PhD Yvan Vachez

PhD Carole Carcenac

PhD Robin Magnard

Lydia Kerkerian-Le Goff

PhD Pascal Salin

PhD Marc Savasta
email: marc.savasta@inserm.fr

PhD Sebastien Carnicella

PhD Sabrina Boulet
email: sabrina.boulet@univ-grenoble-alpes.fr

Ibdm Savasta

PhD Lydia Kerkerian -Le Goff

Subthalamic nucleus stimulation impairs motivation: Implication for apathy in Parkinson's disease

Keywords: Deep brain stimulation, Neuropsychiatry, Parkinson's disease, Apathy, Dopamine

2 rescued the self-administration performances of stimulated rats from the second day of 3 treatment, without bringing performances superior to baseline or to Control + Veh rat levels. 2A, STN-DBS + PPX period, repeated measure ANOVA, STN-DBS x PPX 5 interaction: F 1,58 = 4.658, p = .0351; Session x PPX interaction: F 6,348 = 2.412, p = .0269).

(Figure

6 Both STN-DBS and PPX effects remained stable until the end of self-administration 7 experimentation (Figure 2B, two ways ANOVA, STN-DBS x PPX interaction: F 1,64 = 7.178, p 8 = .009). In order to determine whether the decrease of operant performances induced by 9 STN-DBS was due to impairment in the consummatory or preparatory components of 10 motivated behaviors, we evaluated the sensitivity to the motivational properties of sucrose 11 (consummatory behavior) in a two-bottle choice procedure. The preference for the sucrose 12 solution that was used in the operant self-administration experiment over water was high and 13 not altered by STN-DBS or PPX (Figure 2C, two way ANOVA, STN-DBS x PPX interaction:

14 F 1,61 = .966, p = .330). Therefore, the self-administration paradigm and the sucrose 15 preference test indicate that STN-DBS affected the preparatory component of motivated 16 behaviors during the operant task and that PPX specifically corrected this impairment. 17

In addition, STN-DBS-induced preparatory component deficit and subsequent self-18 administration deficiency cannot be attributed to motor impairment, because STN-DBS did 19 not induce any significant locomotor change during the first 20 minutes of the open area test, 20 corresponding to the exploratory phase when locomotor activity is high, facilitating detection 21 of motor deficit (Figure 3A, two way ANOVA, 0-20 min: F 1,48 = .513; p = .477). Exploration

11

Taken together, these results suggest that STN-DBS induced a clear motivational 12 deficit that was alleviated by the D 2 R/D 3 R agonist, reminiscent of apathy in PD.

14

STN-DBS exacerbates motivational deficit in a rodent model of PD apathetic

15 symptoms and affective disorders 16 We sought to confirm whether STN-DBS would also affect motivation in a 17 pathological context. We used a rodent model of PD that we developed 55 and that exhibits 18 strong motivational deficits, without locomotor alterations.

19

Before STN-DBS was switched-ON, rats were trained again for 10 days in the 20 operant task (only the last 3 days of training are represented in figure 4A). All groups 21 obtained equivalent performance levels (Figure 4A, 3 last days of Pre STN-DBS period, 22 repeated measure ANOVA, Session x STN-DBS x PPX interaction, F 2,32 = 1.088, p = .3490).

23 As previously demonstrated, 55 6-OHDA rats showed a marked and significant decrease in 1 50% from day one of stimulation (Figure 4A, STN-DBS period, repeated measure ANOVA, 2 STN-DBS effect F 1,15 = 4.249, p = .0571), as observed in non-lesioned/control stimulated rats 3 (Figure S2A, two way ANOVA, STN-DBS effect: F 1,42 = 10.48, p = .002 and no STN-DBS x 4 Lesion interaction: F 1,42 = 1,112, p = .298). As for non-lesioned animals, PPX completely 5 alleviated this STN-DBS-induced deficit from the second day of treatment (STN-DBS + PPX 6 period, repeated measure ANOVA, STN-DBS x PPX x Session interaction effect F 9,108 = 7 2.712, p = .0069). Both STN-DBS and PPX effects remained stable until the end of the 8 experiment (Figure 4B, two way ANOVA, STN-DBS x PPX interaction: F 1,16 = .826, p = .377).

9 We also assessed the consummatory components of motivated behaviors, with the two-10 bottle choice procedure and we found that neither STN-DBS nor PPX modified the 11 preference for sucrose (Figure 4C, two way ANOVA, STN-DBS x PPX interaction: F 1,20 = 12 .341, p = .567).

13

PPX or STN-DBS tended to reduce ambulatory activity in the open area test while 14 when they were combined, on average they promoted a high level of activity but with high 15 variability (Figure 5A, two way ANOVA, STN-DBS x PPX interaction: F 1,14 = 5.759, p = .031).

16 These effects are likely to reflect changes in behavioral activity rather than alteration of Combining a relevant in vivo model of neuropsychiatric PD symptoms, chronic 4 bilateral STN-DBS in awake and freely moving animals and a pharmacological approach, we 5 provide strong evidence for a direct deleterious effect of STN-DBS on motivation. STN-DBS 6 induces a significant deficit in motivated behavior, due neither to a reward sensitivity deficit 7 related to a potential anhedonic effect, nor to motor deficits. This behavioral effect was 8 completely reversed by the activation of D 2 R/D 3 R with PPX, revealing the potential critical 9 role of DA in this STN-DBS motivational effect.

10 It is essential to better understand the pathophysiology of neuropsychiatric symptoms of PD 11 and how they respond to current treatments such as STN-DBS. Here, we tried to be as close 12 as possible to the clinical situation in PD, especially for the stimulation conditions. While most 13 preclinical studies have been performed with acute STN-DBS that was applied daily only 14 during the tasks and experiments (e.g., 41,49 ), the novel stimulation system we used allowed 15 continuous STN-DBS for several days. In addition, we used monopolar stimulation. Indeed, it 16 is the most widely applied in PD patients [START_REF] Figure | PPX completely 2 rescued the self-administration performances of stimulated rats from the second day of 3 treatment, without bringing performances superior to baseline or to Control + Veh rat levels. 4 (Figure 2A, STN-DBS + PPX period, repeated measure ANOVA[END_REF] as well as in studies describing apathy under STN-17 DBS 17,[START_REF]Comparison of the effects of STN-DBS (A) or PPX treatment (B) in control and 22 lesioned animals on the mean sucrose deliveries over the last 3 days of sucrose self-23 administration experiment. Data shown as means  SEM[END_REF]48,[61][62][63][64] . Furthermore, a preclinical study in rat has clearly demonstrated that it 18 reduces tissue damage compared to bipolar stimulation. 65 Moreover, although monopolar 19 stimulation is more likely to affect the surrounding STN fibers than bipolar stimulation, it was 20 applied at intensities below 225 A to minimize current spreading as previously 21 demonstrated. 66 The motivational dimension of apathy especially concerns hum-drum daily 22 tasks that are neither challenging nor particularly effortful. [START_REF] Teflon | Stereotaxic coordinates (relative to bregma, 5 according to the stereotaxic atlas of Paxinos and Watson 6 ) were: AP, -3.8 mm; L, ±2.4 mm[END_REF] A sucrose self-administration 23 procedure with a 2.5 % solution, in a fixed ratio 1 schedule, in non-food-deprived rats, is an 24 appropriate way to operationalize a simple effort with a relatively moderate rewarding 25 outcome. This approach has allowed us to demonstrate a clear effect of STN-DBS on 26 motivated behaviors during an operant task. The decrease of baseline ambulatory activity 27 detected during the open area test, unrelated to motor impairment, further suggests that the 28 STN-DBS-induced operant deficit may be considered as a decrease in the maintenance of 1 behavioral activity, as observed in apathy. [START_REF]We also assessed the consummatory components of motivated behaviors, with the two-10 bottle choice procedure and we found that neither STN-DBS nor PPX modified the 11 preference for sucrose (Figure 4C, two way ANOVA[END_REF] While we previously provided strong evidence 2 that the dopaminergic denervation can account for the loss of motivation in PD, here we 3 report that STN-DBS by itself could induce or exacerbate this motivational impairment via 4 DA-driven mechanisms, independent of the hypodopaminergy induced by DRT reduction.

5 Furthermore, the present study provides a missing link, reconciling apparently contradictory 6 clinical observations on the origin of post-operative apathy in stimulated PD patients.

7

The myriad of reported neuropsychiatric side effects of STN-DBS in PD patients 3 has 8 triggered several animal studies to understand their phenomenology. These studies have 9 demonstrated the possible implication of STN-DBS in PD-related depression 49,67 or potent 10 effects on impulsive behaviors, 68 but few of them tackled the effects on pure motivational 11 processes. As far as we know, only one report mentions data that could be interpreted as a 12 transient reduced motivation under STN-DBS. 69 However, another study showed no effect of 13 STN-DBS on operant activity in a fixed-ratio schedule of reinforcement and even described 14 an increase in performance in obtaining a sucrose pellet during a progressive ratio 15 schedule. 41 Major differences between this previous study and our protocol are likely to 16 account for these discrepancies. Indeed, Rouaud et al., used acute bipolar stimulation 17 conditions compared to our chronic monopolar STN-DBS; interestingly, psychiatric side 18 effects of STN-DBS strongly depend on electrode polarity. For example, acute depressive 19 states following regular monopolar STN-DBS are greatly reduced by switching to the scarcer 20 bipolar STN-DBS. 70,71 Moreover, the design of the operant task was different enough to yield 21 diverging results. In particular, our study avoided food-restriction. Whereas all the studies 22 having inactivated the STN found an increase in motivation for food in restricted animals, one 23 study demonstrated that this effect was completely occluded in rats fed ad libitum. 37 Finally, 24 while the conditioned place preference revealed an effect of STN-DBS on the rewarding 25 properties of sucrose in this study, 41 we did not find any modification of this parameter during 26 the two bottle choice test.

27

The STN can be functionally and anatomically subdivided into motor, associative and 28 limbic sub-territories. [72][73][74] Then it is part of the three respective basal ganglia loops. 73,74 In PD

F o r P e e r R e v i e w
1 patients, DBS is tailored to stimulate the motor part of the STN, and so to impact the whole 2 motor circuitry. However, an inaccurate electrode placement within the STN can engage the 3 circuitries involving its associative or limbic part rather than the motor one, 75 potentially 4 leading to non-motor side effects, 70, 74 apathy in particular. In line with this assumption, a very 5 recent study succeeded in alleviating post STN-DBS apathy in patients by displacing the 6 electrode from the limbic STN to the motor part, 23 demonstrating that apathy under STN-DBS 7 could rely on a mechanism caused by the stimulation of the limbic part of the STN. Our data 8 suggest that the dopaminergic system might be involved in this mechanism, at least in part.

9

In patients, apathy occurring after STN-DBS can be treated by dopaminergic 10 agonists, including those targeting the D 2 R and D 3 R. 18,19 This, and the fact that 11 methylphenidate can alleviate fatigue 76 , which is a related and/or confounding symptom of 12 apathy 77 , has prompted the hypothesis that a strong hypodopaminergic state revealed by 13 reduction of DRT, rather than STN-DBS per se, may be responsible for the resurgence of 14 apathy. 13 Using a pharmacological protocol allowing exploration of the chronic effects of PPX 15 and known to increase D 2 R and D 3 R expression, 59 we demonstrated that PPX, a D 2 R/D 3 R 16 agonist, fully rescued the motivational deficit induced by STN-DBS in control and lesioned 17 rats. It should be noted that this pharmacological protocol was not able to reverse the loss of 18 motivation due to the dopaminergic lesion, indicating that even if both the lesion and STN-19 DBS effects on motivation could be driven by dopaminergic mechanisms, these could be 20 significantly different. This suggests that STN-DBS may impact on behavior via its own 21 effects on DA transmission, which is consistent with previous animal model studies showing 22 that STN-DBS modulates DA system. 58,[78][79][80][81][START_REF] Mm | For all measurements, masks from these different striatal and mesencephalic sub-3 regions were drawn with the computer analysis system to ensure that appropriate 4 comparisons were made between homologous anatomical regions. For each striatal sub-5 region, optical densities (OD) were measured and averaged. The OD value obtained for an 6 unlabeled area (the corpus callosum) was used as the background and was subtracted from 7 each of the OD values measured. OD were expressed as percentages relative to the mean 8 optical density values obtained for the homologous regions of the sham-operated animals. 9 10 Histological control of electrodes implantation 11 Sections through the subthalamic nucleus were stained with Cresyl violet and analyzed 12 under a light microscope (Nikon, Eclipse 80i[END_REF] The three so-called basal ganglia limbic, 23 associative and motor loops including the STN are not completely segregated; the different 24 structures involved are functionally interconnected 83 by an ascending striato-midbrain-striatal 25 spiraling circuitry. 84 Moreover, beyond this complex organization, which has yet to be 26 demonstrated in rats, SNc and VTA are overlapping structures 85 and the whole striatum is 27 characterized by a dense local microcircuitry 86,87 which could also contribute to this 28 interconnection. Thus, STN-DBS may modulate DA transmission within the basal ganglia

F o r P e e r R e v i e w
1 and, through these subcortical interactions, disrupt the complex chain of events that 2 transforms intentions into adapted action, and thereby influence non-motor functions. [START_REF]Both STN-DBS and PPX effects remained stable until the end of self-administration 7 experimentation (Figure 2B, two ways ANOVA[END_REF][START_REF]We also assessed the consummatory components of motivated behaviors, with the two-10 bottle choice procedure and we found that neither STN-DBS nor PPX modified the 11 preference for sucrose (Figure 4C, two way ANOVA[END_REF] 3 Nonetheless, we cannot exclude a potential involvement of the structures surrounding the 4 STN . It is unlikely that the electric field emanating from the electrode is restrained to the 5 STN borders and DBS of some of these structures in the vicinity of the STN, such as the 6 lateral hypothalamus, can also modulate DA transmission 88 . We previously found that short 7 STN-DBS can decrease the level of D 2 R and D 3 R within the NAc. 58 Given the critical 8 implication of the D 2 R and D 3 R in motivational processes, 89-92 our data suggest a causal 9 involvement of these receptors in the motivational deficit that we observed. In addition, D 2 R 10 and D 3 R are expressed in several other non-motor structures impacted by PPX treatment 93 11 and some of them are part of down-stream circuits with metabolic activities known to be 12 affected by STN-DBS. 22,94 Thus, beyond the NAc, both STN-DBS and PPX effects could 13 differentially engage some common system responsible for behavior changes highlighted in 14 this study. However, dopaminergic agonists do not always efficiently alleviate apathy under 15 STN-DBS 22 . Non dopaminergic lesions frequently occur during the course of disease 95, 96 , 16 and serotonin for example is proposed to participate in PD apathy 97 . In rats, STN-DBS has 17 been shown to decrease serotonin release promoting depressive like behavior. 49,98 Thus, 18 we cannot completely exclude the implication of other neurotransmitters in the loss of 19 motivation observed in patients or in this study.

20

As previously reported, 99 PPX at 0.3 mg/kg induces hyperlocomotricity in the open 21 area test in control rats. This hyperactivity is unlikely to be responsible for the reversion of 22 the self-administration deficit observed in PPX-treated STN-DBS rats since first, the level of 23 non-stimulated treated rats remains similar to that of control rats, and second, PPX induced 24 hypoactivity in non-stimulated lesioned rats whereas it also reversed the operant deficit 25 induced by STN-DBS in those lesioned rats. This hypoactivity promoted by PPX in lesioned 26 rats has previously been reported during the 120 minutes post injection window in control 27 animals 99 and could rely on a sedating effect mediated by the presynaptic D 3 R 100 . We chose 28 to separate the behavioral test from injection by 3h to avoid this effect. Since we previously 19 to be involved in such processes 109 .

20

STN-DBS has been shown to prevent cocaine or heroin re-escalation intake in rats 40, 21 44 and to reduce dopamine dysregulation syndrome or addictive behavior such as 22 pathological gambling. 110-114 Thus, STN-DBS is currently explored as an effective treatment 23 for addiction. 45,115 However, our results suggest that this apparent beneficial effect could be 24 underlain by a general motivational deficit. In rats, inhibiting the STN can abolish affective 25 responses for salient reward 109 and STN-DBS can induce depression-like behaviors. 49,67,116 26 Furthermore, PD patients for whom addictive behavior was reduced under STN-DBS can 27 also co-express apathy. 14,110 These convergent facts are raising the question of the nature of 28 this apparent "anti-addictive" effect of STN-DBS. This neurosurgical treatment could rather

Affiliations :

1 Inserm, U1216, F-38000 Grenoble, France.

2 Univ. Grenoble Alpes, Grenoble Institut des Neurosciences, GIN, F-38000 Grenoble, France. 3 Aix Marseille Univ, CNRS, IBDM, Marseille, France.

Corresponding authors:

Dr Sabrina Boulet 3 that efficiently alleviates the motor symptoms of Parkinson's disease (PD). [START_REF] Figure | PPX completely 2 rescued the self-administration performances of stimulated rats from the second day of 3 treatment, without bringing performances superior to baseline or to Control + Veh rat levels. 4 (Figure 2A, STN-DBS + PPX period, repeated measure ANOVA[END_REF] However, a 4 plethora of psychiatric manifestations and cognitive deficits have been recently identified as 5 an integral part of the clinical picture of the disease and STN-DBS has been suggested to 6 influencethese symptoms, for better or for worse. 2, 3 Apathy, which can be simplistically 7 defined as a loss of motivation or a reduction in goal-directed behaviors accompanied by loss 8 of emotions and flattening of affect, [START_REF] Teflon | Stereotaxic coordinates (relative to bregma, 5 according to the stereotaxic atlas of Paxinos and Watson 6 ) were: AP, -3.8 mm; L, ±2.4 mm[END_REF][START_REF]0557, p = .814), but caused a significant increase in locomotor activity in the open 7 area test (Figure 3A, PPX effect[END_REF][START_REF]Both STN-DBS and PPX effects remained stable until the end of self-administration 7 experimentation (Figure 2B, two ways ANOVA[END_REF] is the most frequently observed non-motor 9 complication of PD and deeply contributes to worsen the patient's quality of life 2, 5, 7-12 . 10 Importantly, apathy has been reported to occur, or be exacerbated, in some patients under 11 STN-DBS, as blunted affects [START_REF]Time course of the study, groups of animals and histological controls 9 After learning the sucrose self-administration task, lesion and implantation of electrodes, rats 10 were divided into a stimulated and a non-stimulated group according to their performances, 11 to constitute initially equal performance groups. Then, after 4 days of subthalamic nucleus 12 deep brain stimulation (STN-DBS) with further sucrose self-administration, pramipexole 13 (PPX) or vehicle (Veh) was injected twice a day into half of the stimulated (Non lesioned rats: 14 STN-DBS + PPX, n = 8; STN-DBS + Veh, n = 13; lesioned rats: Lesion + STN-DBS + PPX, n 15 = 5; Lesion + STN-DBS + Veh, n = 5) and non-stimulated rats (Control + PPX[END_REF][START_REF]We also assessed the consummatory components of motivated behaviors, with the two-10 bottle choice procedure and we found that neither STN-DBS nor PPX modified the 11 preference for sucrose (Figure 4C, two way ANOVA[END_REF][10][13][14][15][16] . Yet, the clear repercussion of STN-DBS on apathy 12 remains to be elucidated.

13

Because dopaminergic replacement therapy (DRT) is reduced during STN-DBS, 14 apathy in patients under STN-DBS is commonly attributed to the resurgence of pre-existing 15 symptoms revealed by DRT reduction or withdrawal. 17 This assumption is also supported by 16 1) the alleviation of apathy after STN-DBS by dopaminergic agonists, especially those 17 targeting the D2 and D3 DA receptors (D 2 R and D 3 R ) 18, 19 and 2) functional imaging studies 18 in PD patients [START_REF]We also assessed the consummatory components of motivated behaviors, with the two-10 bottle choice procedure and we found that neither STN-DBS nor PPX modified the 11 preference for sucrose (Figure 4C, two way ANOVA[END_REF]17 and some preclinical data indicating that at least some forms of apathy are 19 related to the degenerative process and DA loss. 20 20 However, in several studies, the occurrence of apathy in patients under STN-DBS 21 was not correlated with the reduction of DRT but with DBS-induced changes in glucose 22 metabolism within the associative and limbic circuitry 15,[START_REF]Comparison of the effects of STN-DBS (A) or PPX treatment (B) in control and 22 lesioned animals on the mean sucrose deliveries over the last 3 days of sucrose self-23 administration experiment. Data shown as means  SEM[END_REF]22 or with incorrect location of 23 electrodes in the associative or limbic part of the STN. 23 Thus, apathy in patients under STN-24 DBS was also proposed to be a major side-effect of STN-DBS itself. 15,[START_REF]Comparison of the effects of STN-DBS (A) or PPX treatment (B) in control and 22 lesioned animals on the mean sucrose deliveries over the last 3 days of sucrose self-23 administration experiment. Data shown as means  SEM[END_REF][22][23] There is now an 25 abundance of experimental and clinical studies demonstrating that 1) the STN is involved in 26 reward and motivational processes, [24][25][26][START_REF]number of 3 forelimb adjusting steps measured. The test was carried out three times for each paw. 4 5 Open area 6 Rats were placed in a dimly lit white Perspex (Castorama, Saint Martin d'Hères, France)[END_REF][START_REF] Interconnection | STN-DBS may modulate DA transmission within the basal ganglia 1 and, through these subcortical interactions, disrupt the complex chain of events that 2 transforms intentions into adapted action, and thereby influence non-motor functions[END_REF][29][30][START_REF] Benabid | Deep brain stimulation of the 4 subthalamic nucleus for the treatment of Parkinson's disease[END_REF] 2) manipulating the STN can modify motivational 27 behaviors 32-37 and 3) STN-DBS can alleviate dopamine dysregulation syndrome in some PD 1 patients 14, 38, 39 and be a conceivable approach to treat addictive disorders [40][41][START_REF] Drui | Loss of dopaminergic nigrostriatal neurons 5 accounts for the motivational and affective deficits in Parkinson's disease[END_REF][43][44][45] . Yet, a 2 possible direct impact of STN-DBS on motivation in PD is not unanimously supported.

3

This lack of consensus concerning the origin of apathy in patients under STN-DBS is 4 a critical issue. Whereas the prevalence of apathy before STN-DBS is about 25% 13,46,47 , this 5 percentage ranges from 8 % to 60 % during STN-DBS 14,17,[START_REF]Comparison of the effects of STN-DBS (A) or PPX treatment (B) in control and 22 lesioned animals on the mean sucrose deliveries over the last 3 days of sucrose self-23 administration experiment. Data shown as means  SEM[END_REF]48 , according to the diagnostic 6 approach used, whether established according to cut-off scores on severity scales, 7 instruments rated by caregivers, or clinical diagnostic criteria 47 . Thus, it considerably 8 compromises the benefits of STN-DBS on motor symptoms. [START_REF]0557, p = .814), but caused a significant increase in locomotor activity in the open 7 area test (Figure 3A, PPX effect[END_REF] Investigating this question in 9 patients is difficult because it is impossible to avoid the impact of the progressive process of 10 degeneration and reduction of DRT during STN-DBS. In addition, the animal studies that 11 have sought to explore the limbic and mood effects of STN-DBS may not have combined 12 appropriate behavioral approaches to assess motivation with bilateral and continuous STN-13 DBS 41, 49, 50 as clinically applied most of the time in studies reporting onapathy [START_REF]0557, p = .814), but caused a significant increase in locomotor activity in the open 7 area test (Figure 3A, PPX effect[END_REF]15,19,[START_REF]Comparison of the effects of STN-DBS (A) or PPX treatment (B) in control and 22 lesioned animals on the mean sucrose deliveries over the last 3 days of sucrose self-23 administration experiment. Data shown as means  SEM[END_REF][51][52][53] .

14

In the present study, we explored a potential effect of bilateral STN-DBS on 15 motivation, by using a wireless micro-stimulation system enabling chronic continuous 16 stimulation in rats during several weeks 54 . We first investigated the consequences of bilateral 17 chronic STN-DBS in naïve rats and then in a preclinical model of neuropsychiatric symptoms 18 related to PD that we have developed, using bilateral but partial denervation of the dorsal 19 Rats were trained to self-administer a 2.5% sucrose solution before and after SNc lesion and 20 electrodes implantation (Only the self-administration after electrode implantation is 21 represented). After 10 to 15 days, stable performances were obtained (less than 20% 22 performance variation over three consecutive sessions) and STN-DBS was turned ON.

23 Pharmacological procedures were applied after a new stabilization period: PPX (Sigma, 0.3 24 mg.kg -1, in 0.9% NaCl, 1 ml/kg) or vehicle was administered (sub-cutaneous) twice a day, 3h 25 before the beginning of behavioral tests (i.e. injection at 7 am; test at 10 am) for and then at 26 5 pm, during 20 days. This protocol, known to increase the expression of D 2 R and D 3 R, 59 27 was chosen to explore the chronic effects of PPX. 60 STN-DBS and PPX treatment were 1 uninterrupted until rat euthanasia. After several days of sucrose self-administration, rats were 2 submitted to a two-bottle choice procedure, as well as to a stepping test and 3 locomotor/ambulatory activity evaluation in an open area 55 (Figure 1A). At the end of the 4 experiment, rats were euthanized and brains were processed for histological control of lesion Combining a relevant in vivo model of neuropsychiatric PD symptoms, chronic 4 bilateral STN-DBS in awake and freely moving animals and a pharmacological approach, we 5 provide strong evidence for a direct deleterious effect of STN-DBS on motivation. STN-DBS 6 induces a significant deficit in motivated behavior, due neither to a reward sensitivity deficit 7 related to a potential anhedonic effect, nor to motor deficits. This behavioral effect was 8 completely reversed by the activation of D 2 R/D 3 R with PPX, revealing the potential critical 9 role of DA in this STN-DBS motivational effect.

10 It is essential to better understand the pathophysiology of neuropsychiatric symptoms of PD 11 and how they respond to current treatments such as STN-DBS. Here, we tried to be as close 12 as possible to the clinical situation in PD, especially for the stimulation conditions. While most 13 preclinical studies have been performed with acute STN-DBS that was applied daily only 14 during the tasks and experiments (e.g., 41,49 ), the novel stimulation system we used allowed 15 continuous STN-DBS for several days. In addition, we used monopolar stimulation. Indeed, it 16 is the most widely applied in PD patients 1 as well as in studies describing apathy under STN-17 DBS 17,[START_REF]Comparison of the effects of STN-DBS (A) or PPX treatment (B) in control and 22 lesioned animals on the mean sucrose deliveries over the last 3 days of sucrose self-23 administration experiment. Data shown as means  SEM[END_REF]48,[61][62][63][64] . Furthermore, a preclinical study in rat has clearly demonstrated that it 18 reduces tissue damage compared to bipolar stimulation. 65 Moreover, although monopolar 19 stimulation is more likely to affect the surrounding STN fibers than bipolar stimulation, it was 20 applied at intensities below 225 A to minimize current spreading as previously 21 demonstrated. 66 The motivational dimension of apathy especially concerns hum-drum daily 22 tasks that are neither challenging nor particularly effortful. [START_REF] Teflon | Stereotaxic coordinates (relative to bregma, 5 according to the stereotaxic atlas of Paxinos and Watson 6 ) were: AP, -3.8 mm; L, ±2.4 mm[END_REF] A sucrose self-administration 23 procedure with a 2.5 % solution, in a fixed ratio 1 schedule, in non-food-deprived rats, is an 24 appropriate way to operationalize a simple effort with a relatively moderate rewarding 1 behavioral activity, as observed in apathy. [START_REF]We also assessed the consummatory components of motivated behaviors, with the two-10 bottle choice procedure and we found that neither STN-DBS nor PPX modified the 11 preference for sucrose (Figure 4C, two way ANOVA[END_REF] While we previously provided strong evidence 2 that the dopaminergic denervation can account for the loss of motivation in PD, here we 3 report that STN-DBS by itself could induce or exacerbate this motivational impairment via 4 DA-driven mechanisms, independent of the hypodopaminergy induced by DRT reduction.

5 Furthermore, the present study provides a missing link, reconciling apparently contradictory 6 clinical observations on the origin of post-operative apathy in stimulated PD patients.

7

The myriad of reported neuropsychiatric side effects of STN-DBS in PD patients 3 has 8 triggered several animal studies to understand their phenomenology. These studies have 9 demonstrated the possible implication of STN-DBS in PD-related depression 49,67 or potent 10 effects on impulsive behaviors, 68 but few of them tackled the effects on pure motivational 11 processes. As far as we know, only one report mentions data that could be interpreted as a 12 transient reduced motivation under STN-DBS. 69 However, another study showed no effect of 13 STN-DBS on operant activity in a fixed-ratio schedule of reinforcement and even described 14 an increase in performance in obtaining a sucrose pellet during a progressive ratio 15 schedule. 41 Major differences between this previous study and our protocol are likely to 16 account for these discrepancies. Indeed, Rouaud et al., used acute bipolar stimulation 17 conditions compared to our chronic monopolar STN-DBS; interestingly, psychiatric side 18 effects of STN-DBS strongly depend on electrode polarity. For example, acute depressive 19 states following regular monopolar STN-DBS are greatly reduced by switching to the scarcer 20 bipolar STN-DBS. 70,71 Moreover, the design of the operant task was different enough to yield 21 diverging results. In particular, our study avoided food-restriction. Whereas all the studies 22 having inactivated the STN found an increase in motivation for food in restricted animals, one 23 study demonstrated that this effect was completely occluded in rats fed ad libitum. 37 Finally, 24 while the conditioned place preference revealed an effect of STN-DBS on the rewarding 25 properties of sucrose in this study, 41 we did not find any modification of this parameter during 26 the two bottle choice test.

27

The STN can be functionally and anatomically subdivided into motor, associative and 28 limbic sub-territories. [72][73][74] Then it is part of the three respective basal ganglia loops. 73,74 In PD Yvan Vachez, PhD 1,2 , Carole Carcenac, PhD ,1,2 , Robin Magnard, PhD ,1,2 , Lydia Kerkerian -Le Goff, 5

PhD 3 , Pascal Salin, PhD 3 , Marc Savasta, PhD 1,2 , Sebastien Carnicella, PhD

3 Figure

 3 Figure 1B provides the different electrode positions in left and right STN for the

17

 17

1

  18 STN-DBS induces a motivational deficit in normal rats that is reversed by the D2 R/D 3 R 19 agonist pramipexole 20 Before STN-DBS was switched ON, rats were trained for 10 days in the operant task 21 (only the last 3 days of training are represented in figure 2A). Groups were formed to have 22 equivalent performance levels (Figure 2A, Pre STN-DBS period, repeated measure ANOVA, 23 Session x STN-DBS x PPX interaction, F 2,122 = 1.334, p = .2672). From day one of 24 stimulation, STN-DBS induced a dramatic decrease of about 40% in instrumental responding 25 for the sucrose solution in both stimulated groups before pharmacological treatment (STN-26 DBS + Veh and STN-DBS + PPX) as compared with the pre STN-DBS levels (Figure 2A, 27 STN-DBS period, repeated measure ANOVA, STN-DBS effect: F 1,54 = 9.615, p = .0031; Figure S2A, two way ANOVA, STN-DBS effect: F 1,42 = 10.480, p = .002). PPX completely

221

  and locomotor activity then progressively decreased to a basal level during the last 2 periods 23 (20 to 60 min). Although it did not reach significance, STN-DBS tended to reduce ambulatory 24 activity during this phase. (Figure3A, STN-DBS effect, Two way ANOVA, 20-40 min: F 1,48 = 25 3.311, p = .075; 40-60 min: F 1,48 = 3.525, p = .067) This effect of STN-DBS is unlikely to 26 reflect a motor deficit but rather a decrease in general behavioral activity, because 27 ambulatory speed during this test phase was not changed by the stimulation (Figure3B, 28 STN-DBS effect, Two way ANOVA, F 1,48 = .196, p = .660). Moreover, fine motor skills of left and right limbs assessed using a stepping test were not affected by the stimulation, 2 confirming the absence of any motor deficit induced by chronic and bilateral STN-DBS 3 (Figure3C). As STN-DBS, PPX had no impact on fine motor skills (Figure3C, two way 4 ANOVA, STN-DBS x PPX interaction: left to right moves, left limb: F 1,55 = .110, p = .742; right 5 limb F 1,55 = .000442, p = .983; right to left moves, left limb: F 1,55 = .0135, p = .908; right limb: 6 F 1,55 = .0557, p = .814), but caused a significant increase in locomotor activity in the open 7 area test (Figure 3A, PPX effect, Two way ANOVA, 20-40 min: F 1,48 = 5.686, p = .021; 40-60 8 min: F 1,48 = 5.047, p = .030). Interestingly, the combined effect of the behavioral hypoactivity 9 induced by STN-DBS and hyperactivity due to PPX resulted in normalization of the general 10 activity during the last 2 phases of the open area test (Figure 3A).

  24 self-administration of sucrose as compared to non-lesioned animals (FigureS2A, two way 25 ANOVA, Lesion effect: F 1,42 = 26.592, p = < .001; FigureS2B, two way ANOVA, Lesion 26 effect: F 1,50 = 26.368, p = < .001) which was not reversed by PPX treatment alone (Figure27S2B, two way ANOVA, PPX effect: F 1,50 = .150, p = .701). Despite this important instrumental 28 deficit induced by the lesion, STN-DBS was still able to reduce operant behavior by about

17

  motors skills per se, first because neither PPX nor STN-DBS affected the ambulatory speed 18 during the open area test (Figure 5B, Two way ANOVA, STN-DBS x PPX interaction F 1,48 = 19 .214, p = .650) and second, adjustments during the stepping test were not impacted by either 20 treatment (Figure 5B, two ways ANOVA, STN-DBS x PPX interaction: left to right moves, left 21 limb: F 1,18 = .184, p = .673; right limb: F 1,18 = .653, p = .983; right to left moves, left limb: F 1,18 22 = .0017, p = .915; right limb: F 1,18 = .764, p = .393).

23

  Taken together, these results suggest that STN-DBS exacerbates the loss of 24 motivation induced by the dopaminergic lesion and reduce behavioral activity, effects that 25 were alleviated by PPX.

1

  demonstrated that the 6-OHDA lesion modifies D 3 R expression within the dorsolateral part of 2 the striatum 101 , local imbalance between pre and post synaptic D 2 R and/or D 3 R expression 3 or activity 102 could be responsible for DA transmission blunting, thus promoting this 4 hypoactivity. Very intriguingly, in some rats of both the control and lesioned groups, the 5 combination of STN-DBS and PPX induced a very high-level of activity that could emphasize 6 the importance of individual traits, as demonstrated for the expression or severity of impulse 7 control disorder in PD[103][START_REF] Favier | Pramipexole 11 reverses Parkinson's disease-related motivational deficits in rats[END_REF][105] or compulsive drug taking. 106 Thus, depending on complex 8 interactions with dopaminergic treatments and according to pre-existing individual traits, 9 STN-DBS could also lead to the development of hyperdopaminergic states reminiscent of 10 hypomanic behaviors also observed in PD patients under STN-DBS. 62, 74, 107, 108 11 Altogether, these data bring coherence to clinical observations that seemed 12 contradictory until today: apathy in PD patients, at least its motivational dimension, could be 13 induced by STN-DBS itself and attenuated by activation of D 2 R/D 3 R, regardless of (or in 14 addition to) DRT reduction and the DA-mediated hypofunction. More detailed molecular 15 analysis or techniques such as optogenetics could enable us to clarify the structures or sub-16 territories involved in this STN-DBS induced DA-driven loss of motivation. While it is beyond 17 the scope of the present study, it would be also of interest to further investigate the effect of 18 STN-DBS on the emotional and affective component of apathy, as STN has been proposed

1

  induce a state of negative affect, comparable to a certain extent to the blunted dopaminergic 2 transmission observed during prolonged drug consumption[117][118][119] , increasing the risk of 3 addictive behavior resurgence in the same way as drugs of abuse withdrawal syndrome 4 promotes relapse.120 5 Thus, this new insight calls for reconsideration of the role of STN-DBS on mood in PD 6 to improve patient care and quality of life, and more broadly, to rethink the use of STN-DBS 7 in psychiatry.

23

  This work was supported by the Institut National de la Santé et de la recherche Médicale, Fondation de France, Région Rhône-Alpes (Arc n°2), Neurodis, Grenoble Alpes Université, 4 CNRS, Aix-Marseille Université and France Parkinson. The authors declare no conflict of 5 interest.

6 7 8

 68 Acknowledgments We would like to thank Fiona Hemming for her critical reading, as well as Séverine Lirzin and 9 Olivier Mainard for the maintenance and their technical help concerning the micro-stimulator 10 system. 11 12 Author contributions 13 YV, SB, SC and MS were responsible for the conception and the design of the study. YV, 14 SB, CC and RM carried out the stereotaxic surgeries and the post-surgery monitoring of the 15 animals. YV, CC and SB performed the neuroanatomical analysis of electrode implantation 16 and lesions. YV and SB carried out the behavioral experiments and analysis. LKLG and PS 17 provided micro-stimulators as well as training and advice for their use. YV and SB wrote the 18 paper with the help of the other authors.

FIGURE 1 :

 1 Experimental design and electrode implantation.

8

  (A) Time course of the study, groups of animals and histological controls 9 After learning the sucrose self-administration task, lesion and implantation of electrodes, rats 10 were divided into a stimulated and a non-stimulated group according to their performances, 11 to constitute initially equal performance groups. Then, after 4 days of subthalamic nucleus 12 deep brain stimulation (STN-DBS) with further sucrose self-administration, pramipexole 13 (PPX) or vehicle (Veh) was injected twice a day into half of the stimulated (Non lesioned rats: 14 STN-DBS + PPX, n = 8; STN-DBS + Veh, n = 13; lesioned rats: Lesion + STN-DBS + PPX, n 15 = 5; Lesion + STN-DBS + Veh, n = 5) and non-stimulated rats (Control + PPX, n = 22; 16 Control + Veh, n = 22; Lesioned rats: Lesion + PPX, n = 4; Lesion + Veh, n = 6) for 20 days.

17

 17

  During this time, sucrose self-administration was continued for several days and then 18 followed successively by other behavioral tests: the two-bottle choice test, the stepping test 19 and finally the open area test. (B) Positions of bilateral DBS electrode tips in left and right 20 STNs of stimulated animals in representative coronal sections (anteroposterior relative to 21 Bregma: -3.60 mm; -3.80 mm; -4.16 mm), reproduced from Paxinos and Watson. 121 Each bar 22 represents the placement of one electrode. (C) and (D) Example of electrode trace within the 23 left STN stimulated at 100 μA (C) or 225 μA (D), after Cresyl violet staining. Scale bar: 1 mm.24 IC, Internal Capsule; LH, Lateral Hypothalamus; ZI, Zona Incerta.

25 ( 1 FIGURE 4 : 11 FIGURE 5 :

 2514115 E) and (F) Representative photomicrographs of coronal sections stained for tyrosine 26 hydroxylase (TH) in striatal (bottom, 1.6 mm to bregma) and mesencephalic (top, -5.6 mm to 27 bregma) regions from naïve (top) and lesioned (bottom) rats. 121 Scale bar = 1 mm. (G, H) 28 Quantification of TH ImmunoReactivity (IR) loss in the midbrain (G) and the striatum (H), 29 expressed as the percentage difference compared to the mean value obtained for sham 30 operated animals. The SNc bilateral lesion produced an important but partial denervation of STN-DBS exacerbates the deficit of preparatory behaviors of the existing 2 motivational deficit in a rodent model of the neuropsychiatric symptoms of PD. (A) Time 3 course of the sucrose self-administration experiment. STN-DBS decreased the number of 4 sucrose deliveries. PPX induced a complete reversion of this deficit. (B) Mean sucrose 5 deliveries over the last 5 days of experiment. (C) STN-DBS and PPX did not alter the 6 preference for sucrose in 6-OHDA rats. Lesion + Veh: n = 6; Lesion + PPX: n = 4; Lesion + 7 STN-DBS + Veh: n = 5; Lesion + STN-DBS + PPX: n = 5. Data shown as means  SEM. 8 Lesion + Veh versus Lesion + STN-DBS + Veh: *p < .05; Lesion + STN-DBS + Veh versus 9 Lesion + STN-DBS + PPX: # p < .05. 10 Subthalamic nucleus deep brain stimulation (STN-DBS) or pramipexole (PPX) 12 individually promote hypoactivity but do not impair motor abilities. (A) Total distance traveled 13 during a 1h open area test. STN-DBS induced a non-significant tendency towards decreased 14 basal locomotor activity. In non-stimulated rats, PPX induced a dramatic decrease in the 15 distance traveled. The combination of both treatments increased activity. (B) STN-DBS and 16 PPX did not change the mean ambulatory speed during the open area test. (C) STN-DBS 17 and/or PPX did not alter the fine motor skills of front limbs as demonstrated by adjusting 18 steps in the course of the stepping test. Lesion + Veh: n = 6; Lesion + PPX: n = 4; Lesion + 19 STN-DBS + Veh: n = 5; Lesion + STN-DBS + PPX: n = 5. Data shown as means  SEM. 20 Lesion + PPX versus Lesion + STN-DBS + PPX: *p < .05.Yvan Vachez, PhD 1,2 , Carole Carcenac, PhD 1,2 , Robin Magnard, PhD 1,2 , Lydia Kerkerian -Le Goff, PhD 3 , Pascal Salin, PhD 3 , Marc Savasta, PhD 1,2 , Sebastien Carnicella, PhD 1,2 and Sabrina Boulet, PhD[START_REF] Figure | PPX completely 2 rescued the self-administration performances of stimulated rats from the second day of 3 treatment, without bringing performances superior to baseline or to Control + Veh rat levels. 4 (Figure 2A, STN-DBS + PPX period, repeated measure ANOVA[END_REF]2

1 F2

 1 and Dr Marc Savasta INSERM U1216, Grenoble Institute of Neuroscience, Group "Pathophysiology of Motivation", Grenoble Université -Site Santé La Tronche -BP 170, 38042 Grenoble, France. Phone: 33 4 56 52 06 70; Email addresses: Sabrina.boulet@univ-grenoble-alpes.fr ; marc.savasta@inserm.fr Running title: STN-DBS and Apathy in Parkinson's Disease Relevant conflicts of interest/financial disclosures: Nothing to report. Number of characters in the title: 130 Number of characters in the running title: 41 Number of words in the Abstract: 250 Number of words in the body of the manuscript: 4387 Number of figures: 5 Subthalamic nucleus deep brain stimulation (STN-DBS) is a neurosurgical treatment

  and Apathy in Parkinson's Disease 4

3 9 10

 39 19 striatum (DS) by 6-hydroxydopamine (6-OHDA) lesion of SNc.20, 55 Because D 2 R and D 3 R 20 agonists, such as pramipexole (PPX), can alleviate pre and post STN-DBS apathy in PD 21 patients 18, 19, 56 as well as in preclinical models 20, 57 and because we reported that STN-DBS 22 reduces the level of D 2 R and D 3 R in the nucleus accumbens (NAc) of rats, 58 our working 23 hypothesis was that post-STN-DBS apathy may be due to an alteration of DA transmission 24 induced by STN-DBS itself. Thus, we also investigated the effect of STN-DBS with or without 25 chronic treatment with PPX. Experiments were performed on adult male Sprague-Dawley rats (Janvier, Le Genest-Saint-4 Isle, France) weighing approximately 350g (8 weeks old) at the time of surgery. Animals 5 were individually housed under standard laboratory conditions (12 h light/dark cycle, with 6 lights on at 7 am) with food and water available ad libitum during all the experimental 7 procedures. Protocols used complied with the European Union 2010 Animal Welfare Act and 8 the French directive 2010/63. Bilateral 6-OHDA lesions, bilateral implantation of electrodes and deep brain 11 stimulation of subthalamic nucleus 12 See supplemental informations 13 14 Experimental design 15 After self-administration training and both surgeries, rats were subjected to a sequence of 16 behavioral tests, with one resting day between the different tests (Figure1A). In each 17 experiment, all conditions were counter-balanced among the different test chambers and 18 each apparatus was thoroughly cleaned after each trial or session.

5

  and implantations.

6 7

 6 See supplemental data for full description of behavioral procedures, quantification of the 8 extent of the striatal DA denervation and control of electrode implantation.

9 10

 9 Data and statistical analysis 11 Data are shown as means  SEM and were analyzed by one or two-way ANOVAs, repeated 12 or not as specified in Results. Concerning operant sucrose self-administration, the different 13 experimental periods (Pre-STN-DBS, STN-DBS and STN-DBS + PPX) were analyzed 14 independently by distinct repeated measure ANOVAs for figures 2A and 4A. When indicated, 15 post hoc analyses were carried out with the Student Newman-Keuls procedure.

3 Figure

 3 Figure 1B provides the different electrode positions in left and right STN for the

17

 17

  18 STN-DBS induces a motivational deficit in normal rats that is reversed by the D2 R/D 3 R 19 agonist pramipexole 20 Before STN-DBS was switched ON, rats were trained for 10 days in the operant task 21 (only the last 3 days of training are represented in figure 2A). Groups were formed to have 22 equivalent performance levels (Figure 2A, Pre STN-DBS period, repeated measure ANOVA, 23 Session x STN-DBS x PPX interaction, F 2,122 = 1.334, p = .2672). From day one of 24 stimulation, STN-DBS induced a dramatic decrease of about 40% in instrumental responding 25 for the sucrose solution in both stimulated groups before pharmacological treatment (STN-26 DBS + Veh and STN-DBS + PPX) as compared with the pre STN-DBS levels (Figure 2A, 27 STN-DBS period, repeated measure ANOVA, STN-DBS effect: F 1,54 = 9.615, p = .0031;

  25 outcome. This approach has allowed us to demonstrate a clear effect of STN-DBS on 26 motivated behaviors during an operant task. The decrease of baseline ambulatory activity 27 detected during the open area test, unrelated to motor impairment, further suggests that the 28 STN-DBS-induced operant deficit may be considered as a decrease in the maintenance of

1

  induce a state of negative affect, comparable to a certain extent to the blunted dopaminergic 2 transmission observed during prolonged drug consumption[117][118][119] , increasing the risk of 3 addictive behavior resurgence in the same way as drugs of abuse withdrawal syndrome 4 promotes relapse.120 5 Thus, this new insight calls for reconsideration of the role of STN-DBS on mood in PD 6 to improve patient care and quality of life, and more broadly, to rethink the use of STN-DBS 7 in psychiatry.

23

  This work was supported by the Institut National de la Santé et de la recherche Médicale, Fondation de France, Région Rhône-Alpes (Arc n°2), Neurodis, Grenoble Alpes Université, 4 CNRS, Aix-Marseille Université and France Parkinson. The authors declare no conflict of 5 interest.

6 7 8

 68 Acknowledgments We would like to thank Fiona Hemming for her critical reading, as well as Séverine Lirzin and 9 Olivier Mainard for the maintenance and their technical help concerning the micro-stimulator 10 system. 11 12 Author contributions 13 YV, SB, SC and MS were responsible for the conception and the design of the study. YV, 14 SB, CC and RM carried out the stereotaxic surgeries and the post-surgery monitoring of the 15 animals. YV, CC and SB performed the neuroanatomical analysis of electrode implantation 16 and lesions. YV and SB carried out the behavioral experiments and analysis. LKLG and PS 17 provided micro-stimulators as well as training and advice for their use. YV and SB wrote the 18 paper with the help of the other authors.

FIGURE 1 :FIGURE 2 :

 12 FIGURE 1: Experimental design and electrode implantation. (A) Time course of the study, groups of animals and histological controls After learning the sucrose self-administration task, lesion and implantation of electrodes, rats were divided into a stimulated and a non-stimulated group according to their performances, to constitute initially equal performance groups. Then, after 4 days of subthalamic nucleus deep brain stimulation (STN-DBS) with further sucrose self-administration, pramipexole (PPX) or vehicle (Veh) was injected twice a day into half of the stimulated (Non lesioned rats: STN-DBS + PPX, n = 8; STN-DBS + Veh, n = 13; lesioned rats: Lesion + STN-DBS + PPX, n = 5; Lesion + STN-DBS + Veh, n = 5) and non-stimulated rats (Control + PPX, n = 22; Control + Veh, n = 22; Lesioned rats: Lesion + PPX, n = 4; Lesion + Veh, n = 6) for 20 days. During this time, sucrose self-administration was continued for several days and then followed successively by other behavioral tests: the two-bottle choice test, the stepping test and finally the open area test. (B) Positions of bilateral DBS electrode tips in left and right STNs of stimulated animals in representative coronal sections (anteroposterior relative to Bregma: -3.60 mm; -3.80 mm; -4.16 mm), reproduced from Paxinos and Watson.119 Each bar represents the placement of one electrode. (C) and (D) Example of electrode trace

FIGURE 3 :

 3 FIGURE 3: Subthalamic nucleus deep brain stimulation (STN-DBS) and pramipexole (PPX) promote hypo and hyper locomotor activity respectively but do not impair motor abilities. (A) Total distance travelled during a 1h open area test. STN-DBS induced a non-significant tendency towards decreased basal locomotor activity that was counteracted by treatment with pramipexole. In control rats, pramipexole induced a dramatic increase in the distance traveled. (B) STN-DBS and pramipexole did not change the mean ambulatory speed during the last 2 periods of the open area test. (C) STN-DBS and/or pramipexole did not alter the fine motor skills of front limbs as demonstrated by adjusting steps in the course of the stepping test. Control + Veh: n = 22; Control + PPX: n = 22; STN-DBS + Veh: n = 6; STN-DBS + PPX: n = 8. Data shown as means ± SEM. Control + PPX versus Control + Veh: *p < .05. 190x275mm (300 x 300 DPI)

FIGURE 4 :

 4 FIGURE 4: STN-DBS exacerbates the deficit of preparatory behaviors of the existing motivational deficit in a rodent model of the neuropsychiatric symptoms of PD. (A) Time course of the sucrose self-administration experiment. STN-DBS induced a decrease of the number of sucrose deliveries. Pramipexole induced a complete reversion of this deficit. (B) Mean sucrose deliveries over the last 5 days of experiment. (C) Subthalamic nucleus deep brain stimulation (STN-DBS) and pramipexole (PPX) do not alter the preference for sucrose in 6-OHDA rats. Lesion + Veh: n = 6; Lesion + PPX: n = 4; Lesion + STN-DBS + Veh: n = 5; Lesion + STN-DBS + PPX: n = 5. Data shown as means ± SEM. Lesion + Veh versus Lesion + STN-DBS + Veh: *p < .05; Lesion + STN-DBS + Veh versus Lesion + STN-DBS + PPX: #p < .05. 190x275mm (300 x 300 DPI)

FIGURE 5 :

 5 FIGURE 5: Subthalamic nucleus deep brain stimulation (STN-DBS) and pramipexole (PPX) individually promotes hypoactivity but do not impair motor abilities. (A) Total distance travelled during a 1h open area test. STN-DBS induced a non-significant tendency towards decreased basal locomotor activity. In nonstimulated rats, pramipexole induced a dramatic decrease in the distance traveled. The combination of both treatments increased activity. (B) STN-DBS and pramipexole did not change the mean ambulatory speed during the open area test. (C) STN-DBS and/or pramipexole did not alter the fine motor skills of front limbs as demonstrated by adjusting steps in the course of the stepping test. Lesion + Veh: n = 6; Lesion + PPX: n = 4; Lesion + STN-DBS + Veh: n = 5; Lesion + STN-DBS + PPX: n = 5. Data shown as means ± SEM. Lesion + PPX versus Lesion + STN-DBS + PPX: *p < .05. 190x275mm (300 x 300 DPI)

FIGURE S1 :FIGURE S2 :

 S1S2 FIGURE S1: Example of electrode trace and tissue state in animals stimulated at 100 μA (A) or 225 μA (B), after Cresyl violet staining. Scale bar: 1 mm. 190x275mm (150 x 150 DPI)

  Supplementary information accompanies this paper.

	F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F F o r o r P e P e e r e r R e R v e v i e i w e w
	22
	23
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	25

  [START_REF] Figure | PPX completely 2 rescued the self-administration performances of stimulated rats from the second day of 3 treatment, without bringing performances superior to baseline or to Control + Veh rat levels. 4 (Figure 2A, STN-DBS + PPX period, repeated measure ANOVA[END_REF]2 and Sabrina Boulet, Veh: n = 6; Lesion PPX: n = 5. Control Veh versus Lesion Veh: ### p < .001; Control PPX 2 versus Lesion PPX: ## p < .01.

		Movement Disorders	Page 66 of 68
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		F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F F o r o r P e P e e r e r R e R v e v i e i w e w
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		John Wiley & Sons

1


Rongrong Liu
email: rongrong.liu@unistra.fr

Florent Nageotte
email: nageotte@unistra.fr

Philippe Zanne
email: zanne.philippe@unistra.fr

Michel De Mathelin
email: demathelin@unistra.fr

Birgitta Dresp- Langley
email: birgitta.dresp@unistra.fr

Wearable wireless biosensors for spatiotemporal grip force profiling in real time

Keywords: wearable sensor technology, human grip force, functional analysis, image guided surgical training, simulator task, robot assistance, spatio-temporal skill profiling, human-robot interaction, real time

The temporal evolution of individual grip force profiles of a novice using a robotic system for minimally invasive endoscopic surgery is analyzed on the basis of thousands of individual sensor data recorded in real time through a wearable wireless sensor glove system. The spatio-temporal grip force profiles from specific sensor locations in the dominant hand performing a four-step pick-and-drop simulator task reveal skill-relevant differences in force deployment by the small finger (fine grip force control) and the middle finger (gross grip force contribution) by comparison with the profiles of a highly proficient expert. Cross-disciplinary insight from systems neuroscience, cognitive behavioral science, and robotics, with implications for biologically inspired AI for human-robot interaction, highlight the functional significance of spatio-temporal grip force profiling.

Introduction

Wearable wireless sensor systems are currently developed for the monitoring of health data, exercise activities, and other performance data. Unlike conventional approaches, such devices enable the convenient, continuous, and unobtrusive monitoring of a user's behavioral signals in real time. In previous work [START_REF] Batmaz | Novice and expert behavior while using a robot controlled surgery system[END_REF][START_REF] De Mathelin | Sensors for Expert Grip Force Profiling: Towards Benchmarking Manual Control of a Robotic Device for Surgical Tool Movements[END_REF], we have used a wireless sensor glove system for human-robot interaction within a system prototype designed for minimally invasive endoscopic surgery. Functionally motivated analyses of thousands of human grip force data, collected from various sensor locations in the dominant and non-dominant hands of experts, trainees, and novice surgeons in image-guided task simulations, have shown specific differences between the grip force profiles of individual users as a function of task skill level and expertise in using the robotic system. Experts and non-experts employ different grip-force strategies reflected by differences in total amount of grip force deployed by the same fingers of the same hand. Here in this work, we show further functional analyses of thousands of individual grip force data leading the way towards task-specific grip force monitoring in real time, as clarified in the discussion further below. Task specific sensor data from a complete novice and a highly proficient expert user are exploited here in terms of spatio-temporal variations in average grip force, computed on the basis of data recorded from three task-relevant sensor locations in the dominant hand: 1) the middle phalanx of the middle finger, which mostly contributes to gross force deployment [START_REF] Vm | Multifinger prehension: an overview[END_REF][START_REF] Kinoshita | Grip posture and forces during holding cylindrical objects with circular grips[END_REF][START_REF] Kinoshita | Contributions and co-ordination of individual fingers in multiple finger prehension[END_REF], necessary for lifting weighty objects but useless for skillful execution of the robotic precision task here, 2) the middle phalanx of the small finger, which allows for fine grip force control [START_REF] Vm | Multifinger prehension: an overview[END_REF][START_REF] Kinoshita | Grip posture and forces during holding cylindrical objects with circular grips[END_REF][START_REF] Kinoshita | Contributions and co-ordination of individual fingers in multiple finger prehension[END_REF][START_REF] Ml | Multi-finger prehension: control of a redundant mechanical system[END_REF], critically important in subtle precision grip tasks like the one studied here, and 3) the middle phalanx of the ring finger, which is among the least important in grip force control across a variety of tasks and weights lifted, as discussed previously [START_REF] Vm | Multifinger prehension: an overview[END_REF][START_REF] Kinoshita | Grip posture and forces during holding cylindrical objects with circular grips[END_REF][START_REF] Kinoshita | Contributions and co-ordination of individual fingers in multiple finger prehension[END_REF][START_REF] Ml | Multi-finger prehension: control of a redundant mechanical system[END_REF]. The spatio-temporal grip force profiles of the novice and the expert are compared across ten successive task sessions for repeated execution of the pick-and-drop robotic simulator task with four critical steps:

1. Activate and move tool towards object location; requires movement ahead in depth along a virtual z-axis in 2D image plane 2. Open and close tool-tip grippers to grasp and lift object 3. Move tool with object to target location; requires lateral movement along x-axis of 2D image plane 4. Open tool-tip grippers to drop object in box.

To accomplish these steps in minimal task time and with maximal precision (i.e. no tool trajectory corrections, no incidents) requires skillful manipulation of the task-relevant (left or right) handle of the robotic system (see Materials and Methods). The novice's dominant hand is the right hand, the expert's dominant hand is the left hand.

Materials and Methods

The robotic system is designed for bi-manual endoscopic surgical interventions; the simulator task from this study here solicits only one, in the present case the dominant hand.

Slave Robotic System

The slave robotic system is built on the Anubis® platform of Karl Storz and consists of three flexible, cable-driven sub-systems for robot-assisted endoscopic surgery, described in detail in our previous work [START_REF] Batmaz | Novice and expert behavior while using a robot controlled surgery system[END_REF] available at: https://www.mdpi.com/1424-8220/19/20/4575/htm.

Master/Slave Control

The slave robot is controlled at the joint level by a position loop running at 1000 Hz on a central controller. The master side consists of two specially designed interfaces, which are passive mobile mechanical systems. Each of the two handles has 3 DoF, which translate for controlling instrument insertion, rotate around a horizontal axis for controlling instrument rotation, and rotate around a final axis (moving with the previous DoF) for controlling instrument bending. Each handle is also equipped with a trigger and with a small four-way joystick for controlling additional DoF. In the experiments here, the trigger is operated with the index finger of a given hand for controlling grasper opening and closing. A high-level controller running in real-time Linux OS communicates with the master interfaces and provides reference positions to the slave central controller. The user sits in front of the master console and looks at the endoscopic camera view displayed on the screen in front of him/her at a distance of about 80 cm while holding the two master handles, which are about 50 cm away from each other. Seat and screen heights are adjustable to optimal individual comfort.

The two master interfaces are identical and the two slave instruments they control are also identical. Therefore, for a given task the same movements need to be produced, whatever hand the user is instructed to use (left or right). The master interfaces are statically balanced and all joints exhibit low friction; therefore, only minimal forces are required to produce significant movement in any direction. A snapshot view of a user wearing the sensor gloves while manipulating the system is shown in Figure 1a; Figure 1b shows directions and types of tool-tip and control movements.

Sensor glove design

The robotic system having its own grip style design, a specific wearable sensor glove system with inbuilt Force Sensitive Resistors (FSR)was developed, one for each hand.

Hardware

The gloves designed for the study each contain twelve FSR, in contact with twelve specific locations on the inner surface of the hand [START_REF] Batmaz | Novice and expert behavior while using a robot controlled surgery system[END_REF][START_REF] De Mathelin | Sensors for Expert Grip Force Profiling: Towards Benchmarking Manual Control of a Robotic Device for Surgical Tool Movements[END_REF], including those three for which data are shown here. Two layers of cloth were used and the FSR were inserted between the layers. The FSR were sewn to the cloth around the conducting surfaces (active areas) and did not interact, neither directly with the skin of the subject, nor with the master handles, which provided a comfortable feel when manipulating the system. FSR with a needle and thread. The electrical connections of the sensors are individually routed to the dorsal side of the hand and brought to a soft ribbon cable, connected to a small and very light electrical casing, strapped onto the upper part of the forearm and equipped with an Arduino microcontroller. Each FSR was soldered to 10KΩ pulldown resistors to create a voltage divider, and the voltage read by the analog input of the Arduino is given by (1)

= 3.3/( + ) (1)
where is the resistance of the pull down resistor, is the FSR resistance, and 3.3 is the 3.3 V supply voltage. FSR resistances can vary from 250Ω when subject to 20 Newton (N) to more than 10MΩ when no force is applied at all. The generated voltage varies monotonically between 0 and 3.22 Volt, as a function of the force applied, which is assumed uniform on the sensor surface. In the experiments here, forces applied did not exceed 10N, and voltages varied within the range of [0; 1500] mV. The relation between force and voltage is almost linear within this range. It was ensured that all sensors havesimilar calibration curves. Thus, all following comparisons are directly between voltage levels at the millivolt (mV) scale. Regulated 3.3V is provided to the sensors from the Arduino, and power is provided by a 4.2V Li-Po battery enabling wireless use of the glove system. The battery voltage level is controlled during the whole duration of the experiments by the Arduino, and displayed continuously on the user interface. The glove system is connected to a computer for data storage via Bluetooth-enabled wireless communication running 115200 bits-per-second (bps).

Software

The software of the glove system is divided into two parts: one running on the gloves, and one running on the computer algorithm for data collection. Each of the two gloves is sending data to the computer separately, and the software reads the input values and stores them on the computer according to header values indicating their origin. The software running on the Arduino is designed to acquire analog voltages provided by the FSR every 20 milliseconds (msec) at a 50 Hz rate. In every loop, input voltages are merged with their time stamps and sensor identification. This data package is sent to the computer via Bluetooth.

Results

The raw data from three (see Table 1) of the twelve sensor locations in the dominant hands of the novice and the expert were analyzed. Statistical comparison (2-Way ANOVA) between the original raw data of expert and novice from their first and last task sessions reveal significant interactions between the 'expertise' (2 levels) and 'session' (2 levels) factors for all three sensors considered here (S5, S6, S7). For sensor S5 on the middle finger (gross grip force deployment), the mean (m) grip forces and their standard errors (sem) from the first session yield m=241mV /sem=4.3 for the expert and m=790mV/sem=2.7 for the novice, showing that the latter deploys about three times as much unnecessary gross grip force by comparison with the expert. This expertise-specific difference in proportional gross grip force deployed by the middle finger is even larger in the last session, with m=78mV/sem=4.9 for the expert, and m=640mV/sem=3.6 for the novice. The interaction between the 'expertise' and 'session' factors for sensor S5 is significant with F(1,2880)=28.65; p<.001. For sensor S6 on the ring finger (no meaningful role in grip force control), the differences between the grip force profiles of novice and expert are minimal, as would be expected, in the first session with m=576mV /sem=3.8 for the expert and m=504mV/sem=2.4 for the novice, and in the last session with m=474mV /sem=4.5 for the expert and m=445mV/sem=3.3 for the novice. The interaction between the 'expertise' and 'session' factors for sensor S6 is, however, significant with F(1,2880)=35.86; p<.001, which is explained by the fact that grip forces, i.e. amplitudes in mV, diminish in both users from the first to the last session, but not by the same amounts. For sensor S7 on the small finger (critically important for fine grip force control), the expertise-specific difference between the two user profiles is characterized by the novice deploying largely insufficient grip forces, from the first session with with m=98mV /sem=1.2 to the last with m=78mV/sem=1.6, while the expert produces sufficient grip force for fine movement control from the first session with m=594mV /sem=1.8 to the last with m=609mV/sem=2.2. The interaction between the 'expertise' and 'session' factors for sensor S7 is highly significant with F( 1,2880)=188.53; p<.001. Task times are considerably shorter in the expert (Figure 2), with 10.4 sec in the first session and 7.6 sec in the last indicating a minor practice effect. The novice takes more than twice as long (26 sec) in the first session compared with the expert, with a 30% time gain in the last session (18.8 sec), indicating a temporal training effect. Concerning incidents (trajectory adjustments, grip failures, drop misses), the task videos reveal a total number of 20 in the novice, and only three small trajectory adjustments in the expert's performance.

Discussion

The data reveal expertise-specific differences in the spatio-temporal grip force profiles of an expert and a novice repeatedly performing a 2D image-guided robot-assisted precision task. These differences can be functionally assessed under the light of previous work [START_REF] Vm | Multifinger prehension: an overview[END_REF][START_REF] Kinoshita | Grip posture and forces during holding cylindrical objects with circular grips[END_REF][START_REF] Kinoshita | Contributions and co-ordination of individual fingers in multiple finger prehension[END_REF][START_REF] Ml | Multi-finger prehension: control of a redundant mechanical system[END_REF] on the role of finger grip forces and prehensile synergies, centrally controlled in the human brain for optimizing human motor performance and control. One major difference as a function of task expertise or skill concerns proportional gross grip force deployed by the middle finger, with the novice deploying way too much unnecessary, task-irrelevant gross grip force, while the expert has learnt to skillfully minimize those. Another functionally important, expertise-specific difference concerns precision grip force control by the small finger, critically important in surgical and other precision tasks. Here, the difference between the two users is characterized by the novice deploying insufficient grip forces, with no major evolution between the first and the last task sessions. Concerning grip forces deployed by the ring finger, which plays no major or meaningful role in grip force control, differences between grip force profiles of novice and expert are, indeed and as would be expected, minimal and do not change much across sessions although total grip force magnitudes (mV amplitude) diminishes across sessions in both users. At the beginning, the novice takes more than twice as long performing the precision task by comparison with the expert, but at the end scores a 30% time gain indicating a considerable temporal training effect, especially in the first critical task step, as indicated by the pink boxes in Figure 2. This is readily explained by the fact that the first task step is the most difficult for a novice. It consists of moving the tool along a virtual trajectory towards the object location, and requires movement away from the body in the surgeon's peri-personal space. This implies the perceptual recovery of physically missing depth information along a virtual z-axis in the 2D image plane. Such difficulty results in longer task times and imprecise tool-movements [START_REF] Batmaz | Getting nowhere fast: Trade-off between speed and precision in training to execute image-guided hand-tool movements[END_REF][START_REF] Batmaz | Seeing virtual while acting real: Visual display and strategy effects on the time and precision of eye-hand coordination[END_REF][START_REF] Batmaz | Effects of 2D and 3D image views on hand movement trajectories in the surgeon's peri-personal space in a computer controlled simulator environment[END_REF][START_REF] Dresp-Langley | Towards Expert-Based Speed-Precision Control in Early Simulator Training for Novice Surgeons[END_REF], explaining why the largest training gain in total task time of the novice is observed for task step 1. Finally, the analyses shown here can be run in real-time to monitor manual/bimanual precision tasks, control performance quality, or to prevent risks in robot assisted surgery for systems where excessive grip forces can cause tissue damage [START_REF] Abiri | Multi-Modal Haptic Feedback for Grip Force Reduction in Robotic Surgery[END_REF], which does not apply to this system here. The human hand has evolved as a function of active constraints in harmony with other sensory systems [START_REF] Johansson | Sensory-motor coordination during grasping and manipulative actions[END_REF][START_REF] Dresp-Langley | Combining Visual Contrast Information with Sound Can Produce Faster Decisions[END_REF]. Grip force profiles are a direct reflection of the complex cognitive and behavioral synergies this evolution has produced.

Figure 1 .

 1 Figure 1. Expert wearing the sensor gloves while manipulating the robotic master/slave systema). Direction and type of tool-tip and control movements -b).

Figure 2

 2 shows these profiles for the first and the last individual sessions for sensors 5, 6, and 7.

Figure 2 .

 2 Figure 2. Individual grip force profiles showing average peak amplitudes (mV) from sensors 5, 6, and 7 for fixed successive temporal windows of 2000 milliseconds in a given session, for the first and last of ten sessions of the expert and the novice. Relative durations of each of the four critical task steps within a given session are highlighted by the colored squares.

Table 1 :

 1 Task-relevant sensor locations (dominant hand) from which the spatio-temporal grip force profiles shown here were drawn

	Sensor	Finger	Anatomical reference
	S5	Middle	Middle Phalanx
	S6	Ring	Middle Phalanx
	S7	Pinky	Middle Phalanx

Individual temporal grip force profiles were plotted in terms of average peak amplitudes (AmV) for fixed successive temporal windows of 2000 msec in a given individual session. With one signal per 20 msec and 100 signals per time window of 2000 msec, we have AmV=mVtotal/100, which is the total sum of mV recorded in the time window given divided by the total number of signals in the time window.

Acknowledgments: Material support by CNRS is gratefully acknowledged.

Conflicts of Interest:

The authors declare no conflict of interest.

Funding: This ongoing work is part of a project funded by the University of Strasbourg's Initiative D'EXellence (IDEX).