Restoring motor control and sensory feedback in people with upper extremity amputations using arrays of 96 microelectrodes implanted in the median and ulnar nerves.

OBJECTIVE: An important goal of neuroprosthetic research is to establish bidirectional communication between the user and new prosthetic limbs that are capable of controlling >20 different movements. One strategy for achieving this goal is to interface the prosthetic limb directly with efferent and afferent fibres in the peripheral nervous system using an array of intrafascicular microelectrodes. This approach would provide access to a large number of independent neural pathways for controlling high degree-of-freedom prosthetic limbs, as well as evoking multiple-complex sensory percepts. APPROACH: Utah Slanted Electrode Arrays (USEAs, 96 recording/stimulating electrodes) were implanted for 30 days into the median (Subject 1-M, 31 years post-amputation) or ulnar (Subject 2-U, 1.5 years post-amputation) nerves of two amputees. Neural activity was recorded during intended movements of the subject's phantom fingers and a linear Kalman filter was used to decode the neural data. Microelectrode stimulation of varying amplitudes and frequencies was delivered via single or multiple electrodes to investigate the number, size and quality of sensory percepts that could be evoked. Device performance over time was assessed by measuring: electrode impedances, signal-to-noise ratios (SNRs), stimulation thresholds, number and stability of evoked percepts. MAIN RESULTS: The subjects were able to proportionally, control individual fingers of a virtual robotic hand, with 13 different movements decoded offline (r = 0.48) and two movements decoded online. Electrical stimulation across one USEA evoked >80 sensory percepts. Varying the stimulation parameters modulated percept quality. Devices remained intrafascicularly implanted for the duration of the study with no significant changes in the SNRs or percept thresholds. SIGNIFICANCE: This study demonstrated that an array of 96 microelectrodes can be implanted into the human peripheral nervous system for up to 1 month durations. Such an array could provide intuitive control of a virtual prosthetic hand with broad sensory feedback.

Behavioral and cellular consequences of high-electrode count Utah Arrays chronically implanted in rat sciatic nerve.

OBJECTIVE: Before peripheral nerve electrodes can be used for the restoration of sensory and motor functions in patients with neurological disorders, the behavioral and histological consequences of these devices must be investigated. These indices of biocompatibility can be defined in terms of desired functional outcomes; for example, a device may be considered for use as a therapeutic intervention if the implanted subject retains functional neurons post-implantation even in the presence of a foreign body response. The consequences of an indwelling device may remain localized to cellular responses at the device-tissue interface, such as fibrotic encapsulation of the device, or they may affect the animal more globally, such as impacting behavioral or sensorimotor functions. The objective of this study was to investigate the overall consequences of implantation of high-electrode count intrafascicular peripheral nerve arrays, High Density Utah Slanted Electrode Arrays (HD-USEAs; 25 electrodes mm(-2)). APPROACH: HD-USEAs were implanted in rat sciatic nerves for one and two month periods. We monitored wheel running, noxious sensory paw withdrawal reflexes, footprints, nerve morphology and macrophage presence at the tissue-device interface. In addition, we used a novel approach to contain the arrays in actively behaving animals that consisted of an organic nerve wrap. A total of 500 electrodes were implanted across all ten animals. MAIN RESULTS: The results demonstrated that chronic implantation (⩽8 weeks) of HD-USEAs into peripheral nerves can evoke behavioral deficits that recover over time. Morphology of the nerve distal to the implantation site showed variable signs of nerve fiber degeneration and regeneration. Cytology adjacent to the device-tissue interface also showed a variable response, with some electrodes having many macrophages surrounding the electrodes, while other electrodes had few or no macrophages present. This variability was also seen along the length of the electrodes. Axons remained within the proximity of the electrode tips at the distances required for theoretically effective stimulation and recording (⩽100 µm). SIGNIFICANCE: We conclude from these studies that HD-USEAs do not cause overall global effects on the animals, at least up to the two-month period investigated here. These results demonstrate for the first time that the consequences of high-electrode count intrafascicular arrays compare with other peripheral nerve electrodes currently available for clinical or investigational neuromodulation.

A new high-density (25 electrodes/mm²) penetrating microelectrode array for recording and stimulating sub-millimeter neuroanatomical structures.

OBJECTIVE: Among the currently available neural interface devices, there has been a need for a penetrating electrode array with a high electrode-count and high electrode-density (the number of electrodes/mm(2)) that can be used for electrophysiological studies of sub-millimeter neuroanatomical structures. We have developed such a penetrating microelectrode array with both a high electrode-density (25 electrodes/mm(2)) and high electrode-count (up to 96 electrodes) for small nervous system structures, based on the existing Utah Slanted Electrode Array (USEA). Such high electrode-density arrays are expected to provide greater access to nerve fibers than the conventionally spaced USEA especially in small diameter nerves. APPROACH: One concern for such high density microelectrode arrays is that they may cause a nerve crush-type injury upon implantation. We evaluated this possibility during acute (<10 h) in vivo experiments with electrode arrays implanted into small diameter peripheral nerves of anesthetized rats (sciatic nerve) and cats (pudendal nerve). MAIN RESULTS: Successful intrafascicular implantation and viable nerve function was demonstrated via microstimulation, single-unit recordings and histological analysis. Measurements of the electrode impedances and quantified electrode dimensions demonstrated fabrication quality. The results of these experiments show that such high density neural interfaces can be implanted acutely into neural tissue without causing a complete nerve crush injury, while mediating intrafascicular access to fibers in small diameter peripheral nerves. SIGNIFICANCE: This new penetrating microelectrode array has characteristics un-matched by other neural interface devices currently available for peripheral nervous system neurophysiological research.

Spatial and temporal characteristics of V1 microstimulation during chronic implantation of a microelectrode array in a behaving macaque.

OBJECTIVE: It has been hypothesized that a vision prosthesis capable of evoking useful visual percepts can be based upon electrically stimulating the primary visual cortex (V1) of a blind human subject via penetrating microelectrode arrays. As a continuation of earlier work, we examined several spatial and temporal characteristics of V1 microstimulation. APPROACH: An array of 100 penetrating microelectrodes was chronically implanted in V1 of a behaving macaque monkey. Microstimulation thresholds were measured using a two-alternative forced choice detection task. Relative locations of electrically-evoked percepts were measured using a memory saccade-to-target task. MAIN RESULTS: The principal finding was that two years after implantation we were able to evoke behavioural responses to electric stimulation across the spatial extent of the array using groups of contiguous electrodes. Consistent responses to stimulation were evoked at an average threshold current per electrode of 204 ± 49 µA (mean ± std) for groups of four electrodes and 91 ± 25 µA for groups of nine electrodes. Saccades to electrically-evoked percepts using groups of nine electrodes showed that the animal could discriminate spatially distinct percepts with groups having an average separation of 1.6 ± 0.3 mm (mean ± std) in cortex and 1.0° ± 0.2° in visual space. Significance. These results demonstrate chronic perceptual functionality and provide evidence for the feasibility of a cortically-based vision prosthesis for the blind using penetrating microelectrodes.

Coordinated, multi-joint, fatigue-resistant feline stance produced with intrafascicular hind limb nerve stimulation.

The production of graceful skeletal movements requires coordinated activation of multiple muscles that produce torques around multiple joints. The work described herein is focused on one such movement, stance, that requires coordinated activation of extensor muscles acting around the hip, knee and ankle joints. The forces evoked in these muscles by external stimulation all have a complex dependence on muscle length and shortening velocities, and some of these muscles are biarticular. In order to recreate sit-to-stand maneuvers in the anesthetized feline, we excited the hind limb musculature using intrafascicular multielectrode stimulation (IFMS) of the muscular branch of the sciatic nerve, the femoral nerve and the main branch of the sciatic nerve. Stimulation was achieved with either acutely or chronically implanted Utah Slanted Electrode Arrays (USEAs) via subsets of electrodes (1) that activated motor units in the extensor muscles of the hip, knee and ankle joints, (2) that were able to evoke large extension forces and (3) that manifested minimal coactivation of the targeted motor units. Three hind limb force-generation strategies were investigated, including sequential activation of independent motor units to increase force, and interleaved or simultaneous IFMS of three sets of six or more USEA electrodes that excited the hip, knee and ankle extensors. All force-generation strategies evoked stance, but the interleaved IFMS strategy also reduced muscle fatigue produced by repeated sit-to-stand maneuvers compared with fatigue produced by simultaneous activation of different motor neuron pools. These results demonstrate the use of interleaved IFMS as a means to recreate coordinated, fatigue-resistant multi-joint muscle forces in the unilateral hind limb. This muscle activation paradigm could provide a promising neuroprosthetic approach for the restoration of sit-to-stand transitions in individuals who are paralyzed by spinal cord injury, stroke or disease.

Non-invasive method for selection of electrodes and stimulus parameters for FES applications with intrafascicular arrays.

High-channel-count intrafascicular electrode arrays provide comprehensive and selective access to the peripheral nervous system. One practical difficulty in using several electrode arrays to evoke coordinated movements in paralyzed limbs is the identification of the appropriate stimulation channels and stimulus parameters to evoke desired movements. Here we present the use of a six degree-of-freedom load cell placed under the foot of a feline to characterize the muscle activation produced by three 100-electrode Utah Slanted Electrode Arrays (USEAs) implanted into the femoral nerves, sciatic nerves, and muscular branches of the sciatic nerves of three cats. Intramuscular stimulation was used to identify the endpoint force directions produced by 15 muscles of the hind limb, and these directions were used to classify the forces produced by each intrafascicular USEA electrode as flexion or extension. For 451 USEA electrodes, stimulus intensities for threshold and saturation muscle forces were identified, and the 3D direction and linearity of the force recruitment curves were determined. Further, motor unit excitation independence for 198 electrode pairs was measured using the refractory technique. This study demonstrates the utility of 3D endpoint force monitoring as a simple and non-invasive metric for characterizing the muscle-activation properties of hundreds of implanted peripheral nerve electrodes, allowing for electrode and parameter selection for neuroprosthetic applications.

Multiple factors may influence the performance of a visual prosthesis based on intracortical microstimulation: nonhuman primate behavioural experimentation.

We hypothesize that a visual prosthesis capable of evoking high-resolution visual perceptions can be produced using high-electrode-count arrays of penetrating microelectrodes implanted into the primary visual cortex of a blind human subject. To explore this hypothesis, and as a prelude to human psychophysical experiments, we have conducted a set of experiments in primary visual cortex (V1) of non-human primates using chronically implanted Utah Electrode Arrays (UEAs). The electrical and recording properties of implanted electrodes, the high-resolution visuotopic organization of V1, and the stimulation levels required to evoke behavioural responses were measured. The impedances of stimulated electrodes were found to drop significantly immediately following stimulation sessions, but these post-stimulation impedances returned to pre-stimulation values by the next experimental session. Two months of periodic microstimulation at currents of up to 96 µA did not impair the mapping of receptive fields from local field potentials or multi-unit activity, or impact behavioural visual thresholds of light stimuli that excited regions of V1 that were implanted with UEAs. These results demonstrate that microstimulation at the levels used did not cause functional impairment of the electrode array or the neural tissue. However, microstimulation with current levels ranging from 18 to 76 µA (46 ± 19 µA, mean ± std) was able to elicit behavioural responses on eight out of 82 systematically stimulated electrodes. We suggest that the ability of microstimulation to evoke phosphenes and elicit a subsequent behavioural response may depend on several factors: the location of the electrode tips within the cortical layers of V1, distance of the electrode tips to neuronal somata, and the inability of nonhuman primates to recognize and respond to a generalized set of evoked percepts.

A Novel Method of Fabricating Convoluted Shaped Electrode Arrays for Neural and Retinal Prostheses.

A novel fabrication technique has been developed for creating high density (6.25 electrodes/mm(2)), out of plane, high aspect ratio silicon-based convoluted microelectrode arrays for neural and retinal prostheses. The convoluted shape of the surface defined by the tips of the electrodes could compliment the curved surfaces of peripheral nerves and the cortex, and in the case of retina, its spherical geometry. The geometry of these electrode arrays has the potential to facilitate implantation in the nerve fascicles and to physically stabilize it against displacement after insertion. This report presents a unique combination of variable depth dicing and wet isotropic etching for the fabrication of a variety of convoluted neural array geometries. Also, a method of deinsulating the electrode tips using photoresist as a mask and the limitations of this technique on uniformity are discussed.

Trial-by-trial discrimination of three enantiomer pairs by neural ensembles in mammalian olfactory bulb.

Populations of output neurons in the mammalian olfactory bulb (OB) exhibit distinct, widespread spatial and temporal activation patterns when stimulated with odorants. However, questions remain as to how ensembles of mitral/tufted (M/T) neurons in the mammalian OB represent odorant information. In this report, the single-trial encoding limits of random ensembles of putative single- and multiunit M/T cells in the anesthetized rat OB during presentations of enantiomers of limonene, carvone, and 2-butanol are investigated using simultaneous multielectrode recording techniques. The results of these experiments are: the individual constituents of our recorded ensembles broadly represent information about the presented odorants, the ensemble single-trial response of small spatially distributed populations of M/T neurons can readily discriminate between six different odorants, and the most consistent odorant discrimination is attained when the ensemble consists of all available units and their responses are integrated over an entire breathing cycle. These results suggest that small differences in spike counts among the ensemble members become significant when taken within the context of the entire ensemble. This may explain how ensembles of broadly tuned OB neurons contribute to olfactory perception and may explain how small numbers of individual units receiving input from distinct olfactory receptor neurons can be combined to form a robust representation of odorants.

Encoding mechanisms for sensory neurons studied with a multielectrode array in the cat dorsal root ganglion.

Recent advances in microelectrode array technology now permit a direct examination of the way populations of sensory neurons encode information about a limb's position in space. To address this issue, we recorded nerve impulses from about 100 single units simultaneously in the L6 and L7 dorsal root ganglia (DRG) of the anesthetized cat. Movement sensors, placed near the hip, knee, ankle, and foot, recorded passive movements of the cat's limb while it was moved pseudo-randomly. The firing rate of the neurons was correlated with the position of the limb in various coordinate systems. The firing rates were less correlated to the position of the foot in Cartesian coordinates (x, y) than in joint angular coordinates (hip, knee, ankle), or in polar coordinates. A model was developed in which position and its derivatives are encoded linearly, followed by a nonlinear spike-generating process. Adding the nonlinear portion significantly increased the correlations in all coordinate systems, and the full models were able to accurately predict the firing rates of various types of sensory neurons. The observed residual variability is captured by a simple stochastic model. Our results suggest that compact encoding models for primary afferents recorded at the DRG are well represented in polar coordinates, as has previously been suggested for the cortical and spinal representation of movement. This study illustrates how sensory receptors encode a sense of limb position, and it provides a general framework for modeling sensory encoding by populations of neurons.

Coding of position by simultaneously recorded sensory neurones in the cat dorsal root ganglion.

Muscle, cutaneous and joint afferents continuously signal information about the position and movement of individual joints. How does the nervous system extract more global information, for example about the position of the foot in space? To study this question we used microelectrode arrays to record impulses simultaneously from up to 100 discriminable nerve cells in the L6 and L7 dorsal root ganglia (DRG) of the anaesthetized cat. When the hindlimb was displaced passively with a random trajectory, the firing rate of the neurones could be predicted from a linear sum of positions and velocities in Cartesian (x, y), polar or joint angular coordinates. The process could also be reversed to predict the kinematics of the limb from the firing rates of the neurones with an accuracy of 1-2 cm. Predictions of position and velocity could be combined to give an improved fit to limb position. Decoders trained using random movements successfully predicted cyclic movements and movements in which the limb was displaced from a central point to various positions in the periphery. A small number of highly informative neurones (6-8) could account for over 80% of the variance in position and a similar result was obtained in a realistic limb model. In conclusion, this work illustrates how populations of sensory receptors may encode a sense of limb position and how the firing of even a small number of neurones can be used to decode the position of the limb in space.

High-resolution analysis of the spatio-temporal activity patterns in rat olfactory bulb evoked by enantiomer odors.

Understanding how mammals process olfactory stimuli has motivated the development of tools and techniques which permit the simultaneous study of finely structured spatial and temporal patterns of neural activity. A technique is described that uses an array of 32 penetrating microelectrodes implanted bilaterally into the dorsal aspect of rat olfactory bulb to investigate the responses of mitral and tufted neurons to stimulation with simple enantiomer odor pairs at a number of concentrations. It is shown that stable, simultaneous recordings from up to 49 single- and multi-units can be performed for periods of up to 14 h. We show that such odors evoke unique spatial and fast-temporal activity patterns which may subserve odor discrimination. This technique is extensible to other systems neuroscience investigations of olfactory sensory processing.

High-resolution two-dimensional spatial mapping of cat striate cortex using a 100-microelectrode array.

Much of our understanding of the visuotopic organization of striate cortex results from single-electrode penetrations and serial recording of receptive field properties. However, the quality of these maps is limited by imprecision in quantifying electrode position, combining data from multiple laminae, and eye drift during the measurement of the receptive field properties. We have addressed these concerns by using an array of 100 closely spaced microelectrodes to investigate the two-dimensional visuotopic organization of layer IV in cat striate cortex. This array allowed simultaneous measurement of the receptive field properties of multiple single units on a regularly spaced grid. We found the relationship between cortical and visual space to be locally non-conformal: the receptive field locations associated with a closely spaced line of electrodes appeared randomly scattered in visual space. To quantify the scatter, we fitted a linear transformation of electrode sites onto the associated receptive field locations. We found that the distribution of the difference between the predicted receptive field location and the measured location had standard deviations of 0.59 degrees and 0.45 degrees in the horizontal and the vertical axes, respectively. Although individual receptive field positions differed from the predicted locations in a non-conformal sense, the trend across multiple receptive fields followed the maps described elsewhere. We found, on average, that the 13 mm2 of cortex sampled by the array mapped onto a 5.8-degrees) region of visual space. From the scaling of this map and a combination of the statistics of the receptive field size (2.7+/-1.5 degrees) and scatter, we have explored the impact of electrode spacing on the completeness and redundancy in coverage of visual space sampled by an array. The simulation indicated an array with 1.2-mm spacing would completely sample the region of visual space addressed by the array. These results have implications for neuroprosthetic applications. Assuming phosphene organization resembles the visuotopic organization, remapping of visual space may be necessary to accommodate the scatter in phosphene locations.

High-resolution spatio-temporal mapping of visual pathways using multi-electrode arrays.

The parallel processing of visual information was studied with penetrating microelectrode arrays. We studied the high-resolution visuotopic organization of cat primary visual cortex, and the encoding of simple visual stimuli by ensembles of ganglion cells in the isolated turtle retina. The high-resolution visuotopic organization of visual cortex is non-conformal. Regions of visual cortex separated by 400 mu may have receptive field centers that are separated by as much as 3 degrees, or they may superimpose. Ganglion cells are 'generalists', and are poor specifiers of the color of full field visual stimuli. Groups of 'luminosity' type ganglion cells can assist in the specification of stimulus color, but even individual 'chromatic' ganglion cells are not capable of quality color specification. These basic studies have relevance to the development of visual neuroprostheses based upon electrical stimulation of the retina and cortex.

Selective stimulation of cat sciatic nerve using an array of varying-length microelectrodes.

Restoration of motor function to individuals who have had spinal cord injuries or stroke has been hampered by the lack of an interface to the peripheral nervous system. A suitable interface should provide selective stimulation of a large number of individual muscle groups with graded recruitment of force. We have developed a new neural interface, the Utah Slanted Electrode Array (USEA), that was designed to be implanted into peripheral nerves. Its goal is to provide such an interface that could be useful in rehabilitation as well as neuroscience applications. In this study, the stimulation capabilities of the USEA were evaluated in acute experiments in cat sciatic nerve. The recruitment properties and the selectivity of stimulation were examined by determining the target muscles excited by stimulation via each of the 100 electrodes in the array and using force transducers to record the force produced in these muscles. It is shown in the results that groups of up to 15 electrodes were inserted into individual fascicles. Stimulation slightly above threshold was selective to one muscle group for most individual electrodes. At higher currents, co-activation of agonist but not antagonist muscles was observed in some instances. Recruitment curves for the electrode array were broader with twitch thresholds starting at much lower currents than for cuff electrodes. In these experiments, it is also shown that certain combinations of electrode pairs, inserted into an individual fascicle, excite fiber populations with substantial overlap, whereas other pairs appear to address independent populations. We conclude that the USEA permits more selective stimulation at much lower current intensities with more graded recruitment of individual muscles than is achieved by conventional cuff electrodes.

A technique to prevent dural adhesions to chronically implanted microelectrode arrays.

Minimizing relative movements between neural tissues and arrays of microelectrodes chronically implanted into them is expected to greatly enhance the capacity of the microelectrodes to record from single cortical neurons on a long-term basis. We describe a new surgical technique to minimize the formation of adhesions between the dura and an implanted electrode array using a 12 microm (0.5 mil) thick sheet of Teflon film positioned between the array and the dura. A total of 15 cats were implanted using this technique. Gross examination of 12 implant sites at the time of sacrifice failed to find evidence of adhesions between the arrays and the dura when the Teflon(R) film remained in its initial position. In six implants from which recordings were made, an average of nine of the 11 (81%) connected electrodes in each array recorded evoked neural activity after 180 days post implantation. Further, on average, two separable units were identified on each of the implanted electrodes in these arrays. No significant change was found in the density of cell bodies around implanted electrodes of four of the implanted electrode arrays. However, histological evaluation of the implant sites revealed evidence of meningeal proliferation beneath the arrays. The technique described is shown to be effective at preventing adhesions between implanted electrode arrays and improve the characteristics of chronic recordings obtained with these structures.

A multielectrode array for intrafascicular recording and stimulation in sciatic nerve of cats.

The feasibility of implanting an array of penetrating electrodes into peripheral nerves is studied in acute experiments in the cat sciatic nerve. A novel, silicon-based array of microelectrodes, the Utah Electrode Array, was used, which contains 25 or 100 1-mm long electrodes that project out from a silicon substrate. Electrode arrays of this complexity, when inserted in the peripheral nerve, could cause significant compression of the nerve and block the conduction of action potentials. Using a high velocity insertion technique, the electrode array was implanted into the sciatic nerve. Compound action potentials were evoked by and recorded with cuff electrodes. Compound action potentials recorded 1 h after insertion were only slightly altered from those recorded before insertion. Single units were readily extracted from evoked multiunit neural recordings in response to cutaneous stimulation and limb rotation around joints. Current injections into the nerve through the electrodes evoked muscle twitches with currents in the 10 microA range. Recording and stimulation stability were demonstrated for periods of up to 60 h. We have shown that high density arrays of electrodes can be inserted into the peripheral nerve and can provide a stable recording and stimulating interface to individual peripheral nerve axons. Such an array may be useful in future neuroscience research and potential neuroprosthetic applications.

Population coding in spike trains of simultaneously recorded retinal ganglion cells.

To achieve a better understanding of the parallel information processing that takes place in the nervous system, many researchers have recently begun to use multielectrode techniques to obtain high spatial- and temporal-resolution recordings of the firing patterns of neural ensembles. Apart from the complexities of acquiring and storing single unit responses from large numbers of neurons, the multielectrode technique has provided new challenges in the analysis of the responses from many simultaneously recorded neurons. This paper provides insights into the problem of coding/decoding of retinal images by ensembles of retinal ganglion cells. We have simultaneously recorded the responses of 15 ganglion cells to visual stimuli of various intensities and wavelengths and analyzed the data using discriminant analysis. Models of stimulus encoding were generated and discriminant analysis used to estimate the wavelength and intensity of the stimuli. We find that the ganglion cells we have recorded from are non-redundant encoders of these stimulus features. While single ganglion cells are poor classifiers of the stimulus parameters, examination of the responses of only a few ganglion cells greatly enhances our ability to specify the stimulus wavelength and intensity. Of the parameters studied, we find that the rate of firing of the ganglion cells provides the most information about these stimulus parameters, while the timing of the first action potential provides almost as much information. While we are not suggesting that the brain is using these variables, our results show how a population of sensory neurons can encode stimulus features and suggest that the brain could potentially deduce reliable information about stimulus features from response patterns of retinal ganglion cell populations.

A 100-channel system for real time detection and storage of extracellular spike waveforms.

As extracellular electrode arrays with 100 or more active recording sites become more widely used for simultaneous recording of neural ensembles, practical data acquisition systems that can efficiently accommodate high electrode counts are needed. To reduce the high data rates associated with extracellular recordings from these arrays, various algorithms and systems have been designed to provide complete online detection and classification of extracellular spike waveforms. However, many of these algorithms require significant user supervision to ensure accurate performance. In this paper, we discuss the design and validation of a 100-channel PC-based system that can be used with arrays of extracellular electrodes such as the Utah Electrode Array. Instead of comprehensive online spike analysis, the system performs online detection and storage of the spike waveforms for offline classification. This strategy preserves the data of interest, reduces system complexity, and requires less user supervision during experiments.