes originated from the activation of the cochlear receptors, this review indicates that the vestibular receptors may also take part especially when the sound is in high intensity. This is not surprising since the masseter muscles have strong anatomical and functional connections with the vestibular receptors and since stimulation of the vestibular receptors induces clear short-latency reflex responses on the masseter EMG. These vestibule-masseter reflexes are made up of two short latency inhibitory responses and appear in the unrectified masseteric EMG on both sides. Similar to the reflex responses of the leg and arm muscle EMGs to electrical vestibular stimulation, the authors believe that the first reflex response may have minor importance to postural control but its major role may lie in the fine-tuning voluntary motor output by allowing rapid access of vestibular inputs to jaw muscle motoneurons. The second and more powerful reflex response may be of importance in postural control of masseters by stabilizing the jaw during head movements in space. The final article by Yavuz and colleagues entitled ‘‘Effect of gender, age, fatigue and contraction level on electromechanical delay” deals with the factors that affect the time lag between the onset of the direct EMG response and the onset of force generation. This time lag is referred to as electromechanical delay (EMD) and is studied to find out whether it is one of the responsible factors for age and gender related sports injuries. Working on 15 male and 15 female volunteers at various ages, this study used supramaximal nerve stimulation to elicit action potentials in all muscle fibres so that a standardized approach for EMD studies could be established. This study has found that EMD does increase with age but is not related to the gender of the individuals. While the level of muscle contraction reduced the EMD, muscle fatigue caused the opposite effect.
Acknowledgements K.S.T. holds the Marie Curie Chair of the European Union. We wish to acknowledge support from the Marie Curie Chair funding from the EU (GenderReflex Project; MEX-CT-2006-040317) and Turkish Scientific and Technological Research Organization (TUBITAK - 107S029 - SBAG-3556) project.
Editorial / Clinical Neurophysiology 121 (2010) 1599–1601
References Datta AK, Stephens JA. Synchronization of motor unit activity during voluntary contraction in man. J Physiol 1990;422:397–419. Ellaway PH, Murthy KS. The origins and characteristics of cross-correlated activity between gamma-motoneurones in the cat. Q J Exp Physiol 1985;70:219–32. Keenan KG, Farina D, Maluf KS, Merletti R, Enoka RM. Influence of amplitude cancellation on the simulated surface electromyogram. J Appl Physiol 2005;98:120–31. Nicoll RA. The effects of anaesthetics on synaptic excitation and inhibition in the olfactory bulb. J Physiol 1972;223:803–14. Tucker KJ, Türker KS. A new method to estimate signal cancellation in the human maximal M-wave. J Neurosci Meth 2005;149:31–41. Türker KS. Electromyography: Some methodological problems and issues. Phys Ther 1993;73:698–710. Türker KS, Powers RK. The effects of common input characteristics and discharge rate on synchronization in rat hypoglossal motoneurones. J Physiol 2002;541: 245–60.
1601
Wali FA. General anaesthetics: Effects on transmitter release. Gen Pharm Vas Sys 1985;16:7–11.
Kemal S. Türker Marie Curie Chair of the European Union, Center for Brain Research, Department of Physiology, Faculty of Medicine, Ege University, Bornova, Izmir, Turkey Tel.: +90 2323901810; fax: +90 2323432653 E-mail addresses: [email protected], [email protected] URL: http://www.genderreflex.org Available online 14 May 2010
Contents lists available at ScienceDirect
Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Editorial
Reflexes as tools to study human neuromuscular system Knowledge of the synaptic connections between neurons is a key prerequisite to understanding of the operation of the nervous system. While the anatomy of these connections (‘wirings’) can be obtained by histochemical methods, their functional connections can only be determined using electrophysiological recordings. The functional connection of selected afferent inputs or corticospinal fibres to motoneurons can be studied directly in animal preparations. Compared with the studies on human subjects, experiments on animals have advantages as precise stimulation of selected nerve fibres/neurons and intracellular recordings from accurate sites are possible. Therefore, interpretations of the connections of the stimulated fibres and neurons are straight forward and easy in animal experiments. However, there are also limitations in studies on experimental animals. To prepare animals for experiments, they have to be reduced (anaesthetized, decerebrated, sliced, etc.). It is well recognized that such reductions have severe effects on synaptic potentials (Nicoll, 1972; Wali, 1985). Furthermore, one needs also to remember that active involvement of the supraspinal pathways to discharges of motoneurons is either completely suppressed or altered significantly in these reduced animal preparations. Therefore, although experiments on animals deliver the influence of a stimulated fibre or neuron to another fibre or neuron directly, one needs to accept the results of such experiments with the experimental settings in mind. In particular, describing findings on reduced animal preparations as ‘functional connections of neurons’ should be done with reservations. Although such direct experiments cannot be performed in humans, any experimental findings on conscious human subjects are likely to be more functional since they are not influenced by anesthetics or any other reduction processes and since the activity of supraspinal centres are maintained. To study functional connection of neurons in human subjects it has been customary to use stimulus-evoked changes in the discharge probability and rate of one or more motor units in response to stimulation of a set of peripheral afferents or corticospinal fibers. These are the most common ways to investigate the workings of peripheral and central pathways in human subjects. Although these are indirect methods of studying human nervous system, they are nevertheless extremely useful as there is no other method available yet to record synaptic properties directly in human subjects. The following 10 articles are based on invited lectures at the International Workshop and Conference on Human Reflexes: _ Wiring and Firing of Motoneurons (11–15 May 2009, Izmir, Turkey). The first five articles deal with the issue of estimating the properties of motoneurons using surface EMG and/or single motor unit activity. The last five articles use the surface EMG and single unit techniques to estimate neuronal connections both in healthy individuals and patients with spinal or cerebral injuries.
Although studies on surface EMG can provide much useful information regarding the excitability of the motoneurons especially when it is averaged around the timing of a stimulus, action potentials of individual motor units are more useful as they represent the activation of their motoneurons in a one-to-one basis. That is why researchers have been using intramuscular electrodes along with the surface electrodes to study the characteristics of motoneurons. Intramuscular electrodes however are painful to insert and unpleasant to keep and cannot be used in children and pain patients for ethical reasons. Therefore, there has been a growing interest in obtaining individual motor unit action potentials from the surface EMG records without in the same time inserting intramuscular electrodes. The first two articles deal with this very issue of decomposing the surface EMG into its constitutive motor unit action potentials. Nawab et al.’s article on this subject entitled: ‘‘High-yield decomposition of surface EMG signal” is an eye opener as it shows not only the difficulty of the subject in hand but also how this could be overcome with persistence. This article introduces the development of a five pin active electrode that can record single unit potentials with a certain level of confidence up to very high contraction levels. The article by Farina et al. entitled: ‘‘Decoding the neural drive to muscles from the surface electromyogram” also attempts to decompose surface EMG into its constitutive motor unit action potentials. Due to nonlinear representation of motor unit action potentials to the surface EMG and due to signal cancellation problems (Keenan et al., 2005; Tucker and Türker, 2005), these authors agree with the view that single motor unit discharge rate but not the whole muscle surface EMG represents the drive to a motoneuron pool (see also Fig. 6 in Türker, 1993). To study the discharge rate of single motor units and also without using invasive methods, these authors utilized a special grid electrode system where individual motor unit action potentials are recorded simultaneously in several surface electrodes located at different parts of the muscle of interest. Distinguishing action potentials of different motor units is facilitated by recording the surface EMG from several locations over the muscle using this ‘‘high density surface EMG” system which comprises several closely spaced surface electrodes. The authors have also successfully compared the timing of identified single unit action potentials as recorded on the surface electrode arrays with that of the ‘gold standard’ intramuscular electrodes. They agree however that the surface decomposition approach is limited to isometric contractions, localized portions of active units, and to low forces, in contrast to the more widespread applications of surface EMG recordings. Despite these limitations, surface EMG decomposition allows the accurate detection of relative changes in neural activation.
1388-2457/$36.00 Ó 2010 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2010.04.019
1600
Editorial / Clinical Neurophysiology 121 (2010) 1599–1601
Schmied and Descarreaux’s article entitled: ‘‘Influence of contraction strength on single motor unit synchronous activity” uses an intramuscular technique to obtain data from simultaneously active single motor units so that they can study the changes in the level of common input to pairs of motoneurons at various contraction levels. Using pairs of motor unit discharge data, they compare various synchronization indices to identify the index that is minimally affected by the discharge rate of motor units. They found that the index k’, that is the ratio of the total counts in the peak region of the cross-correlogram to the chance counts in that region (Ellaway and Murthy, 1985), is the index least affected by the contraction strength. In agreement with data obtained in rat motoneurons (Türker and Powers, 2002), the authors also showed that the index SIP (or E) (Datta and Stephens, 1990) was the index least affected by the motor unit firing rates. In their modeling work, Dick Stegeman and colleagues studied the question of translation of cortical oscillations into the EMG signal (‘‘The a-motoneuron pool as transmitter of rhythmicities in cortical motor drive”). They used a model to obtain insight into the corticomuscular signal transmission at various frequencies. Using coherence analysis between simulated motoneuron spike trains and surface EMG signals they have illustrated the importance of common input and found that the coherence between these variables augmented substantially with the increased level of common input. The strength of this coherence encouraged them to suggest that common inputs are needed to make corticomuscular coupling visible and that the coupling strength hardly depended on the frequency in a range from 1 to 100 Hz. They have concluded that the rhythmicities are translated into alpha motoneuron activity without strong non-linearities. The article by Powers and Türker entitled: ‘‘Deciphering the contribution of intrinsic and synaptic currents to the effects of transient synaptic inputs on human motor unit discharge” reviews the current methods of estimating synaptic potentials in human motoneurons. This article compares the methods of quantifying synaptic inputs to human motoneurons and comments on their accuracy based on recording synaptic potentials and their effects on motoneuron discharge in reduced animal preparations. Using the effects of synaptic noise, AHP summation, variations in spike threshold and subthreshold intrinsic currents on the responses of motoneurons, this review also discusses the contributions of the synaptic inputs and intrinsic motoneuron properties to altered motoneuron responses following CNS injury. The article by Maria Knikou entitled: ‘‘Neural control of locomotion and training-induced plasticity after spinal and cerebral lesions” reviews expression and regulation of spinal reflexes at rest and their modulations during locomotion. While summarizing extension and flexion reflexes, Maria reviews the mechanisms that underlie phase-dependent modulation of these reflexes in both healthy subjects and patients with spinal or cerebral lesions. She ties the recovery of walking in patients after locomotor training to the capabilities of these adjustable interneuronal and cortical circuits. She concludes her review with a suggestion that a multidisciplinary approach including rehabilitation and electrical stimulation is needed for maximizing sensorimotor recovery. ElBasiouny et al.’s article on: ‘‘Persistent inward currents in spinal motoneurons: important for normal function but potentially harmful after spinal cord injury and in amyotrophic lateral sclerosis” is an authoritative review on the findings of plateau potential in motoneurons. The authors note that the activation of motoneurons depends on interaction between synaptic inputs to motoneurons and their intrinsic properties. One of the intrinsic properties of motoneurons is the ability of the motoneuronal membrane to generate persistent inward currents (PICs). The authors review the
current findings to illustrate how these PICs can significantly change the motoneuron output to a given input and hence contribute substantially to the activation of motoneurons. In this review it is also indicated how these normally useful currents can turn out to be harmful after spinal cord injury and in ALS. The authors indicate that changes in PICs after injury or disease lead to exaggerated long-lasting reflexes and muscle spasms that contribute eventual neuronal degeneration. Shemmell et al.’s article on ‘‘Stretch sensitive reflexes as an adaptive mechanism for maintaining limb stability” examines the short and long latency stretch reflex in human subjects. They note that short latency stretch reflex’s role in regulation of stiffness in humans is well described and that this reflex generally strengthens the capabilities of a muscle to oppose external perturbations thereby increasing stability of the musculoskeletal system. The role of longer latency stretch reflexes is however less clear. According to these authors, this reflex increases its sensitivity during interactions with compliant environment relative to the sensitivity during interactions with rigid environment. This differential adaptation suggests that the longer latency stretch reflex sensitivity increases to boost limb stability when that steadiness is not provided by the environment. Deriu et al.’s article on ‘‘Reflex responses of masseter muscles to sound” reviews reflexes in the masseter muscle EMG in response to acoustic stimuli. Although previous studies have shown that these reflexes originated from the activation of the cochlear receptors, this review indicates that the vestibular receptors may also take part especially when the sound is in high intensity. This is not surprising since the masseter muscles have strong anatomical and functional connections with the vestibular receptors and since stimulation of the vestibular receptors induces clear short-latency reflex responses on the masseter EMG. These vestibule-masseter reflexes are made up of two short latency inhibitory responses and appear in the unrectified masseteric EMG on both sides. Similar to the reflex responses of the leg and arm muscle EMGs to electrical vestibular stimulation, the authors believe that the first reflex response may have minor importance to postural control but its major role may lie in the fine-tuning voluntary motor output by allowing rapid access of vestibular inputs to jaw muscle motoneurons. The second and more powerful reflex response may be of importance in postural control of masseters by stabilizing the jaw during head movements in space. The final article by Yavuz and colleagues entitled ‘‘Effect of gender, age, fatigue and contraction level on electromechanical delay” deals with the factors that affect the time lag between the onset of the direct EMG response and the onset of force generation. This time lag is referred to as electromechanical delay (EMD) and is studied to find out whether it is one of the responsible factors for age and gender related sports injuries. Working on 15 male and 15 female volunteers at various ages, this study used supramaximal nerve stimulation to elicit action potentials in all muscle fibres so that a standardized approach for EMD studies could be established. This study has found that EMD does increase with age but is not related to the gender of the individuals. While the level of muscle contraction reduced the EMD, muscle fatigue caused the opposite effect.
Acknowledgements K.S.T. holds the Marie Curie Chair of the European Union. We wish to acknowledge support from the Marie Curie Chair funding from the EU (GenderReflex Project; MEX-CT-2006-040317) and Turkish Scientific and Technological Research Organization (TUBITAK - 107S029 - SBAG-3556) project.
Editorial / Clinical Neurophysiology 121 (2010) 1599–1601
References Datta AK, Stephens JA. Synchronization of motor unit activity during voluntary contraction in man. J Physiol 1990;422:397–419. Ellaway PH, Murthy KS. The origins and characteristics of cross-correlated activity between gamma-motoneurones in the cat. Q J Exp Physiol 1985;70:219–32. Keenan KG, Farina D, Maluf KS, Merletti R, Enoka RM. Influence of amplitude cancellation on the simulated surface electromyogram. J Appl Physiol 2005;98:120–31. Nicoll RA. The effects of anaesthetics on synaptic excitation and inhibition in the olfactory bulb. J Physiol 1972;223:803–14. Tucker KJ, Türker KS. A new method to estimate signal cancellation in the human maximal M-wave. J Neurosci Meth 2005;149:31–41. Türker KS. Electromyography: Some methodological problems and issues. Phys Ther 1993;73:698–710. Türker KS, Powers RK. The effects of common input characteristics and discharge rate on synchronization in rat hypoglossal motoneurones. J Physiol 2002;541: 245–60.
1601
Wali FA. General anaesthetics: Effects on transmitter release. Gen Pharm Vas Sys 1985;16:7–11.
Kemal S. Türker Marie Curie Chair of the European Union, Center for Brain Research, Department of Physiology, Faculty of Medicine, Ege University, Bornova, Izmir, Turkey Tel.: +90 2323901810; fax: +90 2323432653 E-mail addresses: [email protected], [email protected] URL: http://www.genderreflex.org Available online 14 May 2010