Targeting the adenosinergic system in restless legs syndrome: A pilot, "proof-of-concept" placebo-controlled TMS-based protocol.

Restless Legs Syndrome (RLS) is a common sleep disorder characterized by an urge to move the legs that is responsive to movement (particularly during rest), periodic leg movements during sleep, and hyperarousal. Recent evidence suggests that the involvement of the adenosine system may establish a connection between dopamine and glutamate dysfunction in RLS. Transcranial magnetic stimulation (TMS) is a non-invasive electrophysiological technique widely applied to explore brain electrophysiology and neurochemistry under different experimental conditions. In this pilot study protocol, we aim to investigate the effects of dipyridamole (a well-known enhancer of adenosinergic transmission) and caffeine (an adenosine receptor antagonist) on measures of cortical excitation and inhibition in response to TMS in patients with primary RLS. Initially, we will assess cortical excitability using both single- and paired-pulse TMS in patients with RLS. Then, based on the measures obtained, we will explore the effects of dipyridamole and caffeine, in comparison to placebo, on various TMS parameters related to cortical excitation and inhibition. Finally, we will evaluate the psycho-cognitive performance of RLS patients to screen them for cognitive impairment and/or mood-behavioral dysfunction, thus aiming to correlate psycho-cognitive findings with TMS data. Overall, this study protocol will be the first to shed lights on the neurophysiological mechanisms of RLS involving the modulation of the adenosine system, thus potentially providing a foundation for innovative "pharmaco-TMS"-based treatments. The distinctive TMS profile observed in RLS holds indeed the potential utility for both diagnosis and treatment, as well as for patient monitoring. As such, it can be considered a target for both novel pharmacological (i.e., drug) and non-pharmacological (e.g., neuromodulatory), "TMS-guided", interventions.

Representation of movement velocity in the rat's interpositus nucleus during passive forelimb movements.

The interpositus nucleus (IN) receives a large amount of sensory information from the limbs and, in turn, elaborates signals for movement control. In this paper, we tried to gather evidence on the possibility that neurons in the IN may elaborate sensory representations of the forelimb kinematics and, particularly, of the movement velocity vector. For this purpose, the forepaw of anesthetized rats was attached to a computer-controlled robot arm displaced passively along two types of trajectories (circular and figure eight), with the limb joints unconstrained. The firing activity of single cells was recorded and related to limb position and the two components of the movement velocity vector, namely, movement speed and direction. By using multiple regression analysis, we found that 12 out of 85 (14%) neurons were modulated by position, 18 out of 85 (21%) neurons were modulated by direction, 24 out of 85 (28%) neurons were modulated by movement speed, and 31 out of 85 (37%) neurons were sensitive to the full movement velocity vector. Most of the neurons modulated only by the speed component of the velocity vector (19 out of 24) were located in the posterior portion of the IN, whereas neurons in the anterior portion were mostly related to both components of the velocity vector. These results suggest that sensory information related to whole-limb movement velocity may be encoded by the IN, indicating also that the posterior interpositus may preferentially represent movement speed.

Neuronal NOS expression in rat's cuneate nuclei following passive forelimb movements and median nerve stimulation.

Nitric oxide (NO) synthase (NOS) has been observed in the Cuneate Nuclei (CN), suggesting a role for NO in the modulation of their neurons' activity. The present study was undertaken to evaluate whether passive movement of forelimb as well as electric stimulation of medial nerve modulate the expression of neuronal isoform of NOS (nNOS) within CN. The experiments were carried out on 21 male Wistar rats, by using two different protocols. In the first group of rats the median nerve was stimulated with high frequency trains (phasic stimulation) or at constant frequency (tonic stimulation); as a control, in the third group, no stimulus was delivered. Moreover, in the second group of rats, we imposed to the animal's left forepaw circular paths at a roughly constant speed (continuous movement), or rapid flexions and extensions of the wrist (sudden movement); as a control, in the third group, no movement was imposed. After the experimental session, free-floating frontal sections of medulla oblongata were processed for nNOS or glutamate (GLU) immunohistochemistry. Phasic stimulation of the median nerve or sudden movements of the forelimb determines a significant decrement of the nNOS-positive neurons within the ipsilateral CN, whereas no effects were observed on GLU positive cells. We have also found a peculiar topographical distribution within IN of nNOS-positive neurons: positive cells were clustered at periphery of some "niches" having circular or elliptical form, with GLU positive cells at center.

Kinematic features of passive forelimb movements and rat cuneate neuron discharges.

We examined the role of main and external cuneate nuclei neurons in processing sensory information during forelimb passive movement. We recorded activity of neurons using circular and figure-eight trajectories, at different speeds, in anaesthetized rats. A multivariate regression analysis was performed to correlate neural discharge to movement direction and speed, the two components of the velocity vector. We found that the activity of the majority of cuneate neurons related to passive movement velocity and that the directional component of the velocity vector accounted for a larger fraction of the variability in the firing rate than the scalar component (speed). These results indicate that cuneate cells can process whole limb afferent information to elaborate a representation of the movement velocity vector.

Anisotropic representation of forelimb position in the cerebellar cortex and nucleus interpositus of the rat.

The relationship between the spatial location of limb and the activity of cerebellar neurons has received little attention and its nature still remains ambiguous. To address this question we studied the activity of Purkinje and nucleus interpositus cells in relation to the spatial location of rat forelimb. A computer-controlled robot arm displaced the limb passively across 15 positions distributed on a parasagittal plane. The limb was upheld for 8 s in each position, which was identified by the Cartesian coordinates of the forepaw. We selected the neurons whose activities were significantly modulated by forepaw position and found that the majority represented preferentially one spatial dimension of the Cartesian plane both in the cerebellar cortex and nucleus interpositus. In particular, the antero-posterior axis was best represented in cerebellar neuronal discharges. This result suggests that the intermediate part of the cerebellum might encode limb position by way of an anisotropic representation of the spatial coordinates of the limb end-point.

Influence of nitric oxide on the activity of cuneate neurons in the rat.

Some neurons of main and external cuneate nuclei are immunoreactive for nitric oxide (NO) synthase, suggesting a role for endogenous NO in the early stages of somatosensory processing. We tested this hypothesis by investigating the possibility that NO modulates cuneate discharge. We observed that both spontaneous and N-methyl-D-aspartate-evoked activities of cuneate neurons were decreased by NO precursor L-arginine. The inhibition of NO synthase, by application of N-nitro-L-arginine methyl ester, instead, abolished the depressant effect induced by L-arginine. Our data suggest a NO modulation of cuneate neurons and provide support for a physiologic role not only in increasing the signal-to-noise ratio in the excited cells but also in a form of surround inhibition.