Indagine sul coinvolgimento del sistema neuropeptidergico nocicettina/recettore NOP a carico della trasmissione nocicettiva

Oggetto di studio in questa tesi è stato il ruolo modulatorio svolto dal neuropeptide nocicettina/orfanina FQ a carico della trasmissione nocicettiva. A scopo introduttivo, sono state illustrate le conoscenze attuali sul sistema nocicettina-NOP; sono state descritte le funzioni, la struttura e la distribuzione del recettore NOP, le azioni farmacologiche finora note e la distribuzione della nocicettina stessa al livello del S.N.C. e in periferia. Lo studio è stato condotto principalmente con due approcci differenti A) E’ stata studiata la capacità della nocicettina esogena o di suoi analoghi agonisti e antagonisti, di modificare la trasmissione nocicettiva. B) Sono state studiate le variazioni a carico del sistema endogeno nocicettina/recettore NOP in seguito a trattamenti di tipo farmacologico. A) E’ stata indagata la capacità della nocicettina e degli analoghi sintetici [Arg14, Lys15]N/OFQ e UFP-101 di modificare la soglia nocicettiva nel ratto, rilevata con il test del tail-flick, a seguito di somministrazione diretta nello spazio subaracnoideo, in confronto con la nocicettina stessa. La somministrazione intratecale del neuropeptide nocicettina (10 nmol/ratto) ha determinato un innalzamento statisticamente significativo delle latenze di risposta al test del tail-flick. L’analogo [Arg14, Lys15]N/OFQ è stato somministrato alla dose di 1 nmole/ratto i.t. provocando un innalzamento massimale delle soglie di latenza per tutto il periodo di osservazione, mentre alla dose 0,2 nmoli/ratto i.t ha provocato un effetto antinocicettivo sottomassimale pur dimostrandosi significativo rispetto ai controlli (p < 0,05 vs controlli a tutti i tempi di rilevazione). Il composto antagonista UFP-101 è risultato capace di antagonizzare l’azione sulla soglia analgesica sia della nocicettina sia dell’analogo [Arg14, Lys15]N/OFQ nel suo dosaggio minore, mentre contro la dose di 1 nmole/ratto i.t ha prodotto solamente una riduzione di effetto. Anche la somministrazione intratecale di MAP-N/OFQ si è dimostrata in grado di modificare la soglia nocicettiva determinata mediante il test del tail-flick, nel ratto, in modo dose dipendente. differentementeuna seconda somministrazione di MAP-N/OFQ dopo 24 ore, si è dimostrata totalmente inefficace nel modificare la soglia nocicettiva nei ratti precedentemente trattati, pur permanendo la loro suscettibilità all’azione analgesica della morfina, mostrando quindi il rapido sviluppo di tolerance al potente peptide nocicettinergico somministrato per via i.t.. Inoltre l’antagonista UFP-101 oltre ad essere ingrado di antagonizzare l’effetto della MAP-N/OFQ, ha mostrato la capacità di ridurre la tolerance sviluppata nei confronti del dendrimero. La somministrazione di MAP-N/OFQ per via i.c.v. ha prodotto variazione della soglia nocicettiva, producendo un innalzamento del volore soglia, dato contrastante con la maggior parte dei dati riguardanti la nocicettina in letteratura. Ha invece replicato l’effetto di antagonismo funzionale nei confronti della morfina, la quale dopo somministrazione di MAP-N/OFQ è risultata essere incapace di modificare la soglia nocicettiva nel ratto. Tale effetto perdura dopo 24 ore, quando una somministrazione di morfina produce un effetto analgesico inversamente proporzionale alla dose ricevuta di MAP-N/OFQ 24 ore prima. E’stato indagato il possibile ruolo neuromodulatorio del neuropeptide nocicettina esogeno, nell’analgesia prodotta da un farmaco di natura non oppiacea. In tal senso si è proceduto ad indagare l’eventuale capacità della nocicettina esogena, somministrata per via intracerebroventricolare e del suo analogo [Arg14, Lys15]N/OFQ, di antagonizzare l’analgesia prodotta dal farmaco paracetamolo. La nocicettina ha evidenziato la capacità di antagonizzare il potere antinocicettivo del paracetamolo fino a bloccarne completamente l’effetto al dosaggio più elevato, mostrando quindi proprietà antagonista dose-dipendente. Inoltre l’UFP-101, che di per se non altera l’analgesia indotta da paracetamolo, è ingrado di antagonizzare l’effetto della nocicettina sul paracetamolo in maniera dose-dipendente. Medesimo è risultato il comportamento dell’analogo della nocicettina, la Arg-Lys nocicettina. B) Sono state indagate le relazioni tra il sistema nocicettina/NOP e le proprietà farmacologiche di un noto farmaco oppiaceo quale la buprenorfina, le cui peculiari caratteristiche farmacodinamiche sano state recentemente collegate alla sua capacità di agire come agonista diretto al recettore NOP. In tal senso si è proceduto ad osservare l’effetto della somministrazione di buprenorfina sull’ assetto recettoriale di NOP, inseguito ad un trattamento prolungato con somministrazione sottocutanea mediante minipompe osmotiche nel ratto, rilevando successivamente, tramite uno studio di binding, le variazioni della densità recettoriale di NOP in alcune aree di interesse per la trasmissione nocicettiva. Sia nell’ippocampo che nel talamo e nella frontal cortex, la somministrazione prolungata di buprenorfina ha causato una riduzione significativa della densità recettoriale di NOP. Come ultimo aspetto indagato, al fine di determinare la presenza del neuropeptide nel liquido cerebrospinale e le sue eventuali modificazioni a seguito di manipolazioni farmacologiche e non farmacologiche, è stata messa a punto una metodica di perfusione dello spazio subaracnoideo nel ratto, che consentisse di ottenere materiale biologico su cui compiere la ricerca e quantificazione della presenza di nocicettina mediante dosaggio radioimmunologico. La perfusione di CSF artificiale arricchito di ione potassio ad una concentrazione pari a 60 mM ha evidenziato la possibilità di stimolare la liberazione della nocicettina nel liquido cerebrospinale di ratto, suggerendo quindi una sua provenienza da elementi eccitabili. E’ stato quindi possibile osservare l’andamento dei livelli di peptide a seguito della stimolazione nocicettiva prodotta da due agenti irritanti con caratteristiche differenti, la carragenina e la formalina. La somministrazione sottocutanea di carragenina (100 µl al 3 %) nella regione subplantare di entrambe le zampe posteriori del ratto non ha determinato alterazioni significative dei livelli di neuropeptide. Invece, la somministrazione di formalina (50 µl al 5 %), dopo un iniziale periodo di 30 minuti, ha causato un incremento significativo della liberazione di N/OFQ a partire dal terzo intervallo di raccolta seguente la somministrazione della sostanza. Questo rispecchia l’andamento di risposta al formalin test ottenuto anche mediante test di natura differente dagli analgesimetrici (es. comportamentale, elettrofisiologico), in quest’ottica l’aumento di nocicettina può essere interpretato come un evento dovuto alla sensibilizzazione centrale all’effetto pronocicettivo.

Sonno e funzioni cognitive: ruolo della microstruttura del sonno NREM

STUDY OBJECTIVE: Cyclic Alternating Pattern (CAP) is a fluctuation of the arousal level during NREM sleep and consists of the alternation between two phases: phase A (divided into three subtypes A1, A2, and A3) and phase B. A1 is thought to be generated by the frontal cortex and is characterized by the presence of K complexes or delta bursts; additionally, CAP A1 seems to have a role in the involvement of sleep slow wave activity in cognitive processing. Our hypothesis was that an overall CAP rate would have a negative influence on cognitive performance due to excessive fluctuation of the arousal level during NREM sleep. However, we also predicted that CAP A1 would be positively correlated with cognitive functions, especially those related to frontal lobe functioning. For this reason, the objective of our study was to correlate objective sleep parameters with cognitive behavioral measures in normal healthy adults. METHODS: 8 subjects (4 males; 4 females; mean age 27.75 years, range 2334) were recruited for this study. Two nocturnal polysomnography (night 2 and 3 = N2 and N3) were carried out after a night of adaptation. A series of neuropsychological tests were performed by the subjects in the morning and afternoon of the second day (D2am; D2pm) and in the morning of the third day (D3am). Raw scores from the neuropsychological tests were used as dependent variables in the statistical analysis of the results. RESULTS: We computed a series of partial correlations between sleep microstructure parameters (CAP, A1, A2 and A3 rate) and a number of indices of cognitive functioning. CAP rate was positively correlated with visuospatial working memory (Corsi block test), Trial Making Test Part A (planning and motor sequencing) and the retention of words from the Hopkins Verbal Learning Test (HVLT). Conversely, CAP was negatively correlated with visuospatial fluency (Ruff Figure Fluency Test). CAP A1 were correlated with many of the tests of neuropsychological functioning, such as verbal fluency (as measured by the COWAT), working memory (as measured by the Digit Span – Backward test), and both delay recall and retention of the words from the HVLT. The same parameters were found to be negatively correlated with CAP A2 subtypes. CAP 3 were negatively correlated with the Trial Making Test Parts A and B. DISCUSSION: To our knowledge this is the first study indicating a role of CAP A1 and A2 on behavioral cognitive performance of healthy adults. The results suggest that high rate of CAP A1 might be related to an improvement whereas high rate of CAP A2 to a decline of cognitive functions. Further studies need to be done to better determine the role of the overall CAP rate and CAP A3 on cognitive behavioral performances.

Social learning and action understanding in human observers: contributions of sensori-motor constraints and prior information

The aim of this thesis was to investigate the respective contribution of prior information and sensorimotor constraints to action understanding, and to estimate their consequences on the evolution of human social learning. Even though a huge amount of literature is dedicated to the study of action understanding and its role in social learning, these issues are still largely debated. Here, I critically describe two main perspectives. The first perspective interprets faithful social learning as an outcome of a fine-grained representation of others’ actions and intentions that requires sophisticated socio-cognitive skills. In contrast, the second perspective highlights the role of simpler decision heuristics, the recruitment of which is determined by individual and ecological constraints. The present thesis aims to show, through four experimental works, that these two contributions are not mutually exclusive. A first study investigates the role of the inferior frontal cortex (IFC), the anterior intraparietal area (AIP) and the primary somatosensory cortex (S1) in the recognition of other people’s actions, using a transcranial magnetic stimulation adaptation paradigm (TMSA). The second work studies whether, and how, higher-order and lower-order prior information (acquired from the probabilistic sampling of past events vs. derived from an estimation of biomechanical constraints of observed actions) interacts during the prediction of other people’s intentions. Using a single-pulse TMS procedure, the third study investigates whether the interaction between these two classes of priors modulates the motor system activity. The fourth study tests the extent to which behavioral and ecological constraints influence the emergence of faithful social learning strategies at a population level. The collected data contribute to elucidate how higher-order and lower-order prior expectations interact during action prediction, and clarify the neural mechanisms underlying such interaction. Finally, these works provide/open promising perspectives for a better understanding of social learning, with possible extensions to animal models.

The future in action: neurophysiological and behavioral evidence of anticipatory motor simulation.

The motor system can no longer be considered as a mere passive executive system of motor commands generated elsewhere in the brain. On the contrary, it is deeply involved in perceptual and cognitive functions and acts as an “anticipation device”. The present thesis investigates the anticipatory motor mechanisms occurring in two particular instances: i) when processing sensory events occurring within the peripersonal space (PPS); and ii) when perceiving and predicting others’actions. The first study provides evidence that PPS representation in humans modulates neural activity within the motor system, while the second demonstrates that the motor mapping of sensory events occurring within the PPS critically relies on the activity of the premotor cortex. The third study provides direct evidence that the anticipatory motor simulation of others’ actions critically relies on the activity of the anterior node of the action observation network (AON), namely the inferior frontal cortex (IFC). The fourth study, sheds light on the pivotal role of the left IFC in predicting the future end state of observed right-hand actions. Finally, the fifth study examines how the ability to predict others’ actions could be influenced by a reduction of sensorimotor experience due to the traumatic or congenital loss of a limb. Overall, the present work provides new insights on: i) the anticipatory mechanisms of the basic reactivity of the motor system when processing sensory events occurring within the PPS, and the same anticipatory motor mechanisms when perceiving others’ implied actions; ii) the functional connectivity and plasticity of premotor-motor circuits both during the motor mapping of sensory events occurring within the PPS and when perceiving others’ actions; and iii) the anticipatory mechanisms related to others’ actions prediction.

Wavelet analysis of heart rate variability related to nocturnal frontal lobe epilepsy seizures

Introduction: Nocturnal frontal lobe epilepsy (NFLE) is a distinct syndrome of partial epilepsy whose clinical features comprise a spectrum of paroxysmal motor manifestations of variable duration and complexity, arising from sleep. Cardiovascular changes during NFLE seizures have previously been observed, however the extent of these modifications and their relationship with seizure onset has not been analyzed in detail. Objective: Aim of present study is to evaluate NFLE seizure related changes in heart rate (HR) and in sympathetic/parasympathetic balance through wavelet analysis of HR variability (HRV). Methods: We evaluated the whole night digitally recorded video-polysomnography (VPSG) of 9 patients diagnosed with NFLE with no history of cardiac disorders and normal cardiac examinations. Events with features of NFLE seizures were selected independently by three examiners and included in the study only if a consensus was reached. Heart rate was evaluated by measuring the interval between two consecutive R-waves of QRS complexes (RRi). RRi series were digitally calculated for a period of 20 minutes, including the seizures and resampled at 10 Hz using cubic spline interpolation. A multiresolution analysis was performed (Daubechies-16 form), and the squared level specific amplitude coefficients were summed across appropriate decomposition levels in order to compute total band powers in bands of interest (LF: 0.039062 - 0.156248, HF: 0.156248 - 0.624992). A general linear model was then applied to estimate changes in RRi, LF and HF powers during three different period (Basal) (30 sec, at least 30 sec before seizure onset, during which no movements occurred and autonomic conditions resulted stationary); pre-seizure period (preSP) (10 sec preceding seizure onset) and seizure period (SP) corresponding to the clinical manifestations. For one of the patients (patient 9) three seizures associated with ictal asystole were recorded, hence he was treated separately. Results: Group analysis performed on 8 patients (41 seizures) showed that RRi remained unchanged during the preSP, while a significant tachycardia was observed in the SP. A significant increase in the LF component was instead observed during both the preSP and the SP (p<0.001) while HF component decreased only in the SP (p<0.001). For patient 9 during the preSP and in the first part of SP a significant tachycardia was observed associated with an increased sympathetic activity (increased LF absolute values and LF%). In the second part of the SP a progressive decrease in HR that gradually exceeded basal values occurred before IA. Bradycardia was associated with an increase in parasympathetic activity (increased HF absolute values and HF%) contrasted by a further increase in LF until the occurrence of IA. Conclusions: These data suggest that changes in autonomic balance toward a sympathetic prevalence always preceded clinical seizure onset in NFLE, even when HR changes were not yet evident, confirming that wavelet analysis is a sensitive technique to detect sudden variations of autonomic balance occurring during transient phenomena. Finally we demonstrated that epileptic asystole is associated with a parasympathetic hypertonus counteracted by a marked sympathetic activation.

Cognitive and affective processes in social actions and decisions

The question of how we make, and how we should make judgments and decisions has occupied thinkers for many centuries. This thesis has the aim to add new evidences to clarify the brain’s mechanisms for decisions. The cognitive and the emotional processes of social actions and decisions are investigated with the aim to understand which brain areas are mostly involved. Four experimental studies are presented. A specific kind of population is involved in the first study (as well as in study III) concerning patients with lesion of ventromedial prefrontal cortex (vmPFC). This region is collocated in the ventral surface of frontal lobe, and it seems have an important role in social and moral decision in forecasting the negative emotional consequences of choice. In study I, it is examined whether emotions, specifically social emotions subserved by the vmPFC, affect people’s willingness to trust others. In study II is observed how incidental emotions could encourage trusting behaviour, especially when individuals are not aware of emotive stimulation. Study III has the aim to gather a direct psychophysiological evidence, both in healthy and neurologically impaired individuals, that emotions are crucially involved in shaping moral judgment, by preventing moral violations. Study IV explores how the moral meaning of a decision and its subsequent action can modulate the basic component of action such as sense of agency.

Role of nitric oxide and endocannabinoids in synaptic plasticity in the perirhinal cortex and in visual recognition memory

Introduction and aims of the research Nitric oxide (NO) and endocannabinoids (eCBs) are major retrograde messengers, involved in synaptic plasticity (long-term potentiation, LTP, and long-term depression, LTD) in many brain areas (including hippocampus and neocortex), as well as in learning and memory processes. NO is synthesized by NO synthase (NOS) in response to increased cytosolic Ca2+ and mainly exerts its functions through soluble guanylate cyclase (sGC) and cGMP production. The main target of cGMP is the cGMP-dependent protein kinase (PKG). Activity-dependent release of eCBs in the CNS leads to the activation of the Gαi/o-coupled cannabinoid receptor 1 (CB1) at both glutamatergic and inhibitory synapses. The perirhinal cortex (Prh) is a multimodal associative cortex of the temporal lobe, critically involved in visual recognition memory. LTD is proposed to be the cellular correlate underlying this form of memory. Cholinergic neurotransmission has been shown to play a critical role in both visual recognition memory and LTD in Prh. Moreover, visual recognition memory is one of the main cognitive functions impaired in the early stages of Alzheimer’s disease. The main aim of my research was to investigate the role of NO and ECBs in synaptic plasticity in rat Prh and in visual recognition memory. Part of this research was dedicated to the study of synaptic transmission and plasticity in a murine model (Tg2576) of Alzheimer’s disease. Methods Field potential recordings. Extracellular field potential recordings were carried out in horizontal Prh slices from Sprague-Dawley or Dark Agouti juvenile (p21-35) rats. LTD was induced with a single train of 3000 pulses delivered at 5 Hz (10 min), or via bath application of carbachol (Cch; 50 μM) for 10 min. LTP was induced by theta-burst stimulation (TBS). In addition, input/output curves and 5Hz-LTD were carried out in Prh slices from 3 month-old Tg2576 mice and littermate controls. Behavioural experiments. The spontaneous novel object exploration task was performed in intra-Prh bilaterally cannulated adult Dark Agouti rats. Drugs or vehicle (saline) were directly infused into the Prh 15 min before training to verify the role of nNOS and CB1 in visual recognition memory acquisition. Object recognition memory was tested at 20 min and 24h after the end of the training phase. Results Electrophysiological experiments in Prh slices from juvenile rats showed that 5Hz-LTD is due to the activation of the NOS/sGC/PKG pathway, whereas Cch-LTD relies on NOS/sGC but not PKG activation. By contrast, NO does not appear to be involved in LTP in this preparation. Furthermore, I found that eCBs are involved in LTP induction, but not in basal synaptic transmission, 5Hz-LTD and Cch-LTD. Behavioural experiments demonstrated that the blockade of nNOS impairs rat visual recognition memory tested at 24 hours, but not at 20 min; however, the blockade of CB1 did not affect visual recognition memory acquisition tested at both time points specified. In three month-old Tg2576 mice, deficits in basal synaptic transmission and 5Hz-LTD were observed compared to littermate controls. Conclusions The results obtained in Prh slices from juvenile rats indicate that NO and CB1 play a role in the induction of LTD and LTP, respectively. These results are confirmed by the observation that nNOS, but not CB1, is involved in visual recognition memory acquisition. The preliminary results obtained in the murine model of Alzheimer’s disease indicate that deficits in synaptic transmission and plasticity occur very early in Prh; further investigations are required to characterize the molecular mechanisms underlying these deficits.

The role of medial parieto occipital cortex in visuospatial attention and reach planning: electrophysiological studies in human and non-human primates

We usually perform actions in a dynamic environment and changes in the location of a target for an upcoming action require both covert shifts of attention and motor planning update. In this study we tested whether, similarly to oculomotor areas that provide signals for overt and covert attention shifts, covert attention shifts modulate activity in cortical area V6A, which provides a bridge between visual signals and arm-motor control. We performed single cell recordings in monkeys trained to fixate straight-ahead while shifting attention outward to a peripheral cue and inward again to the fixation point. We found that neurons in V6A are influenced by spatial attention demonstrating that visual, motor, and attentional responses can occur in combination in single neurons of V6A. This modulation in an area primarily involved in visuo-motor transformation for reaching suggests that also reach-related regions could directly contribute in the shifts of spatial attention necessary to plan and control goal-directed arm movements. Moreover, to test whether V6A is causally involved in these processes, we have performed a human study using on-line repetitive transcranial magnetic stimulation over the putative human V6A (pV6A) during an attention and a reaching task requiring covert shifts of attention and reaching movements towards cued targets in space. We demonstrate that the pV6A is causally involved in attention reorienting to target detection and that this process interferes with the execution of reaching movements towards unattended targets. The current findings suggest the direct involvement of the action-related dorso-medial visual stream in attentional processes, and a more specific role of V6A in attention reorienting. Therefore, we propose that attention signals are used by the V6A to rapidly update the current motor plan or the ongoing action when a behaviorally relevant object unexpectedly appears at an unattended location.

Spatio-temporal models of the functional architecture of the visual cortex

In this work I tried to explore many aspects of cognitive visual science, each one based on different academic fields, proposing mathematical models capable to reproduce both neuro-physiological and phenomenological results that were described in the recent literature. The structure of my thesis is mainly composed of three chapters, corresponding to the three main areas of research on which I focused my work. The results of each work put the basis for the following, and their ensemble form an homogeneous and large-scale survey on the spatio-temporal properties of the architecture of the visual cortex of mammals.

The posterior parietal cortex: a bridge between vision and action

The present work takes into account three posterior parietal areas, V6, V6A, and PEc, all operating on different subsets of signals (visual, somatic, motor). The work focuses on the study of their functional properties, to better understand their respective contribution in the neuronal circuits that make possible the interactions between subject and external environment. In the caudalmost pole of parietal lobe there is area V6. Functional data suggest that this area is related to the encoding of both objects motion and ego-motion. However, the sensitivity of V6 neurons to optic flow stimulations has been tested only in human fMRI experiments. Here we addressed this issue by applying on monkey the same experimental protocol used in human studies. The visual stimulation obtained with the Flow Fields stimulus was the most effective and powerful to activate area V6 in monkey, further strengthening this homology between the two primates. The neighboring areas, V6A and PEc, show different cytoarchitecture and connectivity profiles, but are both involved in the control of reaches. We studied the sensory responses present in these areas, and directly compared these.. We also studied the motor related discharges of PEc neurons during reaching movements in 3D space comparing also the direction and depth tuning of PEc cells with those of V6A. The results show that area PEc and V6A share several functional properties. Area PEc, unlike V6A, contains a richer and more complex somatosensory input, and a poorer, although complex visual one. Differences emerged also comparing the motor-related properties for reaches in depth: the incidence of depth modulations in PEc and the temporal pattern of modulation for depth and direction allow to delineate a trend among the two parietal visuomotor areas.

Rationale for an adjunctive therapy with fenofibrate in pharmacoresistant nocturnal frontal lobe epilepsy (NFLE).

Nocturnal Frontal Lobe Epilepsy (NFLE) is characterized by onset during infancy or childhood with persistence in adulthood, family history of similar nocturnal episodes simulating non-REM parasomnias (sleep terrors or sleepwalking), general absence of morphological substrates, often by normal interictal electroencephalographical recordings (EEGs) during wakefulness. A family history of epilepsy may be present with Mendelian autosomal dominant inheritance has been described in some families. Recent studies indicate the involvement of neuronal nicotinic acetylcholine receptors (nAChRs) in the molecular mechanisms of NFLE. Mutations in the genes encoding for the α4 (CHRNA4) and ß2 (CHRNB2) subunits of the nAChR induce changes in the biophysical properties of nAChR, resulting generally in a “gain of function”. Preclinical studies report that activation of a nuclear receptor called type peroxisome proliferator-activated receptor (PPAR-α) by endogenous molecules or by medications (e.g. fenofibrate) reduces the activity of the nAChR and, therefore, may decrease the frequency of seizures. Thus, we hypothesize that negative modulation of nAChRs might represent a therapeutic strategy to be explored for pharmacological treatment of this form of epilepsy, which only partially responds to conventional antiepileptic drugs. In fact, carbamazepine, the current medication for NFLE, abolishes the seizures only in one third of the patients. The aim of the project is: 1)_to verify the clinical efficacy of adjunctive therapy with fenofibrate in pharmacoresistant NFLE and ADNFLE patients; focousing on the analysis of the polysomnographic action of the PPAR- agonist (fenofibrate). 2)_to demonstrate the subtended mechanism of efficacy by means of electrophysiological and behavioral experiments in an animal model of the disease: particularly, transgenic mice carrying the mutation in the nAChR 4 subunit (Chrna4S252F) homologous to that found in the humans. Given that a PPAR-α agonist, FENOFIBRATE, already clinically utilized for lipid metabolism disorders, provides a promising therapeutic avenue in the treatment of NFLE\ADNFLE.