and the respective search hits were checked. Titles and abstracts related to the retrieved hits identified with the combined search were then checked manually by two examiners independently (CSL and MG, see below). In order to detect published conference and meeting abstracts edited in supplements not available in PubMed, a second independent search was carried out in Web of Science with the above mentioned search terms and then cross-checked. Because of space limitations, only the PubMed search results are shown in the **Figure 1**.

Inclusion criteria:

Exclusion criteria:

Titles and abstracts of the articles retrieved from the combined search were checked for the presence of data of relevant topics (see above). Only articles addressing weakness or paralysis affecting limbs (often also accompanied by loss or reduction of sensory feeling) were considered. Two examiners (CSL and MG) then searched the retrieved titles and abstracts by hand and independently. After retrieval of abstracts fulfilling inclusioncriteria, full text versions of all identified articles were obtained. A cross check of the references from retrieved articles was performed to identify related publications not listed in the examined databases. Data were extracted independently by the two authors (CSL and MG). The two data bases were compared manually and then examined again by both reviewers. Discrepancies were corrected by reference to the original papers.

# Retrospective Reconstruction of the Clinical Global Impression - Improvement (CGI-I) Score

From all selected articles, the manuscript content was checked for clinical descriptions of symptom severity before and after treatment. The narratives were then used to determine an approximate change of FW symptoms, using the principles of the CGI-I scale for a nominal CGI-I score. Narratives were checked independently by two examiners (CSL and MG) and a nominal CGI-I score was established. In case of discrepancies, a consensus decision was reached between the two examiners. The CGI-I score is a 7-point scale which is commonly used to describe changes of a patient's clinical overall improvement related to a specific treatment. It was developed for use in NIMH (National Institute of Mental Health)-sponsored clinical trials to provide a brief, stand-alone assessment of the clinician's view of the patient's global functioning prior to and after initiating a study (Busner and Targum, 2007). CGI-I comprises the following categories: 1 = very much improved; 2 = much improved; 3 = minimally improved; 4 = no change; 5 = minimally worse; 6 = much worse; 7 = very much worse. If single data were available a nominal CGI-I was estimated for each patient reported (case reports and case series). In case of studies not reporting single data, a nominal CGI-I was calculated for the group of patients treated with a certain NIBS method.

### RESULTS

On December 15, 2015, our literature search resulted in the following numbers of hits related to the different search terms: "conversion disorder" n = 2220, "motor conversion disorder" n = 293, "conversion weakness" n = 174, "conversion paralysis" n = 21, "dissociative motor symptoms" n = 184, "dissociative weakness" n = 27, "dissociative paralysis" n = 2, "hysterical conversion" n = 86, "hysterical weakness" n = 19, "hysterical paralysis" n = 49, "hysterical paraplegia" n = 15, "functional weakness" n = 52, "functional paralysis" n = 27, "functional neurological symptoms" n = 33, "medically unexplained neurological symptoms" n = 185, "non-organic weakness" n = 9, "non-organic paralysis" n = 9, "psychogenic weakness" n = 4, "psychogenic paralysis" n = 25. All in all, the literature search retrieved 3141 hits. The combined term search led to the results shown in the **Figure 1**. The search performed in Web of Science allowed the identification of one meeting abstract (Kresojevic et al., 2010) that was not identified using the PubMed database. We could not retrieve any other relevant publications using Web of Science, which were not identified using PubMed. Two articles reported the same patients and therefore had to be excluded (Schönfeldt-Lecuona et al., 2003; Broersma et al., 2013).

#### Electroconvulsive Therapy in FW

We identified n = 2 articles in which ECT was performed in FW (**Table 2**). In both peer-reviewed articles, a single case was reported (Giovanoli, 1988; Gaillard et al., 2012).

#### Case Description

Giovanoli (1988) presented a 61-year old man with a complete right hand paralysis after superficial laceration of the middle finger, 11 months duration prior to ECT. Bilateral ECT (Medcraft B-24) was performed on an outpatient basis 3-times per week for 2 weeks, then twice weekly for 6 weeks (ECT parameters not available). Within the first 10 ECT sessions, a progressive change in color and a decrease in swelling were observed; after the 10th ECT session, approximation of thumb and index finger was possible; after the 19th ECT edema had disappeared and the hand exhibited a full range of motion. One week after completion of

#### TABLE 2 | Electroconvulsive Therapy (ECT) and Transcranial Magnetic Stimulation (TMS) in functional weakness or paralysis (FW).

pat., patient; F8c, figure-8-coil; Cc, Circular coil; mv, mean value; M1, motor cortex; MT, motor threshold intensity; Fr., Frequency; I, Intensity; d, day; s, seconds; (ns), no specified; PMD, psychogenic movement disorder; CCI, clinician's clinical impression; CGI-I, Clinical global impression scale-improvement item: 1 = very much improved; 2 = much improved; 3 = minimally improved; 4 = no change; 5 = minimally worse; 6 = much worse; 7 = very much worse.

ECT fine motor function of the patient's fingers was restored (he was able to button his shirt, tie his shoes and write). At 6 months follow-up and after 1 year, the patient did not use his hand during the examination but it was completely normal in appearance. No specific ECT parameters were stated. CGI-I after ECT was rated: 2 (much improved). The long-term CGI-I was rated: 3 (minimally improved).

Gaillard et al. (2012) presented a 33-year old man with fluctuating quadriplegia, developed 3 years prior to ECT. In total, 35 ECT sessions were performed, initially 2–3 ECT treatments per week. Subsequently, ECTs were performed less frequently (first once per week; then once a fortnight) in order to train motor skills and maintain mobility. Until the ninth ECT the progress in muscular activity allowed the patient to perform movements with increasing complexity. Progress continued with the patient gaining progressively more function, being able to eat without help, and managing all activities of daily living in the perimeter of his room with little help. Until the 25th ECT he was able to walk without help. Relapse occurred after a while, with great symptom fluctuation, dependent on circumstances, but muscular activity remained better than at admission. ECT was performed with increasing intensity until a maximum of 1152 mC in order to reach a seizure of at least 30–40 s. CGI-I after ECT was rated: 2 (much improved). The long term nominal CGI-I was rated: 3 (minimally improved).

### Repetitive Transcranial Magnetic Stimulation (All Variants)

We identified five articles in that TMS/rTMS was performed in FW affecting limb(s) (**Table 2** for characteristics) No articles were identified reporting theta-burst TMS for the treatment of FW. Four articles were published in peer-review journals (Schönfeldt-Lecuona et al., 2006; Chastan and Parain, 2010; Gaillard et al., 2012; Broersma et al., 2015), and a fifth article was retrieved from a conference abstract (Kresojevic et al., 2010). Three articles reported single patients (case reports or case series); only two articles included a larger sample [n = 70 in Chastan et al. (Chastan and Parain, 2010) and n = 11 in Broersma et al. (2015)].

#### Case Description

The study by Schönfeldt-Lecuona et al. (2006) had a prospective design and a clearly defined stimulation protocol based on a biological and functional-anatomical etiological hypothesis (see Discussion). This open-label, non-placebo controlled trial reported four patients (3 FW, 1 malingerer). Patients received 2 weeks (5-sessions/week) of rTMS in supra-threshold intensity [110% resting motor threshold (MT)] over the contra-lateral motor cortex to the paralyzed limb with a focal figure-8 coil and 4000 stimuli per day. Thereafter, once the patient started to independently perform own movements of the fingers, subthreshold rTMS (90% MT) was continued for 4–12 weeks with otherwise the same parameters depending on clinical needs. In all three FW patients rTMS caused a marked amelioration of symptoms over time that was sustained at 1-year follow-up. The estimated nominal group-CGI-I after rTMS was rated (for the 3 FW patients): 2 (much improved). The long term CGI-I was

Gaillard et al. (2012) described the case of a 33-year old man who had developed a quadriplegia (and anasthesia, December 2004) 6 months before admission. rTMS was performed at 1 Hz frequency (intensity and coil type not stated) targeting the motor cortex, right and left, over the arm-hand area, and right and left over the legs' cortical motor area (1000 pulses over each region) with a total of 4000 pulses per day. Initially, treatment was applied five times a week for 8 weeks, and thereafter twice in a week. The authors reported marked, progressive symptom amelioration, so that he was able to walk again. A further deterioration led to a new rTMS treatment leading again to symptom amelioration (not otherwise specified), but the patient was then mobile only with a wheelchair. A third deterioration led to phlebitis, pulmonary embolism and pressure soars, and ECT was performed (the ECT performed in this case was illustrated above). CGI-I after the first rTMS series was rated: 2 (much improved). The long term CGI-I was rated: 4 (unchanged in relation to rTMS beginning).

Kresojevic et al. (2010) presented two cases treated with rTMS. One of them (24-year old man) was suffering from a FW (hemiparesis). This patient was treated in a single session with 12 single pulses using a round coil (at initially 30% maximal stimulator output intensity and increasing intensity in 10% steps up to a maximum of 80% stimulator output) over the vertex. The response to rTMS was stated as immediate ("the patient was able to walk again independently"). At 6-months follow-up, a partial deterioration occurred, but he was still able to walk and minor walking difficulties did not influence his daily activities. The patients' CGI-I after TMS was rated: 2 (much improved). The long term CGI-I after 6 months follow-up was rated: 3 (minimally improved).

Chastan et al. (Chastan and Parain, 2010) presented a retrospective analysis of medical records of 70 FW patients (26 male), who had received TMS. Fifty-seven percent of the patients had paraplegia, 37% had a monoplegia, 3% had a tetraplegia, and 3% a hemiplegia. The stimulation protocol was variable. The TMS was principally used for routine diagnostic purposes in each patient. An average of 30 pulses were delivered at about 0.2–0.25 Hz with a circular coil and an intensity of 100% of maximum stimulator output over the motor cortex ("opposite the correspondence paralysis or on both sides for bilateral paralysis," not otherwise specified). Another session of 30 pulses was sometimes added a few minutes later in case of incomplete improvement. TMS was very effective in 62 patients, with a dramatic improvement in nine, a total recovery in 53 (immediately in 43 patients, within minutes or hours in eight patients, within days in two patients), mild improvement in five, and no effect in three patients. Acute onset of FW was associated with a better outcome (but not age, gender or co-morbid psychiatric disorder). CGI-I after TMS was rated for each reported patient group: 1 (very much improved) for the majority of the FW patients (n = 53; 76%); 2 (much improved) for nine patients (13%); 3 (minimally improved) for five patients (7%); and 4 (unchanged) for three patients (4%). Five to six months after TMS, recurrence of FW occurred in eight patients, six of whom were re-stimulated and responded to

TMS. There was not sufficient information to calculate long-term CGI-I.

Broersma et al. (2015) presented the first study using a placebo-controlled cross-over design and reported 11 patients with FW with at least a flaccid hand paralysis treated with rTMS. Based on the stimulation parameters proposed by Schönfeldt-Lecuona et al. (2006), active rTMS was delivered with a figureof-8 coil at 15 Hz over the motor cortex contralateral to hand paralysis (targeting being guided under neuro-navigation in seven patients) during 30 min once a day, for a total of 10 working days within 2 weeks (**Table 2** for detailed parameters). The placebo condition consisted of small electrical currents applied with a real electromagnetic device (REMP) placed in front of the magnetic coil at otherwise identical parameters. In the study design, the authors attempted to exclude any other additional therapeutic influences that could result from suggestion or afferent feedback due to rTMS-related suprathreshold muscle contraction. To achieve this goal the authors performed the active condition at an intensity of 80% of MT, and the communication with the patients was limited as much as possible. The stimulation condition switched between active and sham after the first 2 weeks of stimulation with a wash-out phase of at least 2 months between both conditions. Because of dropouts, 11 patients received active rTMS and only eight patients received sham rTMS. The primary outcome measure was an objective change in muscle strength as measured by dynamometry after treatment. The secondary outcome measure was the subjective change in muscle strength after treatment. In patients who received both treatments, active rTMS induced a significantly larger median increase in objectively measured muscle strength (24%) compared to sham rTMS (6%). Eight out of 11 patients receiving active rTMS showed an improvement of at least 20% of muscle strength. However, subjective ratings showed no statistical difference between treatments, i.e., patients did not really perceive these objectively measured motor improvements. As the patients' muscle strength improved, the authors suggested that rTMS alone could potentially improve muscle weakness in FW. However, patients did not report subjective improved functioning of the affected hand, which Broersma et al. interpreted as an indication that decreased muscle strength is not the core symptom in FW. They thus propose that rTMS should be applied as add-on therapy to behavioral approaches in FW. There was not sufficient information to calculate nominal CGI-I scores.

#### Nominal CGI-I Score Reconstruction

For patients treated with rTMS we retrieved sufficient information from the physician-estimated functional changes reported in the manuscripts by Schönfeldt-Lecuona et al. (2006) (n = 3), Chastan et al. (n = 70) (Chastan and Parain, 2010), Kresojevic et al. (2010) (n = 1), and Gaillard et al. (2012) (n = 1) that allowed the assessment of 75 FW patients. In the study by Broersma et al. (2015) the main outcome parameter was muscular strength changes assessed by dynamometer. The authors reported that patients were assessed neurologically for sensory deficits, coordination, reflexes and muscle strength at the beginning and end of rTMS treatment. However, the narratives provided in the paper did not allow an estimation of nominal CGI-I scores. Therefore, these patients (n = 11) were excluded from the analysis. For patients treated with ECT we retrieved information from the narratives in the articles by Giovanoli (1988) and Gaillard et al. (2012) that allowed us to assess the two patients reported.

For the patients treated with rTMS the estimated scores showed a satisfactory improvement at the short-term: nominal CGI-I scores were 1 (very much improvement) in 54 of 75 patients (72%) and 2 (much improvement) in 13 patients (17%). Only five of the treated patients (7%) improved minimally (CGI-I = 3), and 3 (5%) remained unchanged (CGI-I = 4). Overall, about 88% of these patients improved markedly (very much or much improvement) after stimulations. In no case did patients worsen in relation to rTMS treatment, and no serious adverse event was reported. A long-term CGI-I could not be estimated for the largest study by Chastan et al. (n = 70) (Chastan and Parain, 2010). FW symptoms recurred in eight patients 5–6 month after rTMS. In 62 patients treatment seem to have caused some amelioration compared to baseline (not stated). In n = 3 cases by Schönfeldt-Lecuona et al. follow-up CGI-I showed a sustained amelioration (Schönfeldt-Lecuona et al., 2006), while in the case by Gaillard the estimated long-term CGI-I was rated 4 (Gaillard et al., 2012). Patients treated with ECT showed a satisfactory response at short-term follow-up as well (ranging CGI-I = 2), but a deterioration of FW symptoms at long-term follow-up (ranging CGI-I = 3 in both cases; **Table 2**).

### DISCUSSION

# Discussion of Literature Search Results

We concentrated our search exclusively on limb weakness and limb paralysis, since other forms of FNS (such as impaired coordination or balance, dystonia, tremor, myoclonus, fainting, tics, hemiballismus, chorea, parkinsonism, bizarre gait, astasia, abasia, aphonia, swallowing difficulty, urinary retention, loss of touch sensation, double vision, blindness, and deafness) might have a different neurobiological etiology and probably other functional-anatomical correlates (Ejareh Dar and Kanaan, 2016). For this reason, we speculate that differential effects of NIBS methods might come into play when treating different forms of FNS.

The literature search identified two case reports with ECT as treatment for FW (Giovanoli, 1988; Gaillard et al., 2012), five articles with TMS (Schönfeldt-Lecuona et al., 2006; Chastan and Parain, 2010; Kresojevic et al., 2010; Gaillard et al., 2012; Broersma et al., 2015), and none for tDCS, with a total of 86 patients. All identified cases and studies reported a shortterm symptom improvement. However, any assumption of a beneficial effect of NIBS in FW has to be seen with caution, as the supporting literature is very sparse and the quality of the small number of identified articles was poor. Major concerns when examining the efficacy of NIBS in FW include the heterogeneity of studies with regard to design and stimulation parameters (paragraph below for more information), the absence of randomized controlled conditions in all but one trial, and the fact that the current literature does not allow a meta-analysis of

outcome data. Most of the included studies were case reports or case series (5 out of 7).

#### Study Designs, Parameters, and Outcomes

For TMS, only one study by Broersma et al. used a prospective, placebo-controlled, cross-over design with an objectively measured outcome using a dynamometer (Broersma et al., 2015). All other identified trials had no sham condition and only used an unstructured physician oriented clinical impression as outcome measure. The study with the hitherto largest sample of 70 patients by Chastan et al. (Chastan and Parain, 2010) was based on a retrospective sample analysis. Moreover, patients were mostly stimulated for diagnostic purposes and about 60% of the patients were children or adolescents. In all studies, FW symptoms and illness duration of the reported patients were heterogeneous and data were insufficient for a retrospective re-analysis, which would have allowed symptom clustering and meta-analysis. In the five identified articles (Schönfeldt-Lecuona et al., 2006; Chastan and Parain, 2010; Kresojevic et al., 2010; Gaillard et al., 2012; Broersma et al., 2015) no detailed information was presented regarding the way patients were informed and the treatments explained. The magnitude of the effect of the explanatory model could therefore not be estimated. The therapeutic effect of the active rTMS in Broersma et al. (2015) was smaller than the one reported by others; the mean increase of muscular strength was only about 20% (dynamometer), but there was no subjective amelioration of symptoms. The stimulation intensity in that study was deliberately kept at 80% MT, and therefore did not trigger any muscle contractions. This may indicate the importance of the patient becoming aware of movement and intact motor pathways as part of subjective symptom improvement. Placebo effects are likely to be involved in the mechanism of action, since in the study of Broersma, six out of nine patients showed a slight improvement after sham rTMS.

The stimulation protocol and parameters used differed considerably between studies. While most studies used lowfrequency stimulation (1 Hz or less), Schönfeldt-Lecuona et al. (2006) and Broersma et al. (2015) delivered rTMS with 15 Hz, considered for rTMS to be high-frequency. Most therapeutic rTMS were performed in a single session**,** but the studies by Schönfeldt-Lecuona et al. and Broersma et al. applied a longer protocol (over weeks).

#### Estimated Functional Improvement

Despite some limitations, we retrospectively managed to judge the efficacy of the investigated stimulation methods (rTMS and ECT) for treating FW patients using the principles of the CGI-I scale. In total, we identified 88 patients with FW affecting limbs that received either rTMS or ECT). For the cases in which a nominal CGI-I could be retrospectively estimated (n = 77) about 90% of them improved markedly (very much or much improvement) after stimulations. Only a minority of the treated patients improved minimally or remained unchanged. In no case did patients worsen significantly after treatment and no serious adverse events were reported. At follow-up, symptom improvement was not quantifiable in terms of CGI-I for the majority of the cases (**Table 2** for detailed information).

#### ECT vs. rTMS

To our knowledge only two cases of ECT treatment in FW of limbs have been published (Giovanoli, 1988; Gaillard et al., 2012) since this technique was established in psychiatry many decades ago. Both published cases reported dramatic improvements of limb movement related to the ECT, thus causing a great improvement of activity of daily living. Besides the known favorable effects on brain function in major depression, no specific mechanism of action has been elucidated for ECT in relation to FW symptoms. One may speculate that the possible mechanism for short term gains is the reduction of stress due to the amelioration of psychological precipitating factors and an improvement in mood after ECT. A major role of a placebo effect in both described cases accounting for the symptom improvement cannot be ruled out. On the other hand, in both cases improvement was not sustained over time, and both patients had a partial relapse after a while. None of the cases postulated or tried a continuation or maintenance ECT, which is recommended for the treatment of major depression when acute ECT effects do not persist (Petrides et al., 2011). A disadvantage of the ECT might be the economical aspect compared with other NIBS methods; the costs of the general anesthesia and the required specialized personal are included. Given the risks of the general anesthesia and (reversible) post-treatment cognitive disturbances, restriction of ECT to the severest and treatmentresistant FW cases should be considered.

Most of the included articles were related to TMS/rTMS (n = 5). rTMS may be the NIBS method that is most appropriate for the use in limb FW for different reasons: (1) rTMS can acutely provoke a muscular contraction or transient movements without needing patients cooperation (or intention) to move. (2) rTMS is relatively easy to apply in FW. This is in contrast to stimulations outside the motor cortex for other indications, in which localization strategies for coil positioning are needed (Schönfeldt-Lecuona et al., 2005). The magnetic pulses applied using intensities above MT (supra-threshold stimulation) will trigger a visually noticeable muscle contraction. Because motor pathways are intact in FW, this technique allows targeting the desired motor area with sufficient precision (Herwig et al., 2001, 2003). (3) Longer lasting rTMS causes plasticity changes in brain areas directly under the magnetic coil (Karabanov et al., 2015), but also trans-synaptic changes in areas far from the stimulation site (Strafella et al., 2003, 2004; Pogarell et al., 2007). (4) rTMS is mostly well tolerated, and has no adverse effects if performed within safety limits (Rossi et al., 2009). rTMS is considered not to be painful (depending on the intensity, frequency and train length of trains applied). (5) rTMS treatment is currently performed by physicians, but can also be performed by trained allied medical professionals (nurses, technicians, psychologists). It does not require any anesthetic, and can be performed in an outpatient setting. (6) The costs per session are lower than ECT, and rTMS devises are common nowadays in neurology and psychiatry departments, and in rehabilitation clinics in many high-income countries.

# Why Might rTMS Work?

#### Psychological Aspects

A crucial effect of supra-threshold TMS/rTMS in contrast to other NIBS methods is the patient's conscious perception of the externally triggered movements of their paralyzed limb. This phenomenon is experienced by all patients treated with rTMS, since prior to every therapeutic trial, single-pulse TMS with different intensities will be applied over the motor cortex in order to establish individual MT. In contrast, tDCS can modulate the excitability of cortical networks but does not directly produce action potentials on stimulated networks, and is therefore unable to trigger muscle contractions. Using ECT in FW, limb movements are actually provoked through the induced seizures, but the patient is not capable of noticing them because ECT is performed under general anesthesia. The patient's awareness of the muscle contractions due to TMS may help through a psychological mechanism: depending on the information received, patients become aware of normal function of neuromuscular structures. In addition, TMS triggering of muscle contraction might make patients aware of the possibility of regaining function. All identified studies showed an excellent response to TMS, except the one by Broersma et al. It was in that study that sub-threshold intensity rTMS was used, which does no provoke a muscular contraction. In addition, rTMS bears a high technical and methodological complexity in terms of technical approaches and calibrating steps that have to be performed prior to the therapeutic application, especially when MRI-guided localization techniques for coil positioning are used. Thus, TMS may generate a placebo effect, which in turn helps the patient to recover function immediately after stimulation. The response to rTMS may also be influenced by the information received and by the style which was used to inform the patients about the treatment strategy and purpose.

#### Neurophysiological Aspects

An increasing body of literature data suggest that focal functional abnormalities in central networks that control motor cortex activity may play a role in the etiology of FW (Geraldes et al., 2008; Liepert et al., 2008, 2009, 2011). The most convincing hypotheses to explain FW affecting limbs include (i) deficient processing of either motor intention or disruption between motor intention and motor execution or (ii) an overactive self-monitoring with enhanced limbic neural activity, which interferes with movement planning, and initiation within frontal regions and thereby disrupting motor execution (Voon et al., 2011). Studies using functional-imaging methods in patients with FW demonstrated enhanced neural activity within the anterior cingulate area or orbito-frontal cortex and reduced neural activity within prefrontal motor areas during movement execution of the paralyzed limb (Marshall et al., 1997; Spence et al., 2000; Stone et al., 2007). These abnormal activation patterns have been interpreted to reflect an active, but unconscious inhibition of movement planning and execution. Focused rTMS protocols with appropriate stimulation parameters might be able to reverse cortical dysfunction and restore activity in suppressed cortical motor areas (Schönfeldt-Lecuona et al., 2003, 2006; Chastan and Parain, 2010; Nielsen et al., 2013). The stimulation site for rTMS in FW is usually obvious with the primary motor cortex being the most plausible candidate region. However, the most challenging issue is still the choice of stimulation protocol (frequency, intensity, inter-train intervals, duration, and number of daily sessions) that can provoke a lasting positive change in cortical network activity. With regard to the question whether complementary stimulations in other cortical regions than the primary motor cortex could enhance therapeutic efficacy of rTMS in FW, no studies could be found.

#### Neuromodulatory Aspects

Single-pulse TMS with short protocols (e.g., performed in only one session for measuring MEPs for diagnostic purposes) might not be causing a durable change in cortical activity. Longlasting changes and thus changes in cortical neuro-plasticity might only be induced performing longer protocols (e.g., for one or more weeks) using high-frequency (>1 Hz) or lowfrequency (≤1 Hz), thus leading to long-term potentiation- or long-term depression-like changes respectively (Pascual-Leone et al., 1994; Chen et al., 1997; Fitzgerald et al., 2006). In most identified trials using TMS in FW, cortical stimulations were performed using repetitive spTMS with frequencies ≤1 Hz for a very short time (<2 or 3 min; Chastan and Parain, 2010; Kresojevic et al., 2010; Gaillard et al., 2012). Therefore, no longlasting cortical effects could be expected, nor any stable changes in motor function due to the used protocols. The immediate and mostly sustained positive responses to the stimulations must therefore have other reasons. Only few studies used rTMS protocols that might potentially cause plasticity changes. We need to stress that not all of these hypotheses of the mechanisms of action of rTMS have been translated into proven clinically relevant changes, and more research is needed to be sure if they have any clinically meaningful effect (McWhirter et al., 2015). Furthermore, given that rTMS has existed as a technique since the 1990s, the number of trials published in this field is amazingly low. Publication bias could be a partial explanation for this, as may be the paucity of clinicians considering rTMS in rehabilitation neurology. However, first rTMS therapeutic trials have been performed to relieve other forms of FNS such as dystonia, myoclonus, tremor, parkinsonism, stereotypies, nonepileptic seizures, functional aphonia, or sensory or visual loss (Chastan et al., 2009, 2011; Dafotakis et al., 2011; Saha et al., 2011; Garcin et al., 2013; Parain and Chastan, 2014; Shah et al., 2015), mostly yielding to symptom amelioration.

# RECOMMENDATION IN EVIDENCE-BASED GUIDELINES AND FUTURE STUDY DESIGNS

In 2014 evidence-based guidelines on rTMS were published and included recommendations for "Motor Conversion Disorders" in general (Lefaucheur et al., 2014). The degree of recommendation on the efficacy of rTMS for motor conversion disorders was: "No recommendation for low or high frequency rTMS of M1 or delivered at the vertex, using a focal or a non-focal coil" (Lefaucheur et al., 2014). The Cochrane library has published recommendations for TMS in the treatment of schizophrenia, depression, obsessive-compulsive disorder, amyotrophic lateral sclerosis, epilepsy and post-traumatic stress disorder (http:// onlinelibrary.wiley.com/cochranelibrary/), but did not address the topic of FW. Regarding ECT or tDCS, no sources were identified that reported evidence-based recommendations for its use in FW or more generally in FNS. Consistent with previous recommendations for the publication of case reports or case series (Lepping et al., 2007), and to allow clinically meaningful analyses from case series, we recommend that the following information should be included in any publication:

(particularly due to the perceptible scalp sensations by active stimulation). Therefore, except when using special placebo coils that provoke scalp effects similar to an active stimulation [as in Broersma et al. (2015) and Rossi et al. (2007)], we suggest that future studies should either be designed as parallel-arm studies (avoiding sham detection in a cross-over design) or as head-to-head studies, comparing active rTMS with usual therapeutic management of FW.

# CONCLUSION

The results of our systematic review provide preliminary evidence that NIBS methods, especially motor cortex rTMS, may be beneficial in the treatment of conversion weakness and paralysis. Most included rTMS studies reported acute beneficial effects on limb function despite heterogeneous protocols. In particular, the crucial influence of an externally triggered muscular contraction should be emphasized. Further rTMS trials should include a control condition, a greater number of sessions, and longer stimulation protocols with proven lasting effects on cortical excitability. However, although advances have been made in the last few years both in diagnostic methods and in the groundwork for a neurobiological model of FW, no definitive rationale for stimulation parameters and for the optimal setting is available. Therefore, further basic research in this area is needed (Aybek et al., 2008). Probably due to practical aspects the future of ECT in this area is expected to be less promising than rTMS. Despite this practical advantage, it remains to be demonstrated that rTMS can have a real therapeutic benefit in the long term, and any impact on the neural mechanisms of FW beyond merely inducing psychological or non-specific placebo effects. Our systematic review contributes to the current knowledge of rTMS application in the treatment of FW, updating the reviews previously published by Pollak et al. (2014) and Parain and Chastan (2014). In summary, the available evidence to date suggests that the application of NIBS in FW is feasible and beneficial. However, due to the small number of published cases in open-label studies, this conclusion should be considered with caution.

# AUTHOR CONTRIBUTIONS

CS, MG, PL made substantial contributions to conception and design of the review, CS, J-PL, MG performed the literaturesearch, analyzed the data, and wrote the manuscript. DN, AS, JL participated in drafting the manuscript, wrote the manuscript, and revisited it critically. BC made substantial contributions to the conception of the review, and revisited the manuscript critically. All authors gave the final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Schönfeldt-Lecuona, Lefaucheur, Lepping, Liepert, Connemann, Sartorius, Nowak and Gahr. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Targeting Neural Endophenotypes of Eating Disorders with Non-invasive Brain Stimulation

Katharine A. Dunlop1, 2, Blake Woodside1, 3, 4, 5 and Jonathan Downar 1, 2, 3, 4, 6 \*

1 Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada, <sup>2</sup> MRI-Guided rTMS Clinic, University Health Network, Toronto, ON, Canada, <sup>3</sup> Department of Psychiatry, University Health Network, Toronto, ON, Canada, <sup>4</sup> Department of Psychiatry, University of Toronto, Toronto, ON, Canada, Eating Disorders Program, University Health Network, Toronto, <sup>5</sup> ON, Canada, <sup>6</sup> Toronto Western Research Institute, University Health Network, Toronto, ON, Canada

The term "eating disorders" (ED) encompasses a wide variety of disordered eating and compensatory behaviors, and so the term is associated with considerable clinical and phenotypic heterogeneity. This heterogeneity makes optimizing treatment techniques difficult. One class of treatments is non-invasive brain stimulation (NIBS). NIBS, including repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS), are accessible forms of neuromodulation that alter the cortical excitability of a target brain region. It is crucial for NIBS to be successful that the target is well selected for the patient population in question. Targets may best be selected by stepping back from conventional DSM-5 diagnostic criteria to identify neural substrates of more basic phenotypes, including behavior related to rewards and punishment, cognitive control, and social processes. These phenotypic dimensions have been recently laid out by the Research Domain Criteria (RDoC) initiative. Consequently, this review is intended to identify potential dimensions as outlined by the RDoC and the underlying behavioral and neurobiological targets associated with ED. This review will also identify candidate targets for NIBS based on these dimensions and review the available literature on rTMS and tDCS in ED. This review systematically reviews abnormal neural circuitry in ED within the RDoC framework, and also systematically reviews the available literature investigating NIBS as a treatment for ED.

Keywords: Eating Disorders (ED), RDoC, rTMS, tDCS, fMRI

# INTRODUCTION

The term "eating disorders" (ED) encompasses a wide variety of disordered eating and compensatory behaviors that inappropriately alter the patient's body shape or weight, or the subjective experience of one's own body shape or weight. According to recent studies, the lifetime prevalence of EDs is 5.7% for females, and 1.2% in males (Golden et al., 2003; Hudson et al., 2007; Smink et al., 2014). The lifetime prevalence of the top three EDs according to the DSM-5 diagnostic criteria is 2.3, 1.7, and 0.8% for adolescent binge eating disorder (BED), anorexia nervosa (AN), and bulimia nervosa (BN), respectively (Golden et al., 2003; Hudson et al., 2007; Smink et al., 2014). BED is associated with recurrent episodes of binging, typically during negative affect (Leehr et al., 2015), and with the absence of inappropriate compensatory behaviors to avoid weight gain. Both AN and BN are associated with disturbances in the subjective experience of one's own body shape or weight; this phenotype is also known as body

#### Edited by:

Ignacio Obeso, Hospital Universitario Puerta del Sur - HM Hospitales, Spain

#### Reviewed by:

Bernd Weber, Rheinische-Friedrich-Wilhelms Universität, Germany Anne Sauvaget, University Hospital of Nantes, France

> \*Correspondence: Jonathan Downar<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 17 November 2015 Accepted: 25 January 2016 Published: 16 February 2016

#### Citation:

Dunlop KA, Woodside B and Downar J (2016) Targeting Neural Endophenotypes of Eating Disorders with Non-invasive Brain Stimulation. Front. Neurosci. 10:30. doi: 10.3389/fnins.2016.00030 dysmorphia. BN is also defined by recurrent episodes of binge eating, with inappropriate compensatory behaviors to avoid weight gain; such behaviors include vomiting, excessive exercise, laxative misuse or diuretic misuse. In contrast, AN is defined by the persistent restriction of food, an intense fear of gaining weight, and a significantly low body weight for one's developmental stage. AN has two subtypes, restricting (ANR) and binge-eating/purging (ANBP), with the latter distinguished from the former by the presence of binges and/or purges.

Despite a low lifetime prevalence rate relative to other psychiatric disorders, EDs carry a significant burden of illness, both socially and individually. Treatment capacity in specialized ED programs is presently inadequate to meet demand (Hart et al., 2011), and for patients who do manage to access specialized programs, economic difficulties and high costs often hamper treatment adherence (Gatt et al., 2014).  According to a recent meta-analysis, the overall standard mortality ratio (SNR) for AN is 5.86, higher than schizophrenia (2.8), bipolar disorder (2.1), and major depression (1.6) (Arcelus et al., 2011). Conventional ED treatments, including pharmacotherapy, and in- and out-patient behavioral therapies, are associated with suboptimal recovery rates (∼50% for AN; ∼45% for BN; ∼50–70% for BED), high relapse rates (ranging from 9 to 65%), and high chronicity (∼20% will develop a chronic disorder; Olmsted et al., 2005; Mitchell et al., 2007; Shapiro et al., 2007; Carter et al., 2012; Hay et al., 2012; Hilbert et al., 2012; Amianto et al., 2015). ANBP, in particular, has the poorest prognosis of the eating disorders (Steinhausen and Weber, 2009). EDs are also highly co-morbid with other psychiatric disorders, such as major depression and obsessive-compulsive disorder, whose presence negatively impacts treatment outcomes (Godart et al., 2003; Crane et al., 2007; Mischoulon et al., 2011). Thus, new treatment approaches are urgently needed, especially for the substantial proportion of ED patients who are unresponsive to conventional treatment strategies.

Neuromodulation technologies are beginning to emerge as a promising new treatment option for treatment resistant ED patients. The potential usefulness of these techniques was recently illustrated in a pilot study using subgenual cingulate deep brain stimulation (DBS) to achieve symptomatic improvements in severe and treatment-refractory AN (Lipsman et al., 2013). Although potentially powerful, DBS remains for the moment a fairly invasive treatment, and is available only to small volumes of patients in specialist neurosurgical centers. A more accessible alternative is non-invasive brain stimulation (NIBS), including techniques such as repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS). rTMS uses rapid pulses of an electromagnetic field to elicit action potentials in the target area of cortex. tDCS uses a weaker intensity electrical stimulus, delivered by scalp electrodes, to modulate cortical excitability in the underlying regions. Both NIBS strategies attempt to alter the cortical excitability of a target brain region to normalize particular disorder-specific phenotypes. Cortical targets are typically selected based on abnormal structural or functional attributes in the disorder relative to healthy controls. Appropriate cortical targeting using NIBS is critical for optimal treatment efficacy (Fox et al., 2013). Therefore, a proper understanding of the neural substrates, as well as the cognitive and behavioral phenotypes accompanying these substrates, is crucial for optimizing future treatments.

Two major issues associated with NIBS as a treatment for ED are the tremendous heterogeneity in the cognitive and behavioral phenotypes of patients within this illness category, and the dynamic course of the illness, in which patients can switch from one ED diagnosis to another over time (Garfinkel et al., 1995; Keel and Mitchell, 1997; Lilenfeld et al., 1998; Sullivan et al., 1998; Strober et al., 2000; Bulik et al., 2005; Milos et al., 2005). This variability within a single diagnosis and this malleability of symptoms is a likely contributor to the limited clinical efficacy of NIBS (and in conventional treatments more generally) for ED.

Two possible solutions to address this heterogeneity are genomic methods, such as phenotypic linkage analyses, as well as neuroimaging methods. Such tools stratify patients on underlying behavioral, genetic, and neuropathological dimensions rather than self-reported symptoms alone. Therefore, these tools may be useful to identify the underlying behavioral and neuropathological endophenotypes related to more basic dimensions of behavior, independent of DSM-5 diagnoses. Such analyses and resulting endophenotypes can also be related to the behavioral and circuit-based dimensions of the recently described Research Domain Criteria (Insel et al., 2010) (RDoC). The RDoC is a recent strategy aimed at integrating basic neuroscientific knowledge with clinical diagnoses by first describing fundamental behaviors, described below, as dimensions. These dimensions are then used to describe the pathological behaviors of psychiatric disorders. By using the RDoC schema in combination with neuroimaging and phenotypic linkage methods, we may be able to identify sufficient stimulatory targets addressing specific phenotypes such as restrictive behavior or binging, regardless of DSM-5 diagnosis. For NIBS treatments, diagnostic systems must be capable of parsing this heterogeneity using endophenotypes so we may select the optimal stimulation target for a particular behavioral marker, or neural substrate.

Here, we will review NIBS as a treatment for the three most prevalent forms of ED: AN, BN, and BED. First, we will posit candidate dimensions as outlined by the RDoC and their underlying behavioral and neurobiological targets associated with ED as potential candidates for NIBS. Second, we will review the available literature on rTMS and tDCS as possible treatments for ED. Lastly, we will discuss the current limitations of the NIBS-ED field, and opportunities of future study and development.

### GOING BEYOND THE DSM-5 DIAGNOSIS: HOW CAN WE MAXIMIZE EFFICACY?

As discussed above, one of the major obstacles in ED diagnosis and treatment is the heterogeneity within each diagnostic category; conversely, comparisons of clinical and psychological features across patients suggest that there is significant overlap between ED diagnoses (Garfinkel et al., 1995; Lilenfeld et al., 1998; Sullivan et al., 1998; Strober et al., 2000). Compounding this problem is the evolution of the illness, such that patients may transition from one diagnostic category to another over time (Bulik et al., 2005). For example, it is estimated that approximately 50% of patients initially diagnosed with ANR will develop binge/purge behaviors, and approximately 30% of BN patients have a history of AN (Keel and Mitchell, 1997). In another study following DSM-IV-diagnosed AN and BN, only one third of subjects retained their original diagnosis after 30 months (Milos et al., 2005). To improve diagnostic consistency and treatment efficacy may require us to identify more stable, more granular, and more biologically based subgroups, or endophenotypes, within the ED population.

Some classification efforts have focused on a single DSM diagnosis. AN has been subdivided into 3 stable classes based on co-occurring symptoms: fat-phobic ANR, fat-phobic ANBP, and non-fat-phobic ANR (Wildes et al., 2013). BN has been subdivided based on personality attributes (affectiveperfectionistic, impulsive and low-comorbid psychopathology clusters Wonderlich et al., 2005) and based on presenting symptoms (binging, purging, and bingeing-purging, Striegel-Moore et al., 2005).

A number of latent class (LCA) and latent profile analyses (LPA) have been performed on symptomatic and personality factors to stratify endophenotypes spanning AN and BN. One symptom-based LCA found optimal fitting for a 4-group classification. ANR, ANBP/BN, ANR without OCD, and BN with only vomiting as purging were the four groups identified (Keel et al., 2004). Another symptom-based LPA identified 4 ED classes: binging with multiple types of compensatory behavior; binging with only vomiting as compensatory behavior; binging without purging; and low/normal weight with excessive exercise (Eddy et al., 2009).

As evidenced above, there now exist a variety of different proposals for how best to subcategorize ED patients, within and across DSM-5 diagnoses. How, therefore, can we converge upon a system that offers maximum clinical usefulness? One potentially fruitful method would be to better characterize the heterogeneity among ED patients in biological terms, using techniques such as positron emission tomography (PET), electroencephalography (EEG) and magnetic resonance imaging (MRI) to identify distinct neurobiological substrates underlying the different subgroups within ED. Clinical endophenotypes could then be tied to neurobiological substrates, which could in turn serve as targets for individually- or phenotypically-tailored treatment strategies.

Such an approach would also allow us to describe illnesses in dimensional rather than categorical terms. For example, the influential RDoC framework (Insel et al., 2010) includes dimensional constructs such as positive valence, negative valence, cognitive systems, social processes, and arousal and regulatory systems (for a review of how RDoC dimensions relate to ED neurobiology, see Wildes and Marcus, 2015, **Table 1**). Many endophenotypes, previously identified as symptom clusters in the ED population, can be framed parsimoniously as the result of pathology affecting these dimensions, either singly or in combination (**Figure 1**). An "RDoC formulation" of our ED TABLE 1 | Overview of the 5 Research Domain Criteria domains as adapted from Insel et al. (2010) and Morris and Cuthbert (2012).

endophenotypes carries the advantage of pointing toward specific cognitive processes, neural pathways, neurotransmitter systems, molecular targets, or genes that might be targeted for therapeutic effect. For the purposes of this review, we will confine our discussion to potential novel uses of NIBS to target specific neural pathways that are associated with RDoC constructs, as they relate to specific endophenotypes within the ED population.

# RDOC DOMAINS AS ED ENDOPHENOTYPES AND NIBS TARGETS

For the following section, a systematic review was completed using PubMed (NIH, http://www.ncbi.nlm.nih.gov/pubmed), with searches containing the following terms: first, clinical terms for the three ED diagnoses in this review (bulimia nervosa, anorexia nervosa, and binge eating disorder), and second, RDoC related terms as discussed in a recent review on RDoC cognitive systems (Morris and Cuthbert, 2012).

#### Negative Valence Systems

Negative valence systems are activated in response to aversive stimuli, and include fear, anxiety and loss-related behaviors. In a recent meta-analysis investigating neural activations for negative and positive affect, negative valence was associated with greater activation in the amygdala and anterior insula (Lindquist et al., 2015). The lateral orbitofrontal cortex (OFC) is also associated with negative valence, particularly during the anticipation and receipt of punishment (Ursu et al., 2008).

A number of studies support the role of negative valence systems in ED, mainly in behaviors associated with negative affect, sensitivity to punishment, anxiety, harm avoidance, and response to the receipt of punishment (**Figure 1**). For example, behavioral measures of negative affect and negative urgency are the two most predictive features before a binge episode in both BED and BN (Bohon and Stice, 2012; Berg et al., 2015; Leehr et al., 2015; Racine et al., 2015). On functional neuroimaging, BN patient reported negative affect is related to neural responsivity during the anticipation of a food reward in both the striatum and insula (Bohon and Stice, 2012). This relation suggests that negative affect and food-reward are inappropriately coupled in this disorder. More generally, BN patients also have higher neural responses to negative body image descriptors (Miyake et al., 2010), in areas associated with the regulation and inhibition of fear and emotional processing circuits, including the dorsomedial prefrontal cortex (DMPFC) (Kühn et al., 2011; Åhs et al., 2015). These findings shed light on the role of negative attentional bias in the psychopathology of bulimic-type disorders.

Restrictive subtypes of ED also show hypersensitivity on measures related to negative valence systems. Behaviorally, exaggerated harm avoidance and sensitivity to punishment are typically associated with forms of AN (Harrison et al., 2010). Similarly, on fMRI, AN patients display increased neural activation in right anterior insula and DLPFC during pain anticipation, and exaggerated responses to punishment (pain and monetary losses) in the DLPFC, and the anterior, mid-, and motor cingulate (Bischoff-Grethe et al., 2013; Strigo et al., 2013; Bar et al., 2015). Cowdrey and colleagues also found an exaggerated response to an aversive taste and sight of food in the insula, striatum and ACC (Cowdrey et al., 2011). Traitanxiety is also a common feature of AN, and is associated with the exaggerated activity of fear-related circuits to food and body-related cues.  Finally, at the receptor level, PET imaging reveals increased striatal dopamine binding potential and altered cingulate serotonergic (increased 5-HT1A, but decreased 5- HT2A) binding potential is associated with harm avoidance in AN (Bailer et al., 2004, 2007; Frank et al., 2005).

#### Summary of Potential Negative Valence Targets

Both bulimic and restrictive-type EDs display some form of negative valence abnormality on behavioral and neuroimaging modalities (**Figure 2**). In ED with a binging component, it appears that negative affect and food-reward responsivity are intimately coupled via the exaggerated response of the amygdala, insula and DMPFC. Restriction-related EDs display a similar pattern in the amygdala, right anterior insula, DLPFC and mPFC accompanying aspects of harm avoidance and receipt

FIGURE 2 | Candidate NIBS targets that address abnormal phenotypes related to the RDoC negative valence dimension.  The dorsolateral prefrontal cortex (DL) is abnormally hyperactive for pain anticipation and the receipt of punishment. The anterior cingulate cortex (ACC) is hyperactive for aversive food stimuli, the receipt of punishment, and anxiety. Finally, the anterior insula (IN) is abnormally hyperactive during anxiety and the anticipation of pain. (B) Candidate negative valence NIBS targets for bulimia nervosa (BN) and binge eating disorder (BED). The ACC is abnormally activated for negative words about the body (Miyake et al., 2010), while the insula is hyperactive during negative affect (Bohon and Stice, 2012).

of punishment. Frontal regions, particularly the medial PFC and DMPFC, are thought to inhibit activity of the basolateral amygdala (BLA) (Cho et al., 2013; Felix-Ortiz et al., 2015).

Hyperactivation of the DMPFC, DLPFC, and anterior insula during negative valence paradigms has two possible interpretations. First, these areas may be inhibiting BLA activity, but insufficiently, in which case excitatory stimulation may be beneficial. Alternatively, these areas may actually be inappropriately driving BLA activity in a top-down fashion, in which case inhibitory stimulation would be preferable. A key study illustrated these opposite mechanisms in emotion regulation in healthy controls vs. MDD patients (Johnstone et al., 2007): during emotional reappraisal, limbic frontal regions suppressed amygdala activity in controls, but counterproductively increased amygdala activity in MDD.

For NIBS interventions, direct suppression of the amygdala is challenging due to its deep location; strategies aimed at damping negative valence systems will therefore likely target in prefrontal cortex and insula. Excitatory prefrontal NIBS has been recently shown to attenuate amygdala-dependent negative processing in healthy controls (Baeken et al., 2010; Guhn et al., 2014), and this strategy may be best in "bottom-up" pathology, where emotional reappraisal/self-regulation systems are underactive rather than pathologically hyperactive (i.e., in BN and BED). Conversely, where negative valence systems are driven by "topdown" pathology, and self-regulation is if anything excessive, inhibitory stimulation may be preferable. Inhibitory NIBS of the DMPFC and lateral OFC both show promise in obsessivecompulsive disorder (Mantovani et al., 2010; Nauczyciel and Drapier, 2012; Dunlop K. et al., 2015), and these strategies may be better suited to AN-R, particularly in cases with comorbid OCD.

### Positive Valence Systems

Positive valence systems encompass neural circuits related to motivation, reward seeking, and habit formation behaviors. According to a recent meta-analysis in healthy controls, positive stimuli are associated with activity in the VMPFC and ACC (Lindquist et al., 2015). All three major EDs, AN, BN, and BED, have been previously shown to be altered in this dimension (**Figure 1**).

From a behavioral perspective, AN patients show diminished sensitivity to conventional reward, as evident on psychometric measures (Harrison et al., 2010; Glashouwer et al., 2014) and delay discounting tasks (Steinglass et al., 2012). From a neurobiological perspective, ANR patients likewise display a blunted neural response to food reward in the insula and striatum (Wagner et al., 2008), decreased response to food images in the insula (Holsen et al., 2012; Oberndorfer et al., 2013b), and altered striatal activation during a reward-learning paradigm (Wagner et al., 2007). In a recent fMRI study of delay discounting in AN patients and healthy controls, AN patients had a marked preference for delayed rewards, associated with lower activation in the striatum and dorsal ACC during decision-making; these behavioral and neural abnormalities normalized to control levels after treatment (Decker et al., 2014). However, another study found that weight restoration did not affect choice behavior on a delay discounting task (Ritschel et al., 2015), suggesting that a preference for delayed over immediate rewards may be an endophenotypic feature in low-BMI individuals. In either case, the identified striatal and prefrontal regions are all involved in the motivational aspect of reward and food-reward processing.

There is also evidence that reward evaluation is altered in AN, in which secondary (contextual) rewards such as exercise and dietary restriction carry higher reward value relative to food or other primary rewards (Schebendach et al., 2007; Klein et al., 2010). The so-called "reward contamination theory" of AN posits a pathological re-configuration of the patient's reward system through stress-induced activation of the mesolimbic dopamine system, via ventral tegmental area opioid receptors. In this framework, AN behaviors essentially represent a maladaptive, but well-entrenched type of habit-formation (Keating et al., 2012; Walsh, 2013).

The findings that support this theory suggest that there is altered motivational salience for disease-related stimuli. For example, AN patients tend to rate physical exercise as "pleasant," more so than food (Giel et al., 2013). In fact, food-reward in AN activates a weight-gain fear response (i.e., negative valence systems) in the amygdala and extrastriate body rather than positive valence systems from the striatum, orbitofrontal cortex, and ACC (Vocks et al., 2011). The DLPFC is also hyperactive in response to images of food and the anticipation of reward, suggesting the presence of enhanced cognitive control over food cues and reward (Ehrlich et al., 2015; Sanders et al., 2015). ANR patients also have a high prevalence of comorbid OCD (Torresan et al., 2013). The level of compulsivity predicts the reactivity of the superior frontal gyrus, ACC and striatum and deactivation of the PFC to images of high-calorie foods (Rothemund et al., 2011), and lowered right DLPFC activity is seen in response to obsessive-compulsive symptom provocation in AN (Suda et al., 2014). Thus, hypofunctioning of primary reward systems (and potentially, hyperfunctioning of secondary/contextual reward systems) may be important target processes in ANR.

In contrast disorders in the BN/BED spectrum are often associated with elevated primary reward valuation and reward sensitivity.  BED patients display hyperactivations in the ventral striatum and inferior frontal gyrus during reward anticipation, and reduced medial PFC activity during a monetary incentive delay task (Balodis et al., 2014, 2013a). On PET imaging, areas like the insula, PFC and ventral striatum, associated with reward-motivation and food-reward processing, have altered serotonergic and dopaminergic binding in BN (Broft et al., 2012; Galusca et al., 2014). An important associated feature may also be deficient behavioral self-regulation and impulsivity. BN patients also show reduced activation in anticipation of a food reward is seen in ACC and right anterior insula; lower ACC activity predicts how much the patient will overeat (Frank et al., 2006; Bohon and Stice, 2011). Parallels have been drawn between the neural substrates of BN/BED and addiction, due to the similar alterations to motivation and reward-related circuitry on fMRI and task-based paradigms between the two psychopathologies (Filbey et al., 2012).

#### Summary of Potential Positive Valence Targets

In terms of positive valence systems, it appears that both restrictive and binging phenotypes of ED display alterations in incentive salience that is potentially modulated by the opioid system (Keating et al., 2012; Giuliano and Cottone, 2015; **Figure 3**). In the case of ANR, conventional primary rewards appear to be devalued in favor of pathological secondary or contextual rewards, such as starvation and excessive exercise. A broader preference for long-term/contextual over immediate primary rewards is also apparent in choice behavior during delay discounting. Neurally, the primary reward systems of the ventral striatum and ventromedial prefrontal cortex appear hypoactive, while contextual or secondary reward systems operating through lateral orbitofrontal and lateral temporal regions appear hyperactive. Hyperactivity in lateral orbitofrontal pathways is also strongly associated with OCD, and with compulsivity in general (Ahmari et al., 2013; Beucke et al., 2013). This finding would be consistent with the broader phenotype of ANR. Neurally-based strategies in ANR might therefore include enhancing primary reward value via medial prefrontal-striatal pathways, or attenuating secondary reward value via lateral prefrontal-striatal pathways. For instances where conventional rewards are less valued than maladaptive ones (restrictive, fat-phobic ED), inhibitory NIBS over lateral networks for maladaptive secondary rewards, and excitatory NIBS over medial networks for conventional rewards, may be a possible therapeutic protocol to realign incentive-salience mechanisms to normal, adaptive functioning.

In the case of binge/purge-related EDs, repeated exposures to the transient reward value of food intake (or the transient antianxiety effect of purging) would cause these behaviors to acquire pathologically high incentive value (especially in the presence of negative urgency), via neural mechanisms that parallel those of addiction. Effective strategies would therefore parallel those for substance addiction: enhancing cognitive/impulse control over urges to binge and purge, or suppressing urge intensity.

NIBS strategies for enhancing cognitive control involve excitatory stimulation of the nodes of the salience network, including the DLPFC, dACC, and insula (Dunlop et al., accepted). Each of these targets have demonstrated efficacy in substance dependence (Mishra et al., 2010; De Ridder et al., 2011; Meng et al., 2014), with effects apparently mediated by enhanced control rather than reduced urge. Recently, excitatory rTMS over the dACC has been reported to reduce symptoms in treatmentresistant binge/purge ED, via enhanced integrity of frontostriatal circuits in the salience network (Dunlop J. et al., 2015).

NIBS may also be capable of suppressing urge, by targeting frontopolar and ventromedial sites. In one preclinical rTMS study, substance use disorder patients underwent inhibitory rTMS over the ventral frontal pole during a task evoked a cue-related craving response. A single session of inhibitory rTMS reduced the severity of craving in this group relative to sham, and stimulation proved capable of engaging core reward nodes in the ventral striatum, as well as the associated ventromedial prefrontal regions (Hanlon et al., 2013, 2015). Urge suppression via inhibitory ventromedial prefrontal stimulation has yet to be attempted in ED, but would be a reasonable strategy to complement excitatory salience-network stimulation in binge/purge-related ED populations.

### Cognitive Systems

The cognitive systems dimension refers to processes responsible for cognitive processing, including attention, perception, memory, language, and cognitive control. In healthy control studies, these behaviors are associated with activity in the DMPFC, DLPFC, and anterior insula (Albares et al., 2014; Cho et al., 2014; Luo et al., 2014; Reineberg et al., 2015). These networks tend to be associated with the central executive and salience resting-state networks (Reineberg et al., 2015), responsible for response selection and inhibition.

Abnormal cognitive control mechanisms are evident in most ED populations (**Figure 1**). On the one hand, BN and BEDtype diagnoses tend to display reduced capacity for impulse and

cognitive control. This is particularly evident for disease-relevant stimuli (Wu et al., 2013), but is also apparent for positive and negative emotional valence images (Tapajóz P de Sampaio et al., 2015), suggesting a broader endophenotype of deficient cognitive and behavioral control. In fact, binge episodes are partially defined by the individual's loss of control during eating, and impulse control disorders (ICD) are common comorbidities (Fernández-Aranda et al., 2008). Purging behaviors are also associated with higher levels of impulsivity, and different forms of purging may represent separate manifestations of compulsivity and impulsivity (Hoffman et al., 2012).

On the other hand, restrictive-type EDs tend to show a different profile of cognitive control abnormalities. Cognitive control capacity may appear above normal levels in certain domains, such as temporal discounting (Steinglass et al., 2012). However, cognitive control may be abnormal in certain specific domains related to the illness; for example, for negative valence images (Tapajóz P de Sampaio et al., 2015), food stimuli (Oberndorfer et al., 2013b; Sanders et al., 2015), or body-imagerelated stimuli (Lee et al., 2014). AN patients also have altered cognitive control depending on the reward valence of the object, as the impulse control networks are overly activated for physical exercise relative to food images in a go/no-go task (Kullmann et al., 2014). AN patients also show a reduced ability to switch to an optimal decision-making strategy, called cognitive flexibility (Zastrow et al., 2009).

From a neural perspective, impulsive-type deficits on response control tasks are related to lower frontostriatal activations. BED patients show reduced activity in the inferior frontal gyrus, ventromedial PFC and insula during the Stroop task, and this diminished activity is associated with poor dietary restraint (Balodis et al., 2013b). BED prefrontal hypoactivity has also been correlated with psychometric measures of attentional impulsiveness and a disease-relevant go/no-go task (Hege et al., 2014). BN patients show hypoactivity in frontostriatal circuitry during cognitive control tasks like the Simon Spatial Incompatibility task; affected areas include the inferior frontal gyrus, striatum, ACC, OFC, DLPFC, and middle frontal gyrus (Marsh et al., 2009, 2011; Celone et al., 2011). On the go/no-go task, adolescent BN and ANBP patients display hyperactivations in the ACC and right DLPFC, albeit without impaired task performance relative to controls (Lock et al., 2011).

AN patients also show hypoactivity in frontostriatal circuits from the medial PFC on a response inhibition task related to cognitive control deficits (Oberndorfer et al., 2011; Wierenga et al., 2014), but hyperconnectivity to a response inhibition task that used exercise-related stimuli as its cue (Kullmann et al., 2014). Additionally, AN patients also display poorer performance on cognitive flexibility tasks, and this performance is reflected by lower activity in frontostriatal circuits through the thalamus, ventral striatum, ACC, middle frontal gyrus, and ventrolateral PFC (Zastrow et al., 2009; Sato et al., 2013; Garrett et al., 2014; Wildes et al., 2014; Lao-Kaim et al., 2015). On resting-state fMRI, higher thalamo-cortical functional connectivity through the DLPFC and anterior PFC is associated with poorer performance on the Stroop task and working memory (Biezonski et al., 2015). Thus, domain-specific abnormalities of cognitive control are evident at both the behavioral and the neural level in AN.

#### Summary of Potential Cognitive Control Targets

Both restricting- and binge/purge-type EDs show deficits on tasks related to cognitive control, including behavioral inhibition, working memory, selective attention, and cognitive flexibility (**Figures 1**, **4**). Generally, BED displays poorer response inhibition and lower activity in the inferior frontal gyrus and ventromedial PFC, both of which are accessible via excitatory forms of NIBS. BN and ANBP display lower activity in the inferior frontal gyrus, ACC, OFC, and DLPFC; all but the OFC are easily accessible for excitatory NIBS. As noted earlier, excitatory NIBS of salience-network nodes in DLPFC, DMPFC, and anterior insula appears to improve cognitive control and impulsivity even in healthy controls (Cho et al., 2014, 2010; Meng et al., 2014). Enhanced cognitive control, via improved frontostriatal connectivity through these salience-network nodes, may mediate recently reported improvements in binge and purge behaviors with excitatory DMPFC-rTMS (Dunlop J. et al., 2015). Similar effects via similar mechanisms should be expected for excitatory rTMS targeting DLPFC and anterior insula.

For AN, neural correlates of cognitive control show considerable variability depending on the task and valence of stimuli. On the one hand, AN patients in some studies show broad deficits of cognitive control and flexibility, and hypoactivity of the frontostriatal circuitry, during many tasks related to cognitive control; hence, excitatory NIBS might be beneficial if combined with cognitive tasks during stimulation. On the other hand, patients sometimes show the reverse pattern of hyperconnectivity and excessive cognitive control/compulsivity in these same circuits, within illnessspecific domains; excitatory stimulation may therefore be unhelpful, or could potentially exacerbate illness. In keeping with this concern, high-frequency DMPFC-rTMS was recently reported to exert a paradoxical inhibitory effect on frontostriatal connectivity in a subpopulation of ED patients with high baseline connectivity; these patients showed symptomatic worsening rather than improvement (Dunlop J. et al., 2015). Thus, targeting cognitive control in AN-R may require a more nuanced approach than is the case for binge-purge symptoms.

#### Social Processing Systems

Social processing systems refer to circuits involved in social communication, and the perception and understanding of oneself and others. Targets identified in healthy controls include the insula, responsible for interoception (Craig, 2002); the temporoparietral junction, for theory of mind-related processing (Saxe and Kanwisher, 2003); and higher-order visual processing regions, for processing one's own and others' faces (Hummel et al., 2013).

This dimension has received less attention in the ED literature relative to positive/negative valence systems and cognitive control (**Figure 1**). However, it may have relevance in AN patients, who show higher levels of alexithymia, deficits in visceral sensory perception or "interoception" (Craig, 2002; Strigo et al., 2013), and distorted perceptions of body shapes (Suchan et al., 2013). AN patients with higher levels of alexithymia show lower ACC, PCC, and right temporoparietal junction (TPJ) activation during social decision-making tasks (Miyake et al., 2009, 2012; McAdams and Krawczyk, 2011). More specifically, ANR patients display altered anterior and dorsal mid-insula activations based on the modality of interoception they are attending to (Kerr et al., 2015). On resting-state fMRI, AN patients also display increased functional connectivity from the anterior insula to the default mode network associated with self-reported problems with interoceptive awareness, suggesting a heightened level of cognitive control toward interoceptive processes (Boehm et al., 2014). AN patients also have altered neural responses to visually-presented body shapes, particularly in areas associated with visual processing and reward: the ventral striatum, extrastriate body area (EBA), DLPFC, parietal regions, medial PFC, and fusiform gyrus (Cowdrey et al., 2012; Spangler and Allen, 2012; Castellini et al., 2013; Fladung et al., 2013; Suchan et al., 2013; Suda et al., 2013; Fonville et al., 2014). Finally, two recent studies have also identified areas of abnormal activation in response to benevolent and malevolent social relationships. During benevolent social relations, AN patients tend to display reductions in DMPFC, possibility related to lowered reward valence for social reward and interaction (McAdams et al., 2015; Via et al., 2015).

In summary, AN patients may show deficits across multiple domains related to self-perception (alexithymia, interoception, and body shape perception) and social function (interpersonal interaction, theory of mind; **Figures 1**, **5**). The latter function has been successfully enhanced with excitatory DMPFC-rTMS in autism-spectrum disorder (Enticott et al., 2011, 2014). During social interactions, AN patients likewise tend to display DMPFC hypoactivity during social interaction, and so excitatory stimulation over this region may worth exploring. For selfperception, relevant targets include anterior insula (alexithymia), posterior insula (interoception), TPJ and EBA (social cue perception, body shape perception). NIBS has successfully targeted each of these regions in other applications (Ciampi de Andrade et al., 2012; Dinur-Klein et al., 2014; Donaldson et al., 2015). Excitatory stimulation of the insula and TPJ may be worth exploring for alexithymia and deficits in interoception. Conversely, inhibitory stimulation of the TPJ and EBA may be worth exploring for aberrant self- and body perception.

# NIBS TECHNIQUES AS THERAPEUTIC INTERVENTIONS IN ED

For the following section, a systematic review was completed using PubMed (NIH, http://www.ncbi.nlm.nih.gov/pubmed),

with searches containing the following terms: first, clinical terms for the three ED diagnoses in this review and related phenotypes (BN, AN, BED, binging, purging, excessive exercise), and second, NIBS related terms (rTMS, TMS, tDCS).

are abnormally hypoactive during interference control, poor dietary restraint, impulsivity, and response inhibition.

# NIBS Overview: rTMS and tDCS

rTMS applies powerful, focused magnetic field pulses over the scalp to elicit action potentials in the underlying region of cortex. Typically, treatment sessions occur once daily, for a total of 20– 30 daily sessions (Carpenter et al., 2012; Solvason et al., 2014). rTMS mechanisms are thought to involve synaptic plasticity via long-term potentiation or depression, with the direction of effect dependent on the stimulation intensity, duration, and pattern (Pascual-Leone et al., 1998; Maeda et al., 2000). Higher frequency stimulation (5–20 Hz) is usually considered to be excitatory, while low frequency (<1 Hz) stimulation is considered inhibitory (Pascual-Leone et al., 1994; Chen et al., 1997). More recently, however, considerable heterogeneity on electrophysiological, neuroimaging, and clinical measures has been found for most if not all patterns of rTMS (Maeda et al., 2000; Eldaief et al., 2011; Dunlop J. et al., 2015; Dunlop K. et al., 2015; Nettekoven et al., 2015).

tDCS, on the other hand, uses a constant, low amplitude current to modulate cortical excitability, rather than eliciting action potentials directly. As with rTMS, sessions typically occur daily, for a total of 10–30 sessions (Meron et al., 2015). While the mechanisms of tDCS are still debated, it is likely that modulated cortical excitability also elicits subtle effects on synaptic plasticity via long-term potentiation and depression (Brunoni et al., 2012). Anodal stimulation is considered excitatory, and cathodal stimulation inhibitory. However, as with rTMS, both types of tDCS display considerable inter-individual variability in their effects (Wiethoff et al., 2014). Newer variants such as transcranial alternating current stimulation (tACS), may exert more consistent, frequency-specific effects (Voss et al., 2014); however, their therapeutic potential is poorly understood at present.

# NIBS as a Treatment for BED and Food Craving

To date, the majority of published NIBS-ED studies have focused on female patients with abnormally high food craving or urge to eat, as opposed to a specific formal DSM-5 diagnosis (**Tables 2**, **3**; McClelland et al., 2013; Grall-Bronnec and Sauvaget, 2014; Val-Laillet et al., 2015). These preclinical studies typically involve a single session of stimulation, with subjectively rated cueinduced craving as the primary outcome. With rTMS, two studies reported contradictory results for 10 Hz stimulation of the left DLPFC rTMS: one study (n = 28) found decreased craving after active vs. sham stimulation (Uher et al., 2005), while the other

FIGURE 5 | Candidate NIBS targets that address abnormal phenotypes related to the RDoC social processing dimension in anorexia nervosa (AN) (Cowdrey et al., 2012; Miyake et al., 2012; Spangler and Allen, 2012; Castellini et al., 2013; Fladung et al., 2013; Suda et al., 2013; Boehm et al., 2014; Fonville et al., 2014; Kerr et al., 2015; McAdams et al., 2015; Via et al., 2015). The anterior cingulate cortex (ACC), posterior cingulate cortex (PCC), and temporoparietal junction (TPJ) are abnormally hypoactive during deficits in social decision-making and alexithymia, while low insula (IN) activity is related to deficits in interoceptive awareness. The dorsolateral prefrontal cortex (DL) is both abnormally hyperactive when the participant views oversized images of themselves, but hypoactive when viewing images depicting body-checking behavior. The fusiform face area (FFA) is both abnormally hyperactive when the participant views highly emotional facial expressions, but hypoactive when viewing distorted body shapes and images depicting body-checking behavior. The extrastriate body area (EBA) is both abnormally hyperactive when the participant views images of their own body, but hypoactive when those images are distorted.

(n = 10) found that active stimulation was no better than sham in terms of cue-induced craving control (Barth et al., 2011). The studies differed in stimulation parameters, however, and enrolled only healthy participants who self-reported having strong food cravings, but did not carry a formal ED diagnosis. Hence, it may be difficult to extrapolate these findings to the effects of a full therapeutic course of 20–30 sessions in patients with pathological deficits of self-control and a formal ED diagnosis.

There is also a growing body of literature investigating DLPFC-tDCS as a method to reduce craving and food intake. In four published studies recruiting individuals with strong food cravings, a single session of anodal right DLPFC/cathodal left DLPFC tDCS was able to reduce cue-induced craving, reduce food intake, and improve the participants' ability to resist food relative to sham-tDCS (Fregni et al., 2008; Goldman et al., 2011; Kekic et al., 2014; Lapenta et al., 2014). Future work involving tDCS should employ multiple sessions as opposed to a single session in a randomized, sham-controlled setting, as a treatment for the inappropriate eating patterns associated with BED. Studies in populations carrying a formal ED diagnosis, with significant functional impairment and distress, are also needed.

# NIBS as a Treatment for BN

The earliest publication of rTMS as a potential treatment for BN is a case report of a patient diagnosed with comorbid depression and BN who achieved an unexpected remission of binge and purge symptoms and depressive improvements after 10 sessions of 20 Hz rTMS over the left DLPFC (Hausmann et al., 2004; McClelland et al., 2013; **Table 2**). Follow-up studies involving high frequency left DLPFC rTMS have been mixed: one group found that a single session reduced the urge to eat, the number of binges 24 h post-rTMS, and salivary cortisol levels (Van den Eynde et al., 2010; Claudino et al., 2011), while another study found no difference between active- and sham-rTMS after 15 sessions of 20 Hz rTMS over the left DLPFC (Walpoth et al., 2008). A more recent study applied a single session of excitatory left DLPFC-rTMS in 8 female patients with BN, and reported reduced subjective ratings of craving post-rTMS, along with lower cerebral oxygenation in the DLPFC on near-infrared spectroscopy (Sutoh et al., 2016). These findings hint at the potential promise of DLPFC-rTMS for BN, which would be in keeping with the much more extensive literature demonstrating that this intervention enhances cognitive control in healthy subjects (Cho et al., 2010), and patient populations (Van den Eynde et al., 2010), with therapeutic effects in mechanistically related disorders such as addiction (Gorelick et al., 2014).

More recently, our group has shifted the rTMS stimulation target from the DLPFC to the DMPFC, as a potential treatment for major depression (Downar et al., 2014; Salomons et al., 2014; Bakker et al., 2015). As with first case report of DLPFC-rTMS for BN, we too found an unexpected remission of chronic treatment refractory binge and purge symptoms in an MDD patient with comorbid BN, following 20 sessions of 10 Hz DMPFC-rTMS. The onset of effect was rapid, occurring in the first week of treatment, and was maintained for 9 weeks post-treatment (Downar et al., 2012). In a follow-up, open-label series of 10 Hz DMPFC-rTMS in 28 ED patients with binge/purge behaviors, we noted ≥ 50% symptom reduction in 57%. On resting-state fMRI, we found increased resting-state functional connectivity in fronto-striatal salience network circuits (through DMPFC, anterior insula, and ventral striatum) specifically in the treatment responders but not non-responders (Dunlop J. et al., 2015), consistent with similar findings for DMPFC-rTMS in MDD and obsessive-compulsive disorder (Salomons et al., 2014; Dunlop K. et al., 2015). These findings suggest that DMPFC-rTMS may improve bulimic symptoms through an improvement of topdown cognitive control over urges, via frontostriatal circuits through salience-network nodes. Future work should include a

 nervosa; B-DMPFC, bilateral dorsomedial prefrontal cortex; BN, bulimia nervosa; ED, eating disorder; EDE-Q, eating disorder examination questionnaire; F, female; FC, food craving; HDRS, Hamilton Scale; L-DLPFC, left dorsolateral prefrontal cortex; L-PFC, left prefrontal cortex; MT, motor threshold; RCT, randomized control trial; RMT, resting motor threshold; rTMS, repetitive transcranial magnetic stimulation.

#### TABLE 3 | Overview of the available ED-tDCS literature.

A, tDCS studies related to food addiction and urges to eat; B, tDCS studies related to AN.

AN, anorexia nervosa; HC, healthy controls; EAT, eating attitudes test; EDI, eating disorder inventory; L-DLPFC, left dorsolateral prefrontal cortex; R-DLPFC, right dorsolateral prefrontal cortex; tDCS, transcranial direct current stimulation; VAS, visual analog scale.

sham-controlled arm, along with behavioral measures to better characterize the cognitive domains mediating the therapeutic effects of DMPFC-rTMS in BN.

#### NIBS as a Treatment for AN

To date, there are few published sham-controlled trials on tDCS and rTMS as treatments for AN (Bainbridge and Brown, 2014; McClelland et al., 2013). One preclinical study in a small sample of AN patients (n = 10) applied a single session of 10 Hz left DLPFC-rTMS, with patients reporting less anxiety and less feeling full and feeling fat (**Table 2**; Van den Eynde et al., 2013). An open-label case series in 5 AN patients applied 20 sessions of excitatory DLPFC-rTMS, reporting improvements in anxiety, feeling fat/full and urge to restrict/exercise over the course of treatment, enduring to 6 months; however, these effects had waned by 12-months post-treatment (McClelland et al., 2016). Another open-label series in 7 AN patients applied 10 sessions of anodal left DLPFC tDCS (**Table 3**), reporting improvements on the Eating Disorders Inventory (EDI) and the Eating Attitude Test (EAT) (Khedr et al., 2014). Although, these early publications are promising, further preliminary work in larger groups, with a longer course and sham control, must be performed to determine whether rTMS and tDCS are efficacious treatments for AN.

#### CONSIDERATIONS FOR FUTURE STUDIES

#### Patient Selection

In an attempt to limit heterogeneity, inclusion criteria for NIBS studies in ED patients are often based on DSM-5 diagnostic categories. However, as noted above, DSM-5 diagnoses still encompass substantial heterogeneity, and may conflate neurobiologically distinct endophenotypes. Future studies enrolling ED patients for NIBS trials should make efforts to at least characterize the underlying phenotypes within the clinical populations they are treating, and ideally should target a specific endophenotype associated with a specific neural substrate. Such studies should also measure behavioral or biological markers of this endophenotype to assess whether the target process was successfully engaged, and whether the engaged process did indeed mediate any observed symptomatic improvements.

#### Intervention Parameters

Several treatment parameters are important to consider when designing NIBS studies in ED. First, treatment parameters (protocol, total number of sessions, and number of sessions per day) needs to be selected, keeping in mind both patient convenience and therapeutic efficacy. In the older MDD-NIBS literature, 20–30 sessions of once daily rTMS is the standard protocol, with sessions lasting up to 45–60 min. However, such schedules are onerous for patients and limit overall clinic capacity. More recent studies have begun to explore much briefer protocols, such as 1–3 min theta-burst stimulation (Li et al., 2014), which have been reported to achieve equivalent or superior outcomes (Bakker et al., 2015). Other protocols, such as quadripulse stimulation (QPS), have been reported to achieve much more consistent effects across individuals (Huang et al., 2005; Tsutsumi et al., 2014). Still other recent MDD trials have delivered multiple sessions per day (up to five sessions a day), to complete the full course in 4–10 days rather than the usual 4– 6 weeks (Holtzheimer et al., 2010; Baeken et al., 2014). Future ED rTMS trials should make use of these innovations to reduce patient burden, increase capacity or consistency, and accelerate the pace of improvement.

#### Concurrent Tasks or Therapies

Another consideration for NIBS trials for ED is whether stimulation should be applied concurrently with psychotherapy or a specific cognitive/behavioral task, as opposed to simply during rest. This is especially the case if NIBS protocols are designed based on RDoC dimensions, and targets cortical regions based on abnormal activation on certain tasks. As discussed above, many areas, including the ACC/mPFC, DLPFC, insula, inferior frontal gyrus, and ventrolateral PFC are hyperactive to some tasks, but hypoactive in others. With stimulation during rest, it is difficult to assess or constrain the activation state of the underlying cortical target. Having the patient perform illness-specific cognitive task has now been shown to enhance (or reduce) the therapeutic effects of rTMS across several different indications. For example, reading trauma-related scripts during rTMS enhanced efficacy for PTSD (Isserles et al., 2013); undergoing rTMS in the presence of substance cues enhances efficacy in addiction (Dinur-Klein et al., 2014). Analogous approaches may be helpful in ED.

#### Treatment Target

A final consideration for ED-NIBS concerns the feasibility of the proposed target. Although, targets such as DLPFC, DMPFC, OFC, and TPJ have now been targeted in a variety of studies, others such as ventromedial prefrontal cortex or anterior insula may be difficult to reach without specially designed coils, and without also stimulating overlying structures. More feasibility studies are needed to assess how well that these areas can be engaged with rTMS and tDCS (Chib et al., 2013).

Another consideration during target selection is determining the appropriate stimulation intensity in the case of rTMS. For example, treatment intensity is determined by measuring the resting motor threshold of region of cortex directly posterior to stimulation site; in these cases, resting motor threshold is determined by the activation of the thumb or big toe for DLPFC and DMPFC, respectively (Schutter and van Honk, 2006; Hallett, 2007). It is therefore unclear for novel stimulatory sites what would be the most appropriate and reliable sites to determine optimal stimulation intensity. Studies using finite element modeling may also be helpful for optimizing stimulator placement and intensity (Nitsche et al., 2012).

The effects of rTMS also dramatically decrease the farther the site is from the scalp surface (Kozel et al., 2000), and so it is likely that stimulation intensity will have to be quite large for deep targets such as anterior insula or VMPFC. If this is the case, it is likely that pain tolerability will be a factor. In addition, trigeminal nerve pain, scalp pain, and headaches are common adverse effects associated with rTMS (Machii et al., 2006; Rossi and Hallett, 2009). Tolerability will need to be maintained when stimulating these novel targets, particularly in scalp regions with trigeminal innervation, such as the frontopolar, orbitofrontal, or temporopolar regions. This may be challenging for more intense rTMS protocols, although helmet-shaped "deep TMS" coil geometries may be somewhat helpful in allowing deep stimulation of these regions while maintaining tolerability (Roth et al., 2007). Certain targets (e.g., OFC, frontopolar cortex) may be more amenable to tDCS, which is relatively painless compared to rTMS. Another non-invasive technique worthy of future investigation is cutaneous non-invasive vagus nerve stimulation, which is also delivered via external electrodes. Its more invasive counterpart, surgically-implanted vagus nerve stimulation has recently shown some efficacy for medication-resistant depression (Ben-Menachem et al., 2015; Grimonprez et al., 2015).

Finally, stimulating multiple targets in a single session might be the optimal way to address all the abnormal behavioral dimensions in a given ED patient. Different ED symptom dimensions map to different cortical targets, and so confining stimulation to a single target may be insufficient to address multi-dimensional ED pathology. For example, in BN, excitatory stimulation of the DMPFC/insula combined with inhibitory stimulation of the VMPFC may be a more optimal strategy for enhancing cognitive control while reducing urge intensity. "Deep TMS" coils have been designed to stimulate multiple targets simultaneously (Dinur-Klein et al., 2014), and multichannel coils allow different protocols at different targets simultaneously (Roth et al., 2014). However, the therapeutic effects of sequential vs. simultaneous stimulation have not yet been compared directly. Further research should be done to describe the safety, tolerability, clinical efficacy, and neural mechanisms of stimulating multiple targets, either sequentially or simultaneously.

# CONCLUSION

Neuroimaging, psychometric, and behavioral findings are converging upon a new approach to classifying psychiatric disorders, including EDs, in terms of endophenotypes or symptom dimensions. New proposed frameworks, such as the RDoC, seek to describe EDs in terms of dysfunction in specific underlying brain functions such as cognitive control, positive and negative valence, and social/self-related cognition. These functions in turn are gradually being linked to specific neurobiological processes, described at multiple levels spanning clinical symptomatology, behavioral task performance, neuroimaging studies of macro-scale network function, and cellular, molecular, and genetic mechanisms. With the advent of anatomically focal NIBS interventions, a "neuroanatomical formulation" of ED pathology may become relevant not only for basic science, but for clinical care.

At present, neuroanatomical, endophenotypic, and RDoC formulations of ED pathology must be considered tentative and preliminary. However, from available literature, it does appear that some of the tremendous and dynamic heterogeneity of symptoms in the ED population can be understood parsimoniously in terms of dysfunction in a few key cognitive systems and their associated neural circuits. For example, in BN and BED, binge and purge behaviors may acquire pathologically strong incentive salience by mechanisms similar to addiction; impaired cognitive control in turn renders binge/purge urges hard to resist, particularly during negative affect. NIBS strategies designed for addiction (e.g., enhancing cognitive control via salience-network stimulation and damping urge intensity via ventromedial stimulation) may be helpful in this setting. In ANR, this strategy may be less helpful; instead, targeting pathologically overactive negative-valence systems may address the excessive valuation of secondary over primary rewards, and the underlying compulsivity. NIBS strategies developed for OCD (such as inhibitory stimulation of the OFC and DMPFC) may be more helpful in this setting. Ancillary NIBS strategies for AN may also target distortions of body image, alexithymia, and deficits of interoception via insular, TPJ, and EBA stimulation. However, it must be acknowledged that nearly all of these approaches are at present theoretically based, and lacking even in preclinical support. The field is urgently in need of future studies in clinical populations, with adequate sample sizes and sham controls, and using endophenotypic markers to validate or refute the proposed mechanisms of action for NIBS in EDs.

To conclude, patients with EDs stand to benefit tremendously from ongoing progress in three areas: symptom characterization, diagnostic formulation, and targeted intervention. Recent initiatives will allow us to make better sense of the heterogeneity of ED pathology, both across individuals and within individuals over time. As we improve our abilities to identify robust symptom clusters, link those clusters to neural substrates, and target those substrates with NIBS interventions, treatment outcomes will improve. These advances need not occur at the expense of existing and well-validated treatment strategies involving medications, psychotherapy, and behavior modification. Rather, they will likely work in a synergistic fashion to complement and facilitate our existing treatment strategies: enhancing the cognitive control that is a prerequisitive for

#### REFERENCES

successful cognitive-behavioral treatments in BN, or reducing the compulsivity and rigidity that hampers behavior modification in AN. Given the considerable patient burden and chronicity of EDs, these advances in treatment options will be a welcome change for patients, families and clinicians alike.

#### AUTHOR CONTRIBUTIONS

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

#### FUNDING

JD has received research support from the Canadian Institutes of Health Research, the National Institutes of Health, the Klarman Family Foundation, the Edgestone Family Foundation, the Toronto General and Western Hospital Foundation, and in-kind support from MagVenture for investigator-initiated studies. BW has received research support from the Ontario Mental Health Foundation, the Price Foundation, The Klarman Foundation, the Canadian Institute for Health Research, and the National Institute for Mental Health. KD has received research support from the Canadian Institutes of Health Research, Vanier Canada Scholarship Program.

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recovered women with anorexia nervosa. Front. Behav. Neurosci. 9:46. doi: 10.3389/fnbeh.2015.00046

Anorexia nervosa: a functional neuroimaging study. PLoS ONE 9:e97998. doi: 10.1371/journal.pone.0097998

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Dunlop, Woodside and Downar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Clinical Value of the Assessment of Changes in MEP Duration with Voluntary Contraction

Marisa Brum1, 2, Christopher Cabib3, 4 and Josep Valls-Solé3, 4 \*

<sup>1</sup> São Bernardo Hospital, Centro Hospitalar de Setúbal, Setúbal, Portugal, <sup>2</sup> Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Portugal, <sup>3</sup> EMG Unit, Neurology Department, Hospital Clinic, University of Barcelona, Barcelona, Spain, <sup>4</sup> Institut d'Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain

Transcranial magnetic stimulation (TMS) gives rise to muscle responses, known as motor evoked potentials (MEP), through activation of the motor pathways. Voluntary contraction causes facilitation of MEPs, which consists of shortening MEP latency, increasing MEP amplitude and widening MEP duration. While an increase in excitability of alpha motorneurons and the corticospinal tract can easily explain latency shortening and amplitude increase, other mechanisms have to be accounted for to explain the increase in duration. We measured the increase in duration of the MEP during contraction with respect to rest in a group of healthy volunteers and retrospectively assessed this parameter in patients who were examined in a standardized fashion during the past 5 years. We included 25 healthy subjects, 21 patients with multiple sclerosis, 33 patients with acute stroke, 5 patients with hereditary spastic paraparesis, and 5 patients with signs suggesting psychogenic paresis. We found already significant differences among groups in the MEP duration at rest, patients with MS had a significantly longer duration, and patients with stroke had significantly shorter duration, than the other two groups. The increase in MEP duration during voluntary contraction was different in patients and in healthy subjects. It was significantly shorter in MS and significantly longer in stroke patients. It was absent in the five patients with suspected psychogenic weakness. In patients with HSP, an abnormally increase in duration occurred only in leg muscles. Our results suggest that the increase in duration of the MEP during contraction may reveal the contribution of propriospinal interneurons to the activation of alpha motorneurons. This mechanism may be altered in some diseases and, therefore, the assessment proposed in this work may have clinical applicability for the differential diagnosis of weakness.

Keywords: motor evoked potential, contraction-induced facilitation, stroke, multiple sclerosis, spastic paraparesis, psychogenic weakness

# INTRODUCTION

Transcranial magnetic stimulation (TMS) gives rise to muscle responses, known as motor evoked potentials (MEP), through activation of the motor pathways (Amassian et al., 1987; Day et al., 1987, 1989; Rothwell et al., 1987, 1991). Voluntary contraction causes facilitation of the MEPs, which consists of shortening the MEP latency, increasing the MEP size and widening the MEP duration resulting in an increase in total MEP area (Mills et al., 1987; Valls-Solé et al., 1994a,b). While latency

#### Edited by:

Ignacio Obeso, Hospitales de Madrid and CEU-San Pablo University, Spain

#### Reviewed by:

Weien Yuan, Shanghai JiaoTong University, China Carmen Terranova, University of Messina, Italy Michele Dileone, Cinac-Hm Hospital Mostoles, Spain

> \*Correspondence: Josep Valls-Solé<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 17 October 2015 Accepted: 21 December 2015 Published: 11 January 2016

#### Citation:

Brum M, Cabib C and Valls-Solé J (2016) Clinical Value of the Assessment of Changes in MEP Duration with Voluntary Contraction. Front. Neurosci. 9:505. doi: 10.3389/fnins.2015.00505 shortening and amplitude increase can be well explained by assuming that voluntary contraction put more motorneurons near the firing threshold, the rationale behind the increase in MEP duration is less clear. On one side, motorneurons should be firing earlier and more synchronously to give rise to the shortening of latency and increase in peak amplitude. However, MEP duration increases beyond the time that the MEP would end if elicited at rest. Therefore, some motorneurons should receive excitatory inputs with latency longer than at rest. This could be due to activation of slow conducting corticospinal fibers or to the generation of excitatory inputs at spinal level (Brasil-Neto et al., 1992; Di Lazzaro et al., 1999, 2001), but it seems contradictory that slow conducting corticospinal fibers are recruited during voluntary contraction. Many sources of inhibition lead to the silent period after synchronized alpha motorneuron firing. After hyperpolarization prevents motorneurons from immediate reactivation and inhibitory inputs from Renshaw cells should reach the motorneuron within few milliseconds (ms) after the peak. Even if some inhibitory activity may be temporarily switched off during contraction, this does not justify the presence of EMG activity at a time when there are no identifiable excitatory inputs.

The EMG activity that follows the MEP during contraction is not usually taken into account in clinical assessment through TMS. The effects of facilitation are usually evaluated by either the onset latency or the size of the MEP. Onset of silent period is usually measured from more stable marks such as the stimulus artifact or MEP latency onset. We argue that measuring the characteristics of such segment of EMG activity might be of some clinical applicability. We reasoned that, when an MEP is elicited during voluntary contraction, inhibitory inputs generated at spinal level or by the descending volleys should counteract the excitatory commands to effectively end the ongoing EMG activity and give way to the silent period. This may take some time and, meanwhile, the EMG activity will not be suppressed. We considered that such "MEPtail," i.e., the EMG activity that follows the MEP peak and ends at the beginning of the silent period, should indicate the relationship between the strength of the excitatory inputs reaching the alpha motorneurons during voluntary contraction and the inhibitory inputs derived from the synchronized activation of alpha motorneurons at the time of the MEP. Therefore, we determined the extent of contraction-induced facilitation of the MEP in healthy subjects, with special attention to the increase in MEP duration, and performed a retrospective analysis of how such parameter was affected in various neurological disorders involving the motor pathway, i.e., multiple sclerosis (MS), stroke, hereditary spastic paraparesis (HSP), and psychogenic weakness.

#### METHODS

We performed a retrospective study of the recordings obtained within the last 5 years in healthy subjects and patients in whom we examined facilitation of the MEP in the first dorsal interosseous muscle (FDI), using a standardized method. We included 25 healthy subjects, 21 patients with multiple sclerosis, 33 patients with acute ischemic stroke, 5 patients with hereditary spastic paraparesis, and 5 patients with the clinical diagnosis of probable psychogenic paresis. In 12 healthy subjects and in the 5 patients with hereditary spastic paraparesis, we recorded the MEP also from the tibialis anterior (TA). The overall inclusion criteria were to have consistently elicitable MEPs at rest to cortical stimulation and a complete study carried out by following the protocol summarized below. Patients with multiple sclerosis were all diagnosed according to the McDonald criteria (Polman et al., 2011). They all had signs compatible with mild to moderate involvement of the motor pathway, with a mean Expanded Disability Status Scale of 3.5 (range between 2 and 6) and no one above four for the functional scale on pyramidal signs (Kurtzke, 1983). Patients with stroke were examined within the first 2 weeks after presentation of the lesion and had mild to severe hemiparesis due to a subcortical ischemic middle cerebral artery infarct. Patients with hereditary spastic paraparesis were all genetically mediated, spastin positive (SPG4), each of them belonging to a different family. Patients with psychogenic weakness had all normal diagnostic tests for possible lesions in the motor pathway and clinical evidence of inconsistent weakness, out of proportion of examination findings.

Retrospective data were all collected following a standardized protocol, which contemplated recording at rest and during a voluntary contraction of about 30% of maximum. The stimuli were applied with a Magstim (Magstim Company, Dyfed, UK), equipped with either a figure-of-8 coil for hand muscles or a circular coil for leg muscles. Subjects were sitting, relaxed, and alert. Silver/silver chloride disk electrodes were used for recording all responses. They were attached bilaterally, in a belly-tendon montage, over the FDI for the study of upper limb muscles, which was done in all subjects, and the TA for the study of lower limb muscles in healthy subjects and patients with HSP. EMG signals were filtered and amplified, and traces were recorded using a KeyPointNet electromyograph. Stimulus intensity was fixed at about 120% resting motor threshold, which, based on the recommendations of Rossini et al. (1994), we determined as the minimum stimulus intensity that gave rise to a MEP of at least 50µV amplitude in at least 50% of trials when TMS was applied to the appropriate scalp location for the target muscle, with the subject at rest. In all instances, we recorded a variable number of MEPs at each stimulation condition (between 2 and 10) and superimposed them at their best fit to facilitate parametric measuring.

In a group of newly recruited 10 healthy subjects, we examined again the effects of facilitation on the MEP to cortical stimulation and added the observation of the effects on the MEP elicited by cervical foraminal stimulation. We asked these subjects to perform two different levels of muscle contraction: mild (10% of maximum voluntary contraction) and strong (30% of their maximum voluntary contraction). At the time of testing, the participants signed an informed consent and the study protocol for retrospective data collection, as well as for the new study in healthy subjects, were approved by the Ethics committee of the Hospital Clinic of Barcelona.

#### DATA ANALYSIS

In each recording, we measured onset latency at the time that the EMG activity became consistently more than 10% above background amplitude, whether at rest or during contraction. Amplitude was measured from the maximum negative peak to the maximum positive peak. Total MEP duration was measured from MEP onset latency to the time at which the activity returned to baseline. We determined the percentage change for each MEP parameter during contraction with respect to rest. For the statistical comparison among groups, we chose to analyze the recordings from the dominant side in healthy subjects and the most impaired side in patients. In the 10 newly recruited subjects, we examined the effects of level of muscle contraction (mild and strong) on the MEP onset latency, peak-to-peak amplitude, and duration. We compared also the effects of cortical to those of foraminal stimulation. The outcome measure in which we focused our study was the increase in duration that takes place during contraction at the tail of the MEP (tail), as a specific aspect of MEP facilitation. This was measured in ms as the difference between the end of the MEP obtained during contraction and the end of the MEP obtained at rest.

All data were analyzed using SPSS 21.0 (IBM UK, London). We used repeated measures ANOVA for the analysis of the effects of level of muscle contraction in healthy subjects (rest, mild contraction and strong contraction). A One-way analysis of variance (ANOVA) was used to determine whether there were significant differences in retrospective data among the three independent groups with a sizeable number of subjects (healthy controls, multiple sclerosis, and stroke patients). In the other 2 groups of patients (HSP and psychogenic patients) the sample was too small to do the analyses. P ≤ 0.05 was considered statistically significant.

# RESULTS

The newly recruited subjects were 5 men and 5 women, with an age ranging from 32 to 65 (44 ± 7). In the subjects recruited retrospectively, mean age was significantly higher in patients with stroke than in the other groups (**Table 1**). Data were available from all subjects initially recruited, with no data gaps or missing information.

TABLE 1 | Demographic and general clinical characteristics of patients included in the retrospective study.

a significantly higher than in the other groups.

\*In leg muscles.

\*\*As for the patients first expression, with no encouragement.

Representative examples of recordings in one of the newly recruited healthy subject are shown in **Figure 1** to cortical and foraminal stimulation. A summary of the mean data is reported in **Table 2**. The effects of contraction on the MEP elicited with cortical stimulation were the expected ones: shortening of onset latency, increase in amplitude and increase in duration. The repeated measures ANOVA for values obtained when comparing the three conditions (rest, mild contraction, and strong contraction) showed statistically significant differences [ANOVA; F(2, 27) = 4.981; p = 0.004]. The post-hoc analysis showed that all significant differences were found when comparing contraction to rest (p < 0.05 for latency, amplitude and duration), but there were no significant differences between data obtained with mild and strong contraction (p > 0.05 for all comparisons). The MEP tail increased a mean of 4.1 ms (SD = 0.8 ms) with mild contraction and 4.4 ms (SD = 0.5 ms) with strong contraction (p > 0.05). With foraminal stimulation there were no significant changes in onset latency, amplitude or duration, although amplitude, and duration increased in a few trials in some subjects (**Table 2**). A burst was consistently seen at a mean latency of 38.7 ms (SD = 5.1 ms), interrupting the post-MEP silent period.

### Comparison of Data among Groups

**Table 3** shows the summary of retrospective data gathered for all groups on the MEP at rest and during contraction as well as the percentage change observed during contraction with respect to rest for onset latency, peak amplitude and MEP duration. Representative recordings of MEPs at rest and during contraction are shown in **Figure 2** for the FDI recordings in each group

of patients. There were already significant differences among groups in the characteristics of the MEPs recorded at rest. The repeated measures one-factor ANOVA, run only on data from the three groups with a sizeable number of subjects (healthy subjects, stroke, and MS patients), showed significant differences in onset latency, peak amplitude, and MEP duration [F(2, 76); p < 0.01 for all of them]. The post-hoc analyses showed that patients with MS and stroke had delayed onset latency and smaller peak amplitude than healthy subjects (p < 0.05 for both groups of patients in both comparisons). In regard to MEP duration, patients with MS had a significantly longer duration, while patients with stroke had significantly shorter duration, than the other two groups (p < 0.005 for all comparisons).

Muscle contraction induced facilitation to different degrees in healthy subjects and patients (**Table 3**). As expected, the

TABLE 2 | Data gathered from 10 newly recruited healthy volunteers on facilitation of the MEP with mild and strong voluntary muscle contractions.

Data are represented as mean and 1 standard deviation (within parenthesis). Data in the columns labeled % are the ratio between contraction and rest for mild and strong contractions. Asterisks refer to a significant difference (p < 0.05) with respect to rest, in the post-hoc analysis after one factor ANOVA.

percentage shortening in onset latency, increase in peak amplitude, and lengthening in MEP duration, including the increase in MEP tail, calculated in healthy subjects were all similar to those reported above on the newly recruited subjects. The effects of contraction on the MEP in patients were not uniform, and the percentage change with respect to rest showed significant differences in all comparisons [ANOVA; F(2, 76); p < 0.05]. The post-hoc analyses indicated a significantly bigger percentage of onset latency shortening, and a significantly larger percentage of peak amplitude increase, in stroke than in MS patients. In MEP duration, differences were found between groups in all comparisons: There was a smaller increase in MS patients than in healthy subjects and stroke patients, and a bigger increase in stroke patients than in healthy subjects and MS patients (p < 0.05 for all comparisons). No significant differences with respect to healthy subjects were observed in hand muscles in patients with HSP but they were observed in leg muscles (reported below). In patients with psychogenic weakness, MEP duration was similar at rest and during voluntary contraction, although a moderate shortening of latency and increase in amplitude was observed in some traces.

A more striking difference among groups was observed in the MEP tail (**Table 4**). The contraction-induced increase in MEP duration beyond the end of the MEP at rest was significantly different among most groups, including psychogenic patients, in the FDI.

#### MEPs from the Tibialis Anterior

Data were gathered from 12 healthy subjects and the 5 patients with HSP. Although we did not run comparative statistics because of the small number of subjects, differences between the two groups of subjects were already clear at rest (**Table 3**) and more so in the percentage increase of MEP duration with voluntary contraction. The mean MEP tail increase was markedly

Data are the mean and one standard deviation (within parenthesis) measured on the MEP recorded in the first dorsal interosseous (except for HV TA and HSP TA, which were recorded in the tibialis anterior). HV, Healthy volunteers; MS, Multiple sclerosis; HSP, Hereditary spastic paraparesis. The number in parenthesis in the first column refers to the number of patients included in the analysis.

Onset latency and duration are measured in ms; peak amplitude in mV. Percentage refers to the percentage of the MEP characteristics during contraction with respect to rest. Superindices a, b, and c refer to significant differences (p < 0.05) found in the comparison among the three groups with a sizeable sample (healthy subjects, patients with stroke, and patients with multiple sclerosis).

<sup>a</sup>Significantly longer than in healthy subjects.

<sup>b</sup>Significantly shorter than in healthy subjects.

<sup>c</sup>Significantly longer/larger in MS than in stroke.

<sup>d</sup>Significantly smaller/shorter in MS than in stroke.

TABLE 4 | Differences in MEP tail among groups of subjects.

Data are the mean calculated among all subjects of the same group. Superindices a and b refer to the statistical significance measured with the post-hoc comparison after onefactor ANOVA including all groups recorded in the same muscle.

<sup>a</sup>Significantly shorter than in control subjects.

<sup>b</sup>Significantly longer than in all other groups.

larger in healthy subjects than in patients (**Table 4**), with a significant difference in the unpaired t-test (p < 0.05). **Figure 3** shows representative TA recordings from healthy controls and patients with HSP.

#### DISCUSSION

The main physiological mechanism for MEP facilitation is, likely, the increase in spinal motorneuronal excitability. During voluntary contraction, the descending volley will meet with

more motoneurons available for firing than at rest (Di Lazzaro et al., 2008). Descending volleys after cortical stimulation in humans have been recorded with epidural electrodes (Di Lazzaro et al., 1999). While a direct (D) wave can be obtained with electrical stimulation, this is usually not so with TMS (Day et al., 1987; Di Lazzaro et al., 1999), where mostly indirect (I) waves are recorded. The I waves have an interpeak latency of about 1.2–2.0 ms, up to about 6 ms after the D wave (Day et al., 1987; Boniface et al., 1991; Nakamura et al., 1997). The ultimate target of such descending volleys is assumedly the alpha motorneuron, but an unknown number of excitatory and inhibitory propriospinal interneurons may lay in between. The number of descending I waves increases little when subjects perform a voluntary contraction (Di Lazzaro et al., 1999). Therefore, the large increase in the MEP duration reveals that facilitation takes place mostly because of summation of inputs from various sources at the alpha motorneuronal level.

Polyphasic MEPs have been reported so far in various diseases. In patients with ALS (Kohara et al., 1999), the authors suggested that polyphasic MEPs were due to activation of additional motor pathways, such as slow monosynaptic pyramidal or even polysynaptic pathways in ALS patients. In patients with DYT11-positive myoclonus-dystonia syndrome (van der Salm et al., 2009), the authors hypothesized that the mutation associated with the disease, SGCE gene, could have caused changes in membrane properties or ion channels, leading to asynchronous discharge timing of spinal motorneurons by the descending corticospinal activity. MEP polyphasia has been also reported in MS, who have already an increase in MEP duration at rest (Kukowski, 1993), compatible with increased temporal dispersion of the impulses reaching the spinal motorneurons. Recently, in patients with idiopathic generalized epilepsy (IGE) and first-degree relatives, Chowdhury et al. (2015) showed increased polyphasia, attributed to abnormal timing and patterning of the descending volleys in the corticospinal tract, and suggested that this was a novel endophenotype in this pathology. No data are available on changes in MEP duration in patients so far and, specifically in the increase in the MEP tail, beyond the end of the MEP at rest. We found that this parameter was the one discriminating better among our groups of subjects.

Patients with MS and HSP had longer duration of MEP already at rest, with little increase during contraction. In patients with stroke, if their damaged corticospinal tract was still excitable, the MEP increased significantly in amplitude and duration, indicating preservation of spinal mechanisms involved in contraction-induced facilitation. The only group studied in which we found no increase in duration was in the patients with psychogenic weakness. This was probably due to voluntary absence of energization of alpha motorneurons by these patients.

The mechanisms involved in the increase in MEP duration with muscle contraction are not clear but our results are consistent with a role for the excitatory pre-motor interneurons that are activated by descending inputs from contralateral corticospinal tract. Patients with MS and HSP, with lesions in the spinal cord, had increased duration at rest likely because of dispersion of the descending volley reaching the alpha motorneuron. These patients could not increase the synchronization of their motorneuronal firing, and the possible implication of the interneurons is masked by the already long duration of the MEP. In patients with stroke, whose spinal interneurons are not altered, the mechanisms of

REFERENCES

contraction-induced facilitation are fully activated, provided that the volley reaches the spinal level. In opposition, patients with psychogenic weakness do not perform the maximum voluntary contraction when requested, and may not set their spinal interneurons excitability at the level required for facilitation of the MEP during contraction.

The main limitation of our study is that it is retrospective and, therefore, it was not specifically designed for the study of the effects of duration. Even though the data reported here were generated after standardized recordings with no missing values, replication of the study is needed before firm conclusions. Meanwhile, we can conclude that the increase in duration of the MEP during contraction beyond the end of the resting MEP may reveal the activation of premotor propriospinal interneurons by descending inputs. This mechanism may be altered in some diseases. The patterns of MEP facilitation with voluntary contraction may differ depending on the disease, and the study of this feature can be of some clinical utility in the differential diagnosis of weakness.

# AUTHOR CONTRIBUTIONS

MB: Review of patients' reports, Analysis of data, and Writing the first draft. CC: Analysis of data, Statistical analysis, and Revision of the manuscript. JV: Conceptual design of the study and Revision and finalization of the manuscript.

### ACKNOWLEDGMENTS

Parts of this work were accomplished thanks to grant 110930 from Fundació Marató TV3.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Brum, Cabib and Valls-Solé. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Combining Robotic Training and Non-Invasive Brain Stimulation in Severe Upper Limb-Impaired Chronic Stroke Patients

Vincenzo Di Lazzaro1, 2 \*, Fioravante Capone1, 2, Giovanni Di Pino1, 2, Giovanni Pellegrino<sup>1</sup> , Lucia Florio<sup>1</sup> , Loredana Zollo<sup>3</sup> , Davide Simonetti <sup>3</sup> , Federico Ranieri 1, 2 , Nicoletta Brunelli 1, 2, Marzia Corbetto1, 2, Sandra Miccinilli <sup>4</sup> , Marco Bravi <sup>4</sup> , Stefano Milighetti <sup>4</sup> , Eugenio Guglielmelli <sup>3</sup> and Silvia Sterzi <sup>4</sup>

#### Edited by:

Marjan Jahanshahi, UCL Institute of Neurology, UK

#### Reviewed by:

Giacomo Koch, Santa Lucia IRCCS, Italy Michelle Nadine McDonnell, University of South Australia, Australia

#### \*Correspondence:

Vincenzo Di Lazzaro<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 13 November 2015 Accepted: 22 February 2016 Published: 08 March 2016

#### Citation:

Di Lazzaro V, Capone F, Di Pino G, Pellegrino G, Florio L, Zollo L, Simonetti D, Ranieri F, Brunelli N, Corbetto M, Miccinilli S, Bravi M, Milighetti S, Guglielmelli E and Sterzi S (2016) Combining Robotic Training and Non-Invasive Brain Stimulation in Severe Upper Limb-Impaired Chronic Stroke Patients. Front. Neurosci. 10:88. doi: 10.3389/fnins.2016.00088 <sup>1</sup> Unit of Neurology, Neurophysiology, Neurobiology, Department of Medicine, Università Campus Bio-Medico di Roma, Rome, Italy, <sup>2</sup> Fondazione Alberto Sordi - Research Institute for Ageing, Rome, Italy, <sup>3</sup> Unit of Biomedical Robotics and Biomicrosystems, Department of Engineering, Università Campus Bio-Medico di Roma, Rome, Italy, <sup>4</sup> Unit of Physical and Rehabilitation Medicine, Department of Medicine, Università Campus Bio-Medico di Roma, Rome, Italy