in the co-activation of agonist and antagonist muscles to guide the limb to the intended action’s endpoint. Indeed, it is perhaps not surprising that changes in proprioceptive accuracy have been associated with musculoskeletal disorders and pain [10]. If hemodynamic effects influence muscle spindle discharge it is important to know the magnitude of this effect, as it might be one of the pathways that link pain, emotional stress, exercise and fatigue with proprioceptive function and thus sensorimotor control. Therefore, one of the central objectives of this study was to examine whether various physiological stressors have the capacity to influence the amount of physiological noise induced in muscle spindles by arterial pulsations and, thereby, potentially lead to clinically significant adverse consequences. The variability of muscle spindle discharge is often ascribed to fusimotor activity; however, it generally does not accurately correlate with the level of fusimotor drive [5]. Changes in muscle hemodynamic parameters may be another important constituent of the variability in muscle spindle discharge, especially in animals with high heart rate. The functional consequences of physiological noise have recently gained increasing attention, as intermediate levels of noise may actually facilitate sensory processing [11]. Hence, a systematic assessment of the contribution of arterial pulsations to discharge variability is required to understand its role in the sensory function of muscle spindles. In the current study we characterize the modulatory effects of arterial pulsations on the spontaneous discharge of human muscle spindles, both at rest and during physiological challenges and manoeuvres. Discharge modulation was quantified in the time and frequency domains to allow detailed comparisons of the effects of arterial pulsations across physiological conditions. In the first experiment, we examined changes in discharge modulation caused by an inspiratory-capacity apnoea, a manoeuvre known to have a profound effect on hemodynamic parameters and muscle sympathetic nerve activity without apparent activation of fusimo- tor neurones in humans [12]. While there is no evidence of a direct modulatory effect of sympathetic nerve activity on muscle spindle discharge in humans [12], such effects have been demonstrated in animals [13,14] and might need to be evaluated in the current study. In a second experiment we investigated whether muscle spindle modulation is changed during acute pain, induced by intramuscular or subcutaneous injection of hypertonic saline. Any change in spindle activity during pain could be caused by local changes in perfusion or by nociceptive reflexes involving fusimotor neurones. It has been previously demonstrated in animal experiments that pain can increase spindle sensitivity [15], but this could not be replicated in humans [16]. In the third experiment we investigated whether cardiac modulation affects spindle discharge during muscle contraction, which is known to increase the discharge of fusimotor neurones. Finally, we will summarise the potential functional consequences such modulation may have on the physiological function of muscle spindles. The robustness of the effect we observed indicates that fluctuations in blood pressure during various physiological conditions have a limited capacity to alter signalling capacity of the afferent population through this mechanism. We also suggest that intermediate levels of physiological noise in some situations could probably facilitate the detection of weak stimuli. Materials and Methods Ethics approval Data were obtained from 40 healthy subjects, 24 males and 16 females ranging in age from 18 to 42 years. Each subject provided informed written consent to the procedures, which were approved Modulation of Human Muscle Spindle Discharge by the human research ethics committee of the University of New South Wales and conformed to the Declaration of Helsinki. Microneurography The common peroneal nerve was located at the fibular head by palpation and electrical stimulation via a surface probe. A tungsten microelectrode (Frederick Haer & Co. Inc., Brunswick, ME, USA) was inserted percutaneously into a motor fascicle of the nerve. Fine manipulation of the microelectrode to isolate single afferents was then performed using auditory feedback of the neural activity while providing mechanical stimuli to the pretibial muscles and tendons of the leg. Unitary recordings were made from muscle spindle afferents. Neural activity was amplified (gain 16104), and filtered at 0.3–3.0 kHz, 50 Hz notch (ISO-80, World Precision Instruments, USA) and digitised at 10–20 kHz sampling rate, depending on bandwidth required by other channels to be recorded. All electrophysiological data were recorded on a computer-based data acquisition system (PowerLab 16SP, ADIn- struments, Bella Vista, Australia). Recorded nerve activity was exported and saved in Igor Pro 5 format (Wavemetrics, USA). Single spike recognition and discharging activity parameter analyses were performed using custom software developed using Igor Pro 5. Discharge activity of single muscle spindle afferents were visually scrutinised during the whole time course of the experiment to detect any inconsistencies and artefacts. Instantaneous discharge rate was calculated as inverse of inter-spike intervals between two successive nerve impulses. R-wave triggered averaging of muscle spindle instanta- neous discharge rate traces was performed on blocks of data with a minimal duration of 30 s. The peak of the R-wave was used as a trigger point. Identification and sample of muscle spindle afferents Identification of the muscle spindle afferents was based on the responses to stretch of the muscle along its line of action, palpation and vibration over the muscle tendons and belly and weak voluntary contractions. Single muscle afferents were identified as group Ia or II spindle afferents according to criteria previously described [17]. Briefly, primary afferents show high dynamic stretch sensitivity, demonstrate an irregular spontaneous or volitionally maintained discharge, and exhibit an off-response at the point of abrupt relaxation following a slow ramping isometric contraction. In contrast, secondary afferents usually exhibit a more regular tonic discharge [18], decelerate during an unloading contraction and do not exhibit an off-response at the termination of a voluntary ramp-and-hold contraction. Measurement of hemodynamic parameters Continuous blood pressure was measured non-invasively using radial arterial tonometry (Colin NIBP, Colin Corp, Japan) and heart rate (HR) via standard Ag–AgCl electrocardiogram (ECG) chest electrodes. Respiration was recorded with a strain gauge transducer attached to a strap around the chest (Pneumotrace, UFI, Morro Bay, CA, USA). The ECG signal was used to generate R-wave triggered averages. Electromyography Surface EMG was recorded via Ag/AgCl electrodes over the tibialis anterior (TA), extensor digitorum longus (EDL) and peronei muscles to confirm that the muscles were relaxed. The surface electrodes were placed over the muscle belly with an inter- electrode distance of 20–25 cm and in optimal positions for picking up surface recordings from the majority of the muscles PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35091