Rochelle Ackerley

Wiklund Katarina

Helena Backlund Fernström

Roger Wasling

Richard Watkins

Åke Johnson

Johan W Vallbo

Wessberg

Katarina Wiklund Fernström

Helena Backlund Wasling

Roger H Watkins

Richard D Johnson

Åke Vallbo

Johan Wessberg

Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

C-tactile (CT) afferents, and the animal homolog C-low threshold mechanoreceptors (CLTMs), are unmyelinated mechanoreceptive afferents that respond to gentle touch and are found exclusively in hairy skin [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Vallbo | A system of unmyelinated afferents for innocuous mechanoreception in the human skin[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Wessberg | Receptive field properties of unmyelinated tactile afferents in the human skin[END_REF][START_REF] Liu | Molecular genetic visualization of a rare subset of unmyelinated sensory neurons that may detect gentle touch[END_REF][START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Li | The functional organization of cutaneous low-threshold mechanosensory neurons[END_REF][START_REF] Lou | Runx1 controls terminal morphology and mechanosensitivity of VGLUT3expressing C-mechanoreceptors[END_REF][START_REF] Vrontou | Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] Mcglone | Discriminative and affective touch: sensing and feeling[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. They are characterized by their exquisite sensitivity to low-force skin indentations (<5 mN; [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]) and a slow stroke over the unit's receptive field evokes optimal responses [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF]). Further, their mean instantaneous firing frequency during skin stroking stimuli correlates well with ratings of pleasantness [START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF], where their responses are optimal to stroking touch applied around human skin temperature [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF]. Hence, due to their response properties, CTs have been implicated in social bonding and affiliation [START_REF] Morrison | The skin as a social organ[END_REF][START_REF] Vrontou | Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo[END_REF][START_REF] Mcglone | Discriminative and affective touch: sensing and feeling[END_REF]).

An under-explored property of CT afferents is their sensitivity to changes in stimulus temperature, which may play a role in shaping affective processing. In humans, a handful of studies have tested the pure thermal sensitivity of CT afferents. [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF] found that two CTs responded with a short burst of spikes to cooling, whereas [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF] and [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF] both observed that heating CT receptive fields produced no or very little activity. Animal studies have shown that CLTMs respond somewhat to cooling, especially when it is rapid [START_REF] Hensel | A quantitative study of sensitive cutaneous thermoreceptors with C afferent fibres[END_REF][START_REF] Iggo | Cutaneous mechanoreceptors with afferent C fibres[END_REF][START_REF] Bessou | Dynamic properties of mechanoreceptors with unmyelinated (C) fibers[END_REF][START_REF] Kumazawa | Primate cutaneous sensory units with unmyelinated (C) afferent fibers[END_REF][START_REF] Shea | Sensory receptors with unmyelinated (C) fibers innervating the skin of the rabbit's ear[END_REF][START_REF] Seal | Injury-induced mechanical hypersensitivity requires C-low threshold mechanoreceptors[END_REF], and one study showed very weak CLTM responses to heating [START_REF] Shea | Sensory receptors with unmyelinated (C) fibers innervating the skin of the rabbit's ear[END_REF]. [START_REF] Bessou | Dynamic properties of mechanoreceptors with unmyelinated (C) fibers[END_REF] and [START_REF] Hahn | Thermal-mechanical stimulus interactions in low-threshold C-fiber mechanoreceptors of cat[END_REF] conducted fuller comparisons between the responses evoked from cooling and those through mechanical stimulation. They both found that touch was a much more effective stimulus for eliciting CLTM responses, where responses to rapid cooling only approached 20% of the maximal response to mechanical stimulation. Both studies also found that mechanical stimulation and cooling evoked similar rates of fatigue in CLTMs and that there was Only two studies have used combined mechano-thermal stimulation to activate CTs/CLTMs, presenting the opportunity to understand how touch and temperature stimuli interact simultaneously, which occurs often in everyday life. [START_REF] Hahn | Thermal-mechanical stimulus interactions in low-threshold C-fiber mechanoreceptors of cat[END_REF] showed that a warm stationary mechano-thermal stimulus resulted in decreased CLTM discharge in cats, as compared to only mechanical stimulation, and that a cool stationary mechano-thermal stimulus elicited CLTM responses, but these were inferior to the sum of separately-applied mechanical and cool stimuli. [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF] used various speeds (0.3-30 cm.s -1 ) of moving cool (18 o C), neutral (32 o C; typical human skin temperature, [START_REF] Arens | The skin's role in human thermoregulation and comfort[END_REF], and warm ( 42o C) probe temperatures to elicit responses in human CTs. This combined mechano-thermal stimulus excited CT afferents most strongly at stroking velocities of 1-10 cm.s -1

, which was correlated with pleasantness ratings (cf. [START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF], but that CT firing frequency was significantly lower during moving touch at cool (33% decrease) and warm (27% decrease) temperatures, as compared to neutral, over all stroking velocities, apart from at 30 cm.s -1

. Thus, touch with a thermal component deviant from skin temperature reduced firing in CLTMs and CTs. However, for cooling, the responses may be more complicated and depend on the exact nature of the stimulation, where cooling alone can elicit low-level firing in CTs/CLTMs, but that combined mechano-cool stimulation may interact, although the mechanisms behind this are unknown.

The previous literature has shown that CTs are primarily mechanoreceptors that likely signal positive affective (pleasant) touch, for example to ameliorate conspecific bonding, and that deviations from typical skin temperature are generally perceived as unpleasant [START_REF] Greenspan | Thermosensory intensity and affect throughout the perceptible range[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF]. Therefore, we aimed to investigate the role of CT afferents in coding changes in both thermal and tactile stimuli, both of which are pertinent in affective interactions. In line with the previous literature, we hypothesized that CT afferent firing would be diminished during heating, whereas the responses to cooling may involve more complex mechanisms. Specifically, two mechanisms may be at work, where CT firing frequency may be suppressed during combined mechano-cool stimulation, but that additional low frequency and long-lasting spiking may be induced by cooling. Stationary and moving stimuli were used to investigate differences between modes of tactile application, where moving stimuli evoke prolonged barrages of CT responses that may be more pertinent in social and affective touch.

Material and Methods

The study was performed using 19 healthy, human participants (age range 21-42 years, 4 males), who gave written, informed consent and were paid for their time. The experimental procedures were approved by the University of Gothenburg ethics committee and performed in accordance with the Declaration of Helsinki. The technique of microneurography [START_REF] Vallbo | Activity from skin mechanoreceptors recorded percutaneously in awake human subjects[END_REF]) was used to record from single CT afferents in the left antebrachial cutaneous nerve, from either the lateral or dorsal branch, which innervates the hairy skin of the forearm. To start, the participants were seated comfortably in an adjustable dental chair, with their left arm immobilized using a vacuum airbag. The experiments were conducted at a controlled, ambient room temperature of 23 o C. A custombuilt preamplifier (Department of Physiology, Umeå University, Sweden) was taped to their upper arm and a ground electrode plate placed under their forearm. The skin just above the cubital fold was palpated to find the ideal place for insertion of the stimulating and recording electrodes, and an uninsulated reference electrode was inserted ~5 cm from this site.

To locate the small diameter antebrachial nerve, an electrical search procedure was used. An uninsulated stimulating electrode (35 or 50 mm length, 200 µm shaft diameter, ~5 µm tip diameter; FHC, Bowdoin, ME) was used to deliver 200 µs square, negative, 1 Hz pulses until the participant reported sensations spreading down the arm (maximum current used ~1.5 mA). Once an ideal depth and location was found, an insulated recording electrode of the same specifications was inserted just distal to the stimulating electrode. The recording electrode was used to access the nerve directly. Once the recording electrode had penetrated the nerve, the experimenter used rapid strokes of the participant's arm to locate single CT units. The mass activity of fast-conducting, myelinated Aβ afferents was typically heard as the stroking occurred, but the experimenter listened for a delayed response to the stroke, which corresponded to slowly-conducting C fiber afferents. Single spikes were identified online by the spike detection algorithms of the data acquisition system, sampled at 12.8 kHz, band-pass filtered (0.2-4 kHz) and stored (using SC/ZOOM; Department of Physiology, Umeå University, Sweden or Spike2; CED, Cambridge, UK). When a single C unit had been located, its responses were assessed by the auditory feedback (via a loudspeaker) and the visualization of spikes (via the traces on a screen) during mechanical stimulation of its receptive field, to determine whether it was a low-threshold (CT) or high-threshold (C-nociceptor) unit.

Single CT units were classified by showing a major deflection in the negative direction (relating to the unmyelinated axon), delayed responses to mechanical stimulation of the skin receptive field (relating to the slow conduction velocity), responding to monofilament indentations of 2.5 mN or less, and prominent responsivity (typically a short barrage of spikes) to gentle brushing, which does not typically activate the high-threshold C mechanoreceptive afferents [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. The conduction velocity of CT units was estimated using a handheld, blunt strain gauge. Responses were recorded to a short, mechanical tap to the center of a unit's receptive field and the conduction velocity was calculated using the distance from this location to the recording electrode (cf. [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Once a CT unit had been located and verified, it was subject to a number of tests involving thermal, mechano-thermal, and electro-thermal stimulation.

Pure thermal (radiant) stimulation

Two different types of stimulation devices were used to deliver radiant thermal stimulation, with no mechanical aspects. Pure cool stimulation was delivered by holding a standard cold pack 3-10 mm above the receptive field, for 30-60 s. Participants reported feeling cooling sensations of the skin. We tested the timecourse of the cooling in 5 separate participants using a calibrated thermistor on the skin in the middle of the stimulus area. The mean detection threshold for cooling was 27 o C and the mean temperature of the skin at 30 s into the stimulation was 22 o C. After 60 s, the final mean skin temperature was 19 o C, hence it is clear that the cooling was an effective stimulus. Pure warm stimulation was delivered using a gold-coated reflector lamp (OSRAM HLX Xenophot 34835, 15V/150W). The power was set to 98 W. The lamp was positioned 8 cm from the participant's forearm. A metal shield with a circular aperture of 1 cm diameter mounted in front of the lamp limited the warmed area to a 9 cm diameter area. The temperature was ramped up and a calibrated thermistor was used to determine the rate of temperature increase at skin level, which was found to be 2 o C/s. The participants readily felt a warming sensation and the heat ramp continued until the participant reported pain. We also tested the timecourse of the warming in 5 separate participants, where the mean detection threshold was 37 o C, the mean temperature of the skin at 30 s was 47 o C, and the mean pain threshold was 49 o C.

Stationary mechano-thermal stimulation

Different devices were used to deliver stationary mechano-thermal stimulation to the skin. The order of stimulation of the different temperatures was randomized and the stimulation duration lasted 1-17 s. Up to 8 repeats of stationary mechano-thermal stimulation were applied, per recorded unit. A circular thermode (skin contact area 660 mm 2 ) was used to deliver thermal stimulation to the unit's receptive field at constant cool (15°C), neutral (32°C, typical human arm skin temperature; [START_REF] Arens | The skin's role in human thermoregulation and comfort[END_REF], or warm (42°C) temperatures. The thermode was constructed of Peltier elements (Melcor CP Series thermoelectric module; Laird, London, UK) mounted on a steel block, which acted as an effective heat sink to maintain the desired temperature. The weight of the device was ~300 g. An accelerometer (EGAX-5; Entran Sensors and Electronics, Fairfield, NJ) was placed on the steel block to indicate skin contact onset and offset. The thermode was held perpendicular to the receptive field, by hand. To keep the thermode in contact with the skin the experimenter supported the thermode, allowing it to exert its inherent load on the receptive field.

A further type of stationary mechano-thermal stimulation was used, namely a lightweight steel metal spatula (15 x 140 x 1 mm), applied to the receptive field. The spatula was held horizontally over the receptive field and the experimenter was trained to deliver a load of ~6.5 g, as measured with a high precision balance. Between stimuli, spatulas were kept in ice water or hot water, and before being applied to the skin, the spatula was dried and left at 120( 4 Directly after the stationary thermal stimulation, manual brush strokes were often applied to assess the responsivity of the unit, while the skin temperature was either cool or warm. This was to further assess whether afterdischarges, i.e. additional impulses that outlast the duration of the stimulus, could be generated, as stroking produces more prominent afterdischarges, compared to static indentation of the receptive field (in CLTMs [START_REF] Iggo | Cutaneous mechanoreceptors with afferent C fibres[END_REF]in CTs: Nordin 1990). Occasionally, the receptive field was cooled with an ice cube, then dried and brushed to evoke spikes, in a similar manner to generate afterdischarges.

Slowly moving mechano-thermal stimulation

A rotary tactile stimulator (Dancer Design, Wirral, UK) was used to move a mechano-thermal probe (contact surface ~5 cm

2 ) across a CT unit's receptive field. The data were obtained in an earlier study [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF], where this device was used to collect CT firing to three constant probe temperatures (cool (18 o C), neutral (32 o C), and warm ( 42o C)), delivered over six stroking velocities (0.1, 0.3, 1, 3, 10, and 30 cm.s -1

). Ackerley et al. ( 2014) presented the thermal responses over velocities 0.3-30 cm.s

-1 and we currently present the results at the stroking velocity of 0.1 cm.s -1 , over all three temperatures. This slow stroking velocity was used as it had a moving element (which is an effective stimulus for eliciting CT responses; [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF]), but that it had a much longer time course while stroking the CT's receptive field. This allowed more detailed analyses of CT firing, as with velocities over 1 cm.s -1

, far fewer spikes are elicited [START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF]).

The contact surface of the probe was a rounded, smooth metallic plate, where its temperature was controlled using a custom-designed thermode consisting of probe-mounted Peltier elements (Melcor CP Series thermoelectric module; Laird, London, UK) interfaced to Melcor PR-59 programmable control modules and thermocouples (0.05 o C resolution). This robotic stimulator provided high-precision computer control over the velocity of stroking and temperatures, delivering a calibrated normal force (0.4 N). The temperatures were presented in a block design, where the presentation order was pseudo-randomized. In each block, three repeats of the 0.1 cm.s -1 stroking velocity were presented that were inter-leaved with other stroking velocities presented in [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF]. There was an inter-stimulus-interval of 30 seconds to allow for recovery of CT afferent response that can show fatigue with repeated stimulation [START_REF] Zotterman | Touch, pain and tickling: an electro-physiological investigation on cutaneous sensory nerves[END_REF][START_REF] Iggo | Cutaneous mechanoreceptors with afferent C fibres[END_REF][START_REF] Bessou | Dynamic properties of mechanoreceptors with unmyelinated (C) fibers[END_REF][START_REF] Hahn | Thermal-mechanical stimulus interactions in low-threshold C-fiber mechanoreceptors of cat[END_REF][START_REF] Iggo | A quantitative study of C-mechanoreceptors in hairy skin of the cat[END_REF][START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Our previous study showed that there was no overall effect of the repetitive thermal stimulation on ambient skin temperature with this period of recovery [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF].

Combined electro-thermal stimulation

The responses of a single CT afferent were recorded during the electrical stimulation of its receptive field, combined with radiant temperature stimuli. This protocol has been found to induce additional spiking which is not time locked to, but presumably indirectly induced by, the electrical stimulation in around half of CT afferents studied [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. The same radiant heat lamp was used to warm the skin to a stable temperature of 44 o C and a piece of dry ice was held a few millimeters above the skin to provide cooling to a stable temperature of 24 o C. In the protocol, the CT unit was first tested twice at neutral skin temperature ( 35o C), then warming was tested, then cooling, and after, the protocol was re-tested during neutral skin temperature once more. The temperature of the skin was measured throughout using a thermocouple attached just next to the CT receptive field. In each test, constant current electrical stimulation (0.5 ms pulse width) was delivered through two uninsulated microneurography electrodes that were inserted obliquely into the skin, just around the receptive field of the CT [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Electrical stimulation of the CT showed that it had a threshold of 1.6 mA for activation and a level of two times the threshold was set throughout the protocols. A standard 2 Hz latency identification protocol was used for each temperature investigation. This was composed of a baseline stimulation period of 0.25Hz repetitive electrical stimulation, 2 Hz stimulation for 3 minutes, followed by 10 pulses of recovery at 0.25 Hz [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. A period of at least 2 minutes of rest was used between protocols, to allow for the recovery of spike conduction to baseline levels. The nerve data were pre-processed to verify the single-unit nature of all units with an offline pattern-matching algorithm and the recorded spikes were inspected in expanded time-scale, using custom-written software implemented in MATLAB (The Mathworks, Natick, MA). Analyses for the electro-thermal stimulation were also carried about in Spike2 (CED; Cambridge, UK). Single spikes were time-stamped and descriptive statistics were gained on the number of spikes generated and their instantaneous firing frequency, over temperature conditions. In the combined electro-thermal stimulation, electrically evoked spikes were identified by virtue of their constant latency using a custom-written script in Spike2 and additional spikes generated during the stimulation that were not time locked to the electrical stimulus were quantified for the 2 Hz stimulation train [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Nonparametric statistical comparisons (Wilcoxon signed Rank tests for comparing medians to a hypothetical value, equal to zero, for no evoked activity and Friedman ANOVAs, with Dunn's multiple comparison post-hoc tests for group comparisons) were made using GraphPad Prism (La Jolla, CA), as some of the data were not normally distributed (using Shapiro-Wilk tests) and significances were sought below the p < 0.05 level (p values are given for significance to three decimal places).

CT spike analysis

Results

A total of 35 CT units were studied (locations in Fig. 1). All units were highly sensitive to innocuous skin deformation and responded readily to slow stroking of the skin. Their force activation thresholds ranged between 0.05-2.5 mN (median 0.7 mN) and they had slow conduction velocities between 0.4-1.5 m.s -1 (mean 0.9 m.s -1

). They responded with a mean peak instantaneous frequency of 70 spikes.s -1 (range 41-90 spikes.s -1

) to these innocuous stimuli. No spontaneous activity was observed in any of the units during these mechanical identification procedures.

Pure thermal (radiant) stimulation CT units were tested for their responses to pure thermal stimuli, with no concomitant mechanical contact. A total of 5 units were tested to radiant cooling, where a few spikes of low frequency (mean 0.4 spikes.s -1 , range 0.1-0.9) occurred during the test period. The responses were not modulated by the onset and termination of cooling, when the dynamic change in skin temperature was greatest. Responses to radiant warming were tested in 17 units and as with the responses to cooling, only infrequent spikes were seen (mean 1.1 spikes.s -1 , range 0.03-3.8). The majority of the units (14/17) produced firing frequencies of <1.5 spikes.s-1 , whereas 3 units showed a slightly higher rate between 2.4-3.8 spikes.s -1

. As per the cooling stimulus, the majority of units were not modulated by the onset and termination of the thermal stimulus. Only one CT showed a modulation to the timing of the warming, where it responded with a low mean discharge rate of 0.1 spikes.s -1 during the first 10 s of warming, which increased to a mean of 3.3 spikes.s -1 for its entire observation period of 25 s. This unit also showed a brief spike afterdischarge when the thermal stimulus was removed, consisting of 14 spikes over 1.8 seconds. Hence, responses to thermal stimuli were far inferior, as compared to responses to light mechanical stimuli.

Stationary mechano-thermal stimulation

Combined stationary mechano-thermal stimulation demonstrated that CT responses were modulated markedly by temperature. A total of 15 CT units were tested with the stationary mechano-thermal stimulation, at cool, neutral and warm probe temperatures. Figure 2A-C shows example responses from a single CT afferent to cool (15°C), neutral (32°C), and warm (42°C) stationary mechano-thermal stimulation, respectively. Here, consistent responses were elicited from each mechanical contact with the skin; however, at the warm temperature, the responses were much fewer, in terms of the number and duration of spiking, and the instantaneous firing frequency (peak 11 spikes.s -1 ; Fig. 2C). In contrast, CT spiking duration was longer and consisted of more spikes at higher frequencies for both cool (peak 18 spikes.s -1 ; Fig. 2A) and neutral (peak 30 spikes.s -1 ; Fig. 2B) stationary touch. Example spikes evoked and instantaneous frequencies of a single CT, to the (A) cool (15o), (B) neutral (32o), and (C) warm (42o) stationary mechano-thermal probe. (A) The cool stationary mechano-thermal stimulus evoked high initial firing frequencies, which then levelled off. Afterdischarge was observed, where the CT continued to fire after the stimulus had been removed. (B) The neutral stationary mechano-thermal stimulation evoked a strong response that declined rapidly within 1.5 s. (C) The warm stationary mechano-thermal stimulation gave a shortduration and weak response, with no afterdischarge. (D) An example of a prolonged afterdischarge in response to 13 s of stationary cool mechano-thermal stimulation. The stimulation evoked an afterdischarge that lasted ~17 s on removal of the probe. The inset shows 20 overlaid CT spikes (horizontal scale, 1 ms; vertical scale, 40 µV). (E) A box-and-whisker plot (median, quartile, minimum-maximum) for the number of CT spikes generated in the first second of contact with the stationary mechano-thermal probe stimulation (n = 15 units) for cool, neutral and warm temperatures. There was a significant decrease in the number of spikes generated during warm stimuli compared to the cool and neutral stimuli. (F) An example of afterdischarge during neutral skin temperature stroking. The inset shows 20 overlaid CT spikes (horizontal scale, 1 ms; vertical scale, 20 µV). N.B. The instantaneous frequency for a myelinated unit in the recording, which responds almost instantaneously, is shown below the delayed CT instantaneous frequency response (scale same as for CT). (G) Overview of the tendency for afterdischarge in CTs and its reproducibility. In n = 15 CT units, the number of CTs responding with an afterdischarge is shown, and whether this occurred again in a subsequent test.

To quantify the group thermo-modulatory effect, the number of spikes during the first second of stimulation were counted, pooled, and compared. A significant effect was found (Friedman ANOVA, F = 17.16, p < 0.001), where a decrease in the number of spikes was seen during the warm, as compared to both the neutral (median decrease 67%; p < 0.001) and cool (median decrease 64%, p = 0.001) mechano-thermal stimuli (Fig. 2E). No significant difference was found between the responses at cool and neutral temperatures (median 8% decrease during cool touch, p = 0.411). A recurring observation during the microneurography experiments was that CTs often produced afterdischarges (cf. Nordin 1990) after the removal of the mechano-thermal stimulus. The most prominent afterdischarges were observed after skin cooling and Figure 2D shows an example where the CT response started during stationary mechano-thermal cooling and continued after the stimulus had left the skin. The afterdischarges from CTs in cooled skin could last many seconds (e.g. ~30 s in Fig. 2D) and occurred at an instantaneous firing rate of ~5 Hz frequency (Fig. 2A,D). The tendency for afterdischarge was quantified in 15 CT units at cool, neutral and warm temperatures, using the stationary mechano-thermal probe. On the group level, the afterdischarge from mechano-cool stimulation lasted for an average of 5.6 s ± 2.9 SEM (range: 0.5-31 s, n = 12 units), at an instantaneous frequency of 5.3 Hz ± 0.5 SEM. The mean coefficient of variation for the afterdischarge frequency was 0.81, which indicates regular neuronal firing (when this value is <1, [START_REF] Taube | Interspike Interval Analyses Reveal Irregular Firing Patterns at Short, But Not Long, Intervals in Rat Head Direction Cells[END_REF]. Afterdischarge was also observed in 3/5 units tested with the cool spatula, and the application of an ice cube then brushing the skin readily evoked a barrage of afterdischarge.

Journal of Neurophysiology

With neutral, as well as warm, mechano-thermal stimulation only small and short-lasting afterdischarges were seen. It consisted of only 1-3 spikes and terminated directly after the removal of the probe. In the 15 units tested, afterdischarges with the mechano-neutral temperature probe were found in only 2 units; moreover these afterdischarges were irreproducible. Similarly, no afterdischarges were observed when the units were tested with the neutral temperature spatula; however, these 2 units showed afterdischarges to cooling. Incidentally, brief afterdischarges were common after stroking the skin at neutral skin temperature (Fig. 2F), although these consisted of only a few spikes that lasted a couple of seconds at most. With warm mechano-thermal stimulation, only 2/15 units showed brief afterdischarge responses, yet neither of these units responded to mechano-cool or -neutral stimulation. Akin to the lack of reproducibility from neutral temperature stimuli, these units did not show reproducible afterdischarge on re-warming (Fig. 2G).

Slowly moving mechano-thermal stimulation

In a separate paradigm, we tested the effects of a slowly moving mechano-thermal stimulus (at a velocity of 0.1 cm.s -1 ) on 10 CT units. Example responses of a single CT, to (A) cool (18oC), (B) neutral (32oC), and (C) warm (42oC) slowly moving (0.1 cm.s-1) mechano-thermal stimuli delivered with the rotary tactile stimulator. Note that the stroking stimulation occurred the whole time in the presented windows, where the probe was also in contact with the skin surrounding the CT, and that the time windows are aligned to the middle of the movement. Box-andwhisker plots (median, quartile, minimum-maximum) for (D) the number of spikes and (E) the mean instantaneous firing frequency for cool, neutral and warm temperatures (n = 10 units). There was a significant decrease in the number of spikes generated during warm stimulation, as compared to cool and neutral stimuli in (D), whereas there was a significant decrease between the neutral and both the cool and warm condition for the mean instantaneous firing frequency in (E) (all p < 0.05). The insets show 20 overlaid CT spikes for each condition (scale bar in C and same for all; horizontal scale, 1 ms; vertical scale, 30 µV).

It can be seen that numerous spikes were evoked to the very slow mechanical stimulus, yet these were not of particularly high firing frequency. Figure 3D andE show the number of spikes and the mean instantaneous firing frequency, respectively, for grouped data from 10 CT units tested at the different temperatures. Significant effects of temperature were found (Friedman ANOVA, F = 14.00, p = 0.002), where decreased numbers of spikes were elicited during slowly moving warm stimulation, as compared to neutral (median decrease 67%, p = 0.001) and cool (median decrease 60%, p = 0.014) temperatures (Fig. 3D). No significant difference was found between the number of evoked spikes during the cool vs. neutral temperature touch (median 16% decrease during cool touch, p = 0.480). Further, a different effect was seen in the mean instantaneous firing frequency of CTs (Friedman ANOVA, F = 10.12, p = 0.003), where an increase was found for the neutral temperature condition, as compared to the cool (median decrease 35%, p = 0.004) and warm (median decrease 47%, p = 0.049) conditions (Fig. 3E). No significant difference was observed between firing frequency in the cool and warm conditions (median 18% decrease during warm touch, p = 0.239).

Combined electro-thermal stimulation

A further paradigm was tested in a single CT to test whether temperature-dependent effects were also found during electrical stimulation, which by-passes the mechanical transduction and encoding of a stimulus. It has been shown that this kind of electrical stimulation may induce background firing in addition to the single spike timelocked to the electrical stimuli [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Figure 4 shows that during the warm stimulus, markedly less additional spikes were generated than at neutral or cool temperatures. There was no marked effect on the time course or duration of spiking, in relation to the electrical pulse, in any condition (not shown), and warming acted to reduce the number of spikes by decreasing the mean rate of additional spiking in this unit.

Discussion

We investigated the responses of CT afferents during pure thermal stimulation and during both mechano-thermal and electro-thermal stimulation. CT afferents were not spontaneously active and showed negligible or weak responses to cool or warm radiant stimulation, hence CTs are not thermoreceptors. Combined mechano-thermal stimulation modulated CT firing differentially, where maximal responses were consistently produced during neutral (skin) temperature touch, as compared to warm touch that reduced CT firing. A different and more complex relationship was found with cool touch. The impulse rate was lower during cool touch, than during neutral temperature touch, and a continued afterdischarge of spikes was often seen on termination of the mechano-cool stimulation, which was rarely found from neutral temperature or warm touch. The long-lasting response to cool stimuli was not related to any perceived pleasantness (cf. [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF]) and the precise mechanism of afterdischarge modulation is unknown, but may be a result of visco-elastic changes in the skin (cf. [START_REF] Iggo | A quantitative study of C-mechanoreceptors in hairy skin of the cat[END_REF].

In our present results, CT afferents were readily activated by gentle mechanical stimulation, which is in agreement with the literature defining them as low-threshold mechanoreceptors [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Vallbo | A system of unmyelinated afferents for innocuous mechanoreception in the human skin[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Wessberg | Receptive field properties of unmyelinated tactile afferents in the human skin[END_REF][START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Previous work on both CTs [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF] and CLTMs [START_REF] Iggo | Cutaneous mechanoreceptors with afferent C fibres[END_REF][START_REF] Hahn | Thermal-mechanical stimulus interactions in low-threshold C-fiber mechanoreceptors of cat[END_REF][START_REF] Kumazawa | Primate cutaneous sensory units with unmyelinated (C) afferent fibers[END_REF] generally agrees that their responses are weak or reduced during warming, hence it is likely that CTs (and CLTMs) do not possess mechanisms to directly encode increases in temperature. This is in line with the hypothesis that CTs are involved in encoding positive affective touch, as heating the skin much over typical skin temperature is not pleasant [START_REF] Greenspan | Thermosensory intensity and affect throughout the perceptible range[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF], may lead to damage, and such stimuli are readily encoded by nociceptors and thermoreceptors (Ackerley and Watkins 2018). Conversely, CTs [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF]) and CLTMs [START_REF] Hensel | A quantitative study of sensitive cutaneous thermoreceptors with C afferent fibres[END_REF][START_REF] Iggo | Cutaneous mechanoreceptors with afferent C fibres[END_REF][START_REF] Bessou | Dynamic properties of mechanoreceptors with unmyelinated (C) fibers[END_REF][START_REF] Hahn | Thermal-mechanical stimulus interactions in low-threshold C-fiber mechanoreceptors of cat[END_REF][START_REF] Kumazawa | Primate cutaneous sensory units with unmyelinated (C) afferent fibers[END_REF][START_REF] Shea | Sensory receptors with unmyelinated (C) fibers innervating the skin of the rabbit's ear[END_REF][START_REF] Seal | Injury-induced mechanical hypersensitivity requires C-low threshold mechanoreceptors[END_REF]) respond somewhat to cooling. There is, however, little detail in the literature about the exact responses and we advance this presently by showing that the number of spikes elicited during cooling is not reflected in an increased firing frequency. CTs do not seem to encode decreases in temperature per se, but their firing may be modulated by the subsequent physical effects from decreasing skin temperature.

An earlier study, using combined moving mechano-thermal stroking at faster velocities (0.3-10 cm.s -1

), found similar maximal instantaneous frequency responses around human skin temperature, as compared to cool (~18 o C) or warm (~42 o C) touch [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF]. We extend the previous findings by showing the same effect for very slowly moving thermal touch (0.1 cm.s -1

) and for stationary thermal touch. We found a general decrease in the propensity for CTs to fire (in both the number of spikes and instantaneous firing frequency) during warm touch, whereas for cool touch, only the firing frequency was reduced. Therefore, the modulatory effect of temperature on CT responses to mechanical stimuli is complex and deviations from neutral skin temperature in either direction diminish the response. However, CT responses were fundamentally different for cool and warm temperatures and a striking feature in response to mechano-cool stimulation was the promotion of afterdischarges. The propensity for afterdischarges, i.e. a response that outlasts a (mechanical) stimulus, has been demonstrated in CTs [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF]) and CLTMs [START_REF] Zotterman | Touch, pain and tickling: an electro-physiological investigation on cutaneous sensory nerves[END_REF][START_REF] Iggo | Cutaneous mechanoreceptors with afferent C fibres[END_REF][START_REF] Bessou | Dynamic properties of mechanoreceptors with unmyelinated (C) fibers[END_REF][START_REF] Iggo | A quantitative study of C-mechanoreceptors in hairy skin of the cat[END_REF][START_REF] Lynn | Primary afferent units from the hairy skin of the rat hind limb[END_REF]. [START_REF] Iggo | Cutaneous mechanoreceptors with afferent C fibres[END_REF] found that these brief afterdischarges were prominent when the skin was stroked and occurred at a low frequency (~5 Hz, similar to the present findings). [START_REF] Iggo | A quantitative study of C-mechanoreceptors in hairy skin of the cat[END_REF] postulated that such afterdischarges were due to restorative movements of the skin, where visco-elastic mechanisms prolong CLTM responses after removal of a (mechanical) stimulus. We found that CTs showed similar afterdischarge properties to CLTMs, but we extend the work by demonstrating that these afterdischarges were far more pronounced when the skin was cooled, which may relate to the tendency for CLTMs to be activated in general by cooling. It seems that small changes in the mechanics of the skin are sufficient to activate CTs/CLTMs and the prolonged afterdischarge in cooling may be a clear consequence of this, where contractile movements during skin cooling, and then re-warming, have a direct and extended effect on the CT receptor. The contraction and expansion of the skin may be related to changes in internal and external temperature, where it also follows that the warmer the skin, the more it expands, and the CT receptor would be less sensitive to the decreased skin movement, as we found presently. However, due to the time lags in thermal changes of the skin, it is likely that CTs do not have a strong relationship between absolute temperatures and their firing (i.e. that only stronger or longer-lasting heating or cooling may change their responses).

As well as using combined mechano-thermal stimuli, we tested a repetitive electrical stimulation paradigm during radiant thermal stimuli, by-passing the mechanotransduction process, and found concurrent results. During cooling 120(4): 1885-1892 doi.org/10.1152/jn.00940.2017 Temperature modulation of C-tactile responses 10 https://www.physiology.org/doi/full/10.1152/jn.00940.2017 and at neutral (skin) temperature, additional spiking (cf. CTs in [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]) was induced, whereas in warming, there was less spiking as compared to neutral. Although we present data here from only one CT, the results were congruent with our other findings, in that temperature changes shape CT responses in the presence of other stimuli. Hence, the electro-thermal stimulation showed that these effects were not simply due to a mechanical stimulus and the additional spiking may relate to visco-elastic changes of the skin affecting the CT receptor, which may also be the case with the afterdischarge phenomenon.

Our findings are in accordance with the affective touch hypothesis [START_REF] Olausson | The neurophysiology of unmyelinated tactile afferents[END_REF][START_REF] Mcglone | Discriminative and affective touch: sensing and feeling[END_REF], which proposes that CTs play a central role in positive somatosensory affective experiences, especially in reinforcing social and inter-personal bonds. Our finding that CTs respond optimally to touch at human skin temperature adds to their role in conveying skin-to-skin interactions (cf. [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF]. CTs fired at significantly lower frequencies to warm or cool touch, than touch at neutral temperature, and the cooling afterdischarges occurred only at low rates (~5 Hz). It is believed that the firing frequency of C-fibers determines their coding intensity, where the actual spike timing is less important. Therefore, the low firing rates seen during cool touch may not provide substantive somatosensory information and/or that low rates may actually correlate with decreases in pleasantness (cf. [START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF].

In conclusion, we find that CT afferents fire optimally to touch, but are sensitive to changes in stimulus temperature. Vigorous responses were generated to combined mechanical-thermal stationary and slowly-moving stimuli at neutral skin temperature (~32 o C). Warm touch (~42 o C) consistently decreased their responses, in terms of mean instantaneous firing frequency and number of spikes. Cool touch (~18 o C) was more complex: CTs fired at a lower instantaneous frequency, but significantly more spikes were generated than for warm touch. An afterdischarge of spikes was also regularly seen with cool touch that outlasted the skin contact, which may relate to visco-elastic mechanisms in the skin modifying the propensity of CTs to fire. In general, human CT and animal CLTM afferents show similar properties in their responses to applied mechanical and thermal stimuli. These findings reinforce the role of CTs in coding innocuous, pleasant touch, but their afterdischarge responses to cool touch should be investigated further.

  www.physiology.org/doi/full/10.1152/jn.00940.2017

Figure 1 :

 1 Figure 1: Location of C-tactile (CT) units.Locations of 35 CT units during the radiant and stationary mechano-thermal stimuli (n = 24), the slowly moving mechano-thermal stimuli (n = 10), and the combined electrothermal stimulation (n = 1).

Figure 2 :

 2 Figure 2: C-tactile (CT) firing to stationary mechano-thermal stimuli over all temperatures.

  /www.physiology.org/doi/full/10.1152/jn.00940.2017

  Figure 3 shows example responses of CT firing to slowly moving cool (18 o C; Fig. 3A), neutral (32 o C; Fig. 3B), and warm (42 o C; Fig. 3C) temperatures. (Figure 3 legend on next page) Journal of Neurophysiology 120(4): 1885-1892 doi.org/10.1152/jn.00940.2017 Temperature modulation of C-tactile responses 8 https://www.physiology.org/doi/full/10.1152/jn.00940.2017

Figure 3 :

 3 Figure 3: C-tactile (CT) responses to a slowly moving mechano-thermal stimulus at cool, neutral, and warm temperatures.

Figure 4 :

 4 Figure 4: Effect of temperature on the non-time locked spikes evoked during 2 Hz electrical stimulation. Spike responses from the electrical stimulation of a CT receptive field during different temperatures (n = 1 unit). (A) Illustration of four successive responses during electrical stimulation at a rate of 2 Hz with the CT spikes marked above the trace, at neutral temperature. Filled circles represent electrically-evoked spike responses and open circles represent the non-electrically evoked spikes. (B) Quantification of the total number of non-time locked spikes evoked during 2 Hz electrical stimulation for 3 mins, showing a marked decrease in the number of spikes evoked during warming, as compared to neutral temperature (which was repeated and hence has error bars, see Methods). The insets show 20 overlaid CT spikes (1 ms; vertical scale, 30 µV).

Acknowledgements

This work was supported by the Swedish Research Council (grant 62X-3548 to J.W.), Sahlgrenska University Hospital (ALFGBG grant 3161 to J.W.) and the Wenner-Gren Foundation (R.D.J.). The authors thank Karin Göthner, Staffan Berg, and Dan Magnusson for excellent technical assistance. The authors declare no competing financial interests.


Rochelle Ackerley
email: rochelle.ackerley@univ-amu.fr

Helena Backlund Wasling

Max Ortiz-Catalan

Rickard Brånemark

Johan Wessberg

Case Studies in Neuroscience: Sensations elicited and discrimination ability from nerve cuff stimulation in an amputee over time

Keywords: artificial touch, prosthetics, somatosensory, amputation, electrical nerve stimulation, hand

The present case study details sensations elicited by electrical stimulation of peripheral nerve axons using an implanted nerve cuff electrode, in a participant with a transhumeral amputation. The participant uses an osseointegrated electromechanical interface, which enables skeletal attachment of the prosthesis and long-term, stable, bidirectional communication between the implanted electrodes and prosthetic arm. We focused on evoking somatosensory percepts, where we tracked and quantified the evolution of perceived sensations in the missing hand, which were evoked from electrical stimulation of the nerve, for over 2 years. These sensations included small, point-like areas of either vibration or pushing, to larger sensations over wider areas, indicating the recruitment of a few and many afferents, respectively. Further, we used a two-alternative forced choice paradigm to measure the level of discrimination between trains of brief electrical stimuli, to gauge what the participant could reliably distinguish between. At best, the participant was able to distinguish a 0.5 Hz difference, and on average, acquired a 3.8 Hz just-noticeable difference at a more stringent psychophysical level. The current work shows the feasibility for long-term sensory feedback in prostheses, via electrical axonal stimulation, where small and relatively stable percepts were felt that may be used to deliver graded sensory feedback. This opens up opportunities for signaling feedback during movements (e.g. for precision grip), but also for conveying more complex cutaneous sensations, such as texture.

Introduction

The restoration of cutaneous sensory signals after amputation may be accomplished through the electrical stimulation of peripheral nerves fibers. [START_REF] Clippinger | A sensory feedback system for an upper-limb amputation prosthesis[END_REF] used electrical stimulation of the median nerve, producing sensations of paresthesia, which were used to elicit pressure sensations during grasping. More recent investigations have used the same approach to produce sensations in guiding prosthetic use [START_REF] Horch | Object discrimination with an artificial hand using electrical stimulation of peripheral tactile and proprioceptive pathways with intrafascicular electrodes[END_REF]Raspopovic et al. 2014;[START_REF] Davis | Restoring motor control and sensory feedback in people with upper extremity amputations using arrays of 96 microelectrodes implanted in the median and ulnar nerves[END_REF][START_REF] Schiefer | Sensory feedback by peripheral nerve stimulation improves task performance in individuals with upper limb loss using a myoelectric prosthesis[END_REF], including the transmission of more natural-feeling sensations, such as pressure and texture [START_REF] Tan | A neural interface provides longterm stable natural touch perception[END_REF][START_REF] Oddo | Intraneural stimulation elicits discrimination of textural features by artificial fingertip in intact and amputee humans[END_REF].

A mechanoreceptor has the propensity to encode basic sensations, including pressure, vibration, and force [START_REF] Vallbo | Properties of cutaneous mechanoreceptors in the human hand related to touch sensation[END_REF][START_REF] Ko | The roles and functions of cutaneous mechanoreceptors[END_REF], as well as more complex facets [START_REF] Connor | Tactile roughness: neural codes that account for psychophysical magnitude estimates[END_REF][START_REF] Weber | Spatial and temporal codes mediate the tactile perception of natural textures[END_REF][START_REF] Pruszynski | Edge-orientation processing in first-order tactile neurons[END_REF]. Single unit intra-neural microstimulation of Aβ mechanoreceptive afferents gives rise to a quantal tactile sensation, where the electrical stimulation of a fast-adapting type 1 (FA1) afferent produces a small sensation of vibration and a slowly-adapting type 1 (SA1) of pressure (Vallbo et al. 1984). Conversely, gross electrical nerve stimulation produces paresthesia and feels unnatural [START_REF] Schady | Peripheral projections of nerve fibres in the human median nerve[END_REF]; however, recent advances have shown that patterned electrical stimulation of many afferents can produce more natural sensations [START_REF] Tan | A neural interface provides longterm stable natural touch perception[END_REF].

Presently, we used a participant with a transhumeral amputation that was performed on the right arm in 2003. Later, an osseointegrated implant was surgically inserted into the humerus bone (OPRA Implant System; Integrum, Sweden), which provides a stable way to attach prostheses to the body. In January 2013, permanent electrodes were placed around the ulnar nerve and implanted on viable muscles. We aimed to produce perceived sensations in the missing right hand, through electrical stimulation of the ulnar

Methods

The experiment was approved by the local ethics committee and performed in accordance with the Declaration of Helsinki. The study was conducted on a single male participant (aged 40 at the start of testing; Fig. 1A), who gave written, informed consent. The ulnar nerve cuff had three electrode stimulation sites (E1, E2, E3), each with a surface area of 1 mm 2 , with a common reference electrode [START_REF] Ortiz-Catalan | Effect on signal-to-noise ratio of splitting the continuous contacts of cuff electrodes into smaller recording areas[END_REF]Fig. 1B). The participant was tested for perceived projected sensations from electrical stimulation through the nerve cuff in 8 experimental sessions, at 2, 3, 5, 12, 16, 18, 23, and 25 months post-surgery. Extra-neural electrical stimulation was delivered through the cuff electrode via a microneurography unit (Umeå University, Sweden), which received pulses from a data acquisition device (National Instruments, Austin, TX). Short, square-wave pulse trains (0.2 ms pulses at 30 Hz for 1 s) were delivered down each electrode (with the current return down the reference, Fig. 1B), while the current was increased from zero until the participant reported feeling a projected sensation in the missing hand (see Fig. 1C for current thresholds). Other electrical pulse frequencies were tested (1,15,30,60,90,120 Hz) and both positive and negative current flows. The current was kept below 350 µA. The participant was asked to describe any sensation and its location; the experimenters noted these and drew a realistic representation of the shape/size of the perceived sensation on hand maps (Figure 2).

The sensation from electrical stimulation, for each electrode tested per experiment, was quantified using a number of measures, namely its: diameter (mm), distinctness of the border (sharp/slightly diffuse/clearly diffuse/points of intense sensation), shape (round/oval/long/irregular), if movement was present (linear/circular/no movement), and the naturalness of the sensation (completely natural/almost natural/possibly natural/rather unnatural/completely unnatural) (Vallbo et al. 1984). The participant was readily able to verbally ascribe these qualities to his perceptions, using a prompt-sheet that gave scale examples (Fig. 3A).

A two-alternative forced choice (2AFC) frequency discrimination protocol was used to determine the difference between two trains of electrical pulses. The participant was asked to attend to the sensation elicited and decide which of pulse trains had the higher frequency. One of the pulse trains was a constant reference frequency (15 Hz, 1.5 s), whereas the other was the test frequency, which was always higher frequency (starting at double the reference, 30 Hz for 1.5 s; Table 1). The order of the highest pulse frequency delivered first was randomized. The participant sat in front of a screen that displayed the question, 'Which is higher frequency (more intense)?'. The participant was required to answer promptly, saying '1' or '2' and he received feedback on his answer. An adaptive transformed-rule up-down staircase was used, which converged on a 67% level of correct responses [START_REF] Levitt | Transformed Up-Down Methods in Psychoacoustics[END_REF]. When the participant https://www.physiology.org/doi/full/10.1152/jn.00909.2017?journalCode=jn answered two consecutive trials correctly, the difference between the baseline and test pulses was halved; however, a wrong answer doubled the difference (Table 1). The experiment was completed when six incorrect answers (reversals, i.e. just-noticeable difference) at a certain level were obtained. Two psychophysical levels were sought for each staircase. The first level, called the 'minimum' level, was the lowest level at which a correct answer was given, and which could not have been reached by answering randomly (p<0.05, using a random-walk simulation of the full protocol). The second, called the 'justnoticeable difference', was taken to be the eventual stable level, gained after six reversals, where the participant could reliably distinguish between paired pulse trains for that sensation. 1: Comparison of frequency increases and decreases for the staircase paired pulse train discrimination protocol. The paradigm starts with the baseline pulses (15 Hz for 1.5 s = 23 pulses) compared to double this (30 Hz for 1.5 s = 46 pulses). For comparison, the frequency is given in Hertz, as well as the equivalent number of pulses.

Results

The participant was tested systematically for sensations evoked from ulnar nerve electrical stimulation per electrode (total 57 tests), up to 25 months post-surgery. The timing of the sensations coincided strictly with the duration of the train of stimulation pulses delivered. The participant was able to locate the sensation on a drawing of the hand precisely (Fig. 2). In the first session, 2 months post-surgery, low currents (mean 15 µA) consistently gave projected sensations emanating out into the ulnar-ventral side of the missing hand. The participant described a variety of sensations, e.g. E1 gave a sensation that was 'like a pen pushing underneath the skin' at trains of 60 Hz. When the frequency was decreased to 30 Hz, this sensation became more like pulsing. The participant described it as a small (1 mm diameter), round point that felt quite natural (Fig. 2). The same tests were repeated later in the experiment and identical sensations were found. Other sensations included a warm/burning sensation (that was not unpleasant or painful) from E2, and a tactile pushing sensation that was small and point-like from E3 (Fig. 2), which turned into a line on increasing the current. Diagrammatic representations of the perceived sensations felt from electrical stimulation of each electrode over time (months postsurgery). The sizes and shapes represent the territories of the actual sensation felt. Note that the labels with asterisks (electrode 1, month 5; electrodes 2 and 3, month 5) showed an elongation of the sensation into lines emanating proximally down the finger when the current was increased (all >20 µA over the initial sensation) and that at month 25, the sensations were all in the same location (although they were of different quality). https://www.physiology.org/doi/full/10.1152/jn.00909.2017?journalCode=jn

There was some constancy between the sensations elicited over the testing sessions, with a preservation of the general innervation territory. Figure 2 shows the locations and details of evoked sensations per electrode, over time. E1 and E2 gave the most consistent sensations between sessions, where E1 typically produced a sensation in the missing palm, whereas stimulation of E2 produced sensations in the ring finger. The sensations, and the current used to evoke them (Fig. 1C), stabilized after 3 months. Different electrical pulse frequencies were tested (e.g. 1-120 Hz) and the participant consistently reported stronger sensations with higher pulse frequencies. At 2 months post-surgery, E2 gave a pushing sensation at 60 Hz stimulation that became more intense at 90 Hz; however, no sensation was felt at 30 Hz. At 3 months post-surgery, stimulation of E1 gave a small point of vibration at 30 Hz, which became stronger at 60 Hz. At 23 months post-surgery, stimulation of E1 produced a buzzing sensation at 60 Hz, which was noticeably weaker at 30 Hz, but became more pulsating at 90 Hz.

Regarding the general characteristics and quality of the projected sensations, the median size of the sensation was 3 mm diameter (min: 1 mm, max: 10 mm, mode: 1 mm), therefore the stimulation was felt as constrained and specific. The shape of the sensation was usually perceived as round or oval, and the borders of the sensations were perceived as quite-to-clearly diffuse. Typically, there was no movement associated with the sensation. The 'naturalness' of the sensation was usually described as 'almost natural' or 'possibly natural'. From 1 year post-surgery, the electrically-evoked sensations were subject to quantitative testing for the level of discrimination achievable between the intensity of pulse trains in a 2AFC paradigm. The participant was able to discriminate as low as a 0.5 Hz difference between two pulse trains (median: 0.9 Hz difference). The median just-noticeable difference was 3.8 Hz (median of 42 trials, range 39-69). Figure 3B details both discrimination levels for each electrode/experiment tested. (A) The different types of questions/responses to quantify each sensation. These were presented diagrammatically in front of the participant, who chose one response from each question that was noted by the experimenter. (B) Discrimination levels (minimum level achieved and the overall just-noticeable difference) over time. We show each level for the month (M) post-surgery and electrode (E) tested. The discrimination difference (Hz) is shown as compared to the 15 Hz (for 1.5 s) baseline.

Discussion

We performed electrical stimulation through a chronically implanted nerve cuff electrode, for over 2 years post-surgery, in an osseointegrated amputee. We found that stimulation of the ulnar nerve gave rise to confined, projected sensations, resembling the sensations generated during single unit intra-neural microstimulation (Vallbo et al. 1984). The participant could clearly indicate a sensation that was welllocalized to a small point and had no problem indicating the precise location on a drawing of a hand. At lower stimulation currents, the character of the percepts resembled sensations commonly elicited when stimulating a single SA1 or FA1 afferent, namely pressure or vibration, respectively. Increasing the stimulus intensity resulted in a spatial elongation of the sensation, e.g. like a line or a larger area [START_REF] Schady | Peripheral projections of nerve fibres in the human median nerve[END_REF]Vallbo et al. 1984;[START_REF] Sanchez Panchuelo | Mapping quantal touch using 7 Tesla functional magnetic resonance imaging and single-unit intraneural microstimulation[END_REF], as expected when more afferent nerve fibers are recruited. https://www.physiology.org/doi/full/10.1152/jn.00909.2017?journalCode=jn

We found relatively stable areas of projected sensations, over time, where there was constancy and conservation of some sensations, especially those produced from E1 in the palm, and E2 in the ring finger. During the initial testing, the sensations varied; however, they settled down from month 5 postsurgery. E3 produced some sensations in the little finger, but for the majority of testing, no particular sensations were elicited from stimulating this electrode. Overall, similarities were found in the projected sensations, but these were not always constant, which may have been due to a number of reasons, including changes at the interface, cortical plasticity, or cognitive effects. The current intensity for sensations was just above that normally used in single unit intra-neural microstimulation studies initially (Vallbo et al., 1984). An increase in the threshold current occurred during the early months of testing, but from month 5, the level was stable (~165 µA). These findings are in good correspondence with the threshold current measurements in the same patient made in a different laboratory over 11 months [START_REF] Ortiz-Catalan | An osseointegrated human-machine gateway for longterm sensory feedback and motor control of artificial limbs[END_REF]. The increase in current after the first 3 months (not shown in [START_REF] Ortiz-Catalan | An osseointegrated human-machine gateway for longterm sensory feedback and motor control of artificial limbs[END_REF], as well as the changes in perceived sensations, likely represented the stabilization of the electrodes around the nerve, where the formation of a fibrous membrane caused an increase in impedance. Further, the stabilization of stimulation is in agreement with different implanted electrodes in other patients [START_REF] Tan | Stability and selectivity of a chronic, multicontact cuff electrode for sensory stimulation in human amputees[END_REF], which independently validates the feasibility of providing long-term sensory feedback in prostheses.

The perceptual results we present are in correspondence with results from the same participant, from tests performed independently in a different laboratory. [START_REF] Ortiz-Catalan | An osseointegrated human-machine gateway for longterm sensory feedback and motor control of artificial limbs[END_REF] showed that the electrodes gave similar general innervation territories, where E1 showed projected sensations arising in the palm, E2 in the ring finger, and E3 in the little finger. An issue raised in the current work was the finding of sensations perceived in the index finger and thumb, which are not ulnar, but median nerve, innervation territory, which was found at month 3 (also cf. [START_REF] Tan | Stability and selectivity of a chronic, multicontact cuff electrode for sensory stimulation in human amputees[END_REF]. In month 2, the participant reported sensations at the wrist (paresthesia/burning), which may have been generated from general ulnar nerve stimulation. These sensations may have occurred for a number of reasons, including that the participant had not felt externally applied stimulation for over 10 years, so there may have been cortical or cognitive effects. It is likely that conflicts may have occurred between top-down vs. bottom-up information processing, where the incoming afference was novel at the time and, as described by the participant, was a welcome sensation, but may have led to a mismatch of sensation localization.

Other studies in amputees implanted with electrodes between 1 week and 3 years have shown similar feasibility for producing sensations through stimulation, although these studies have focused on more specialized activities, such as motor-functional [START_REF] Horch | Object discrimination with an artificial hand using electrical stimulation of peripheral tactile and proprioceptive pathways with intrafascicular electrodes[END_REF]Raspopovic et al. 2014;[START_REF] Tan | A neural interface provides longterm stable natural touch perception[END_REF][START_REF] Davis | Restoring motor control and sensory feedback in people with upper extremity amputations using arrays of 96 microelectrodes implanted in the median and ulnar nerves[END_REF][START_REF] Schiefer | Sensory feedback by peripheral nerve stimulation improves task performance in individuals with upper limb loss using a myoelectric prosthesis[END_REF]) and tactile-discrimination [START_REF] Graczyk | The neural basis of perceived intensity in natural and artificial touch[END_REF][START_REF] Oddo | Intraneural stimulation elicits discrimination of textural features by artificial fingertip in intact and amputee humans[END_REF] tasks. We focused on the somatosensory aspects of nerve stimulation and quantified these on a more fundamental level, using classifications of the sensations, for direct comparability over time on a number of factors (e.g. shape, naturalness), which provides a more comprehensive view of the general type and quality of sensations generated.

Our 2AFC paradigm showed that the participant could use information from electrical stimulation to distinguish differences in frequency between two pulse trains. At best, the participant distinguished 0.5 Hz (1 pulse difference) between trains of electrical stimuli. The overall just-noticeable difference equated to 3.8 Hz (6 pulse difference), which represents a more consistent measure for use in prosthetic feedback in general and can be applied to a number of areas (e.g. signaling grip pressure, texture), although this may be adapted on-line depending on the conditions. We asked which of two trains was of 'higher frequency (more intense)', which could bias the results, as modulating the frequency may have other effects than just changing the intensity of sensation. During testing, the participant felt stronger sensations at higher frequencies, although reported some changes of sensation quality at higher (>90 Hz) frequencies. As our 2AFC paradigm had a baseline frequency of 15 Hz, we do not expect that our results were biased by the question we asked; however, it is clear that modulating the frequency may lead to richer sensations. We show the feasibility of using graded levels of electrical stimulation to signal differences in intensity. For the transmission of more natural-feeling sensations (e.g. texture) it is of benefit to comprehend the minimal level that can be reliably distinguished to convey fine somatosensory qualities. [START_REF] Graczyk | The neural basis of perceived intensity in natural and artificial touch[END_REF] report similar potential for discrimination abilities in amputee nerve stimulation, yet we show that this can vary

Figure 1 :

 1 Figure 1: Set-up of electrodes and current used per experiment.

  (A) The set-up of the electrodes coming out of the osseointegrated implant in the right arm. (B) Photo of the nerve cuff electrode, showing the electrode configuration (E1: electrode 1; E2: electrode 2; E3: electrode 3). Note that the common reference is interconnected at the top and bottom. (C) The current intensity used to evoke a sensation per electrode, over time.

Figure 2 :

 2 Figure 2: Perceived projected sensations over time, from each electrode.

Figure 3 :

 3 Figure 3: Perceived sensations documented through psychophysical testing.

Acknowledgments

We thank Karin Göthner for excellent technical assistance. This work was supported by grants from the Swedish Research Council (62X-3548), Sahlgrenska University Hospital (ALFGBG 3161), and the EU FP7 (NANOBIOTOUCH) to JW. MOC and RB acknowledge grants from VINNOVA and the Promobilia foundation for the development of the osseointegrated interface.