Ipsilateral Inhibition Research Articles

Multimodal sensory control of motor performance by glycinergic interneurons of the mouse spinal cord deep dorsal horn

Sensory feedback is integral for contextually appropriate motor output, yet the neural circuits responsible remain elusive. Here, we pinpoint the medial deep dorsal horn of the mouse spinal cord as a convergence point for proprioceptive and cutaneous input. Within this region, we identify a population of tonically active glycinergic inhibitory neurons expressing parvalbumin. Using anatomy and electrophysiology, we demonstrate that deep dorsal horn parvalbumin-expressing interneuron (dPV) activity is shaped by convergent proprioceptive, cutaneous, and descending input. Selectively targeting spinal dPVs, we reveal their widespread ipsilateral inhibition onto pre-motor and motor networks and demonstrate their role in gating sensory-evoked muscle activity using electromyography (EMG) recordings. dPV ablation altered limb kinematics and step-cycle timing during treadmill locomotion and reduced the transitions between sub-movements during spontaneous behavior. These findings reveal a circuit basis by which sensory convergence onto dorsal horn inhibitory neurons modulates motor output to facilitate smooth movement and context-appropriate transitions.

Abstract TMP96: Maladaptive Plasticity In Dystonia Following Pediatric Basal Ganglia Stroke: Associations Between Structural Connectivity And Cognitive Functioning

Background: Dystonia is one of the most common movement disorders after pediatric basal ganglia stroke, causing significant disability. It presents several months post stroke suggesting a role of maladaptive neuroplasticity in its manifestation. Children with dystonia show additional difficulties in intelligence (IQ), academics, and cognitive inhibition, beyond those associated with stroke. Objective: To examine structural connectivity in children with post-stroke dystonia and its relationship with cognitive outcomes. Method: A prospective study of children with unilateral basal ganglia stroke and dystonia and healthy controls. Diffusion tensor imaging was used to assess white matter integrity in the cingulum and superior longitudinal fasciculus (SLF) using fractional anisotropy (FA) and mean diffusivity (MD). Participants completed measures of IQ and cognitive inhibition and flexibility. Results: Six children with basal ganglia stroke and dystonia (6 female; Median age= 15.98, IQR= 4.32) and 6 neurotypical controls (3 male; Median age= 18.01, IQR= 5.13) were included. Participants were age-matched within 1-3 years. Hemispheres were matched by dominance such that if the stroke was in the dominant hemisphere the control’s dominant hemisphere was matched to the ipsilateral side and vice versa. Mann Whitney-U tests showed children with dystonia had poorer performance on measures of IQ (U= 0, p=. 002, η 2 = .69) and inhibition (U= 4.5, p= .026, η 2 = .39). FA was lower in the ipsilateral cingulum (U= 5, p= .041, η 2 = .36) and MD was higher in the ipsilateral SLF (U= 32, p= .026, η 2 = .42) in the dystonia participants. Significant associations (p< .05) were found between ipsilateral cingulum FA and IQ (r= .56), inhibition (r= .76), and flexibility (r= .60); contralateral cingulum FA and inhibition (p= .59); ipsilateral SLF FA and inhibition (r= .51) and flexibility (r= .58); and ipsilateral SLF MD and inhibition (r= -.54) and flexibility (r= -.55). Associations between the contralateral cingulum FA and IQ (r= .47, p= .060) and flexibility (r= .50, p= .051) trended to significance. Conclusion: Findings support the hypothesis that post-stroke dystonia reflects maladaptive reorganization after injury that is associated with motor and cognitive outcome.

Continuous theta burst stimulation increases contralateral mu and beta rhythms with arm elevation: implications for neurorehabilitation.

The study of the physiological effects underlying brain response to transcranial magnetic stimulation is important to understand its impact on neurorehabilitation. We aim to analyze the impact of a transcranial magnetic stimulation protocol, the continuous theta burst (cTBS), on human neurophysiology, particularly on contralateral motor rhythms. cTBS was applied in 20 subjects over the primary motor cortex. We recorded brain electrical activity pre- and post-cTBS with electroencephalography both at rest and while performing motor tasks, to evaluate changes in brain oscillatory patterns such as mu and beta rhythms. Moreover, we measured motor-evoked potentials before and after cTBS to assess its impact on brain's excitability. On the hemisphere contralateral to the protocol, we did observe a significant increase in mu (p = 0.027) and beta (p = 0.006) rhythms from pre- to post-cTBS, at the beginning of arm elevation. The topology of action planning and motor execution suggests that cTBS produced an inhibitory effect that propagated to the contralateral hemisphere, thereby precluding the expected/desired excitation for therapy purposes. This novel approach provides support for the notion that this protocol induces inhibitory changes in contralateral motor rhythms, by decreasing desynchronization, contradicting the ipsilateral inhibition vs. contralateral disinhibition hypothesis. Our results have implications for personalized cTBS usage as a rehabilitation intervention, suggesting that an unexpected propagation of inhibition can occur.

Spinal V2b neurons reveal a role for ipsilateral inhibition in speed control.

The spinal cord contains a diverse array of interneurons that govern motor output. Traditionally, models of spinal circuits have emphasized the role of inhibition in enforcing reciprocal alternation between left and right sides or flexors and extensors. However, recent work has shown that inhibition also increases coincident with excitation during contraction. Here, using larval zebrafish, we investigate the V2b (Gata3+) class of neurons, which contribute to flexor-extensor alternation but are otherwise poorly understood. Using newly generated transgenic lines we define two stable subclasses with distinct neurotransmitter and morphological properties. These V2b subclasses synapse directly onto motor neurons with differential targeting to speed-specific circuits. In vivo, optogenetic manipulation of V2b activity modulates locomotor frequency: suppressing V2b neurons elicits faster locomotion, whereas activating V2b neurons slows locomotion. We conclude that V2b neurons serve as a brake on axial motor circuits. Together, these results indicate a role for ipsilateral inhibition in speed control.

Selective Inhibition of Volitional Hand Movements after Stimulation of the Dorsoposterior Parietal Cortex in Humans

Inhibition is a central component of motor control. Although current models emphasize the involvement of frontal networks [1, 2], indirect evidence suggests a potential contribution of the posterior parietal cortex (PPC). This region is active during inhibition of upper-limb movements to undesired targets [3], and its stimulation with single magnetic pulses can depress motor-evoked potentials [4, 5]. Also, it has been speculated that alien hand movements caused by focal parietal lesions reflect a release of inhibition from PPC to M1 [6]. Considering these observations, we instructed 16 patients undergoing awake brain surgery to perform continuous hand movements while electrical stimulation was applied over PPC. Within a restricted dorsoposterior area, we identified focal sites where stimulation prevented movement initiation and instantly inhibited ongoing responses (which restarted promptly at stimulation offset). Inhibition was selective of the instructed response. It did not affect speech, hand movements passively generated through muscle electrical stimulation, or the ability to initiate spontaneous actions with other body segments (e.g., the feet). When a patient inadvertently performed a bilateral movement, a bilateral inhibition was found. When asked to produce unilateral movements, this patient presented a contralesional but not ipsilateral inhibition. This selectivity contrasted sharply with the unspecific inhibitions reported by previous studies within frontal regions, where speech and all limbs are typically affected (as we here confirm in a subset of patients) [7-10]. These results provide direct evidence that a specific area in the dorsoposterior parietal cortex can inhibit volitional upper-limb responses with high selectivity.

Time evolution of interhemispheric coupling in a model of focal neocortical epilepsy in mice.

Epilepsy is characterized by substantial network rearrangements leading to spontaneous seizures and little is known on how an epileptogenic focus impacts on neural activity in the contralateral hemisphere. Here, we used a model of unilateral epilepsy induced by injection of the synaptic blocker tetanus neurotoxin (TeNT) in the mouse primary visual cortex (V1). Local field potential (LFP) signals were simultaneously recorded from both hemispheres of each mouse in acute phase (peak of toxin action) and chronic condition (completion of TeNT effects). To characterize the neural electrical activities the corresponding LFP signals were analyzed with several methods of time series analysis. For the epileptic mice, the spectral analysis showed that TeNT determines a power redistribution among the different neurophysiological bands in both acute and chronic phases. Using linear and nonlinear interdependence measures in both time and frequency domains, it was found in the acute phase that TeNT injection promotes a reduction of the interhemispheric coupling for high frequencies (12-30 Hz) and small time lag (<20 ms), whereas an increase of the coupling is present for low frequencies (0.5-4 Hz) and long time lag (>40 ms). On the other hand, the chronic period is characterized by a partial or complete recovery of the interhemispheric interdependence level. Granger causality test and symbolic transfer entropy indicate a greater driving influence of the TeNT-injected side on activity in the contralateral hemisphere in the chronic phase. Lastly, based on experimental observations, we built a computational model of LFPs to investigate the role of the ipsilateral inhibition and exicitatory interhemispheric connections in the dampening of the interhemispheric coupling. The time evolution of the interhemispheric coupling in such a relevant model of epilepsy has been addressed here.

Motor cortex activation during motor imagery of the upper limbs in stroke patients

ABSTRACT Objective: The objective of this study was to analyze the functional brain activation in acute stroke patients during motor execution (ME) and motor imagery (MI) and to discuss the association between damaged brain structure and impaired brain function in stroke patients. Methods: The functional magnetic resonance imaging technique was used to observe activation of the brain during ME/MI of the upper limbs in 12 acute stroke patients (with the left brain damage) and 12 healthy controls. Results: During ME, the stroke patients appeared to be activated more strongly than the healthy controls in the ipsilateral primary motor areas. The MI of the affected hand in the stroke patients was not significantly different from that of the healthy hand. The nonmotor areas, the angular gyrus, and the fusiform gyrus were also activated during ME/MI. Conclusion: Structural damage in the brain is associated with the activation of brain function in acute stroke patients. Ipsilateral inhibition is reduced in stroke patients during ME and the damaged brain needs to recruit more brain areas to complete the desired action due to motion difficulties resulting from brain damage. The participation of nonmotor brain areas in ME/MI indicates that cortical reorganization may contribute to the restoration of motor function following stroke. MI can be used to improve injured brain areas, helping with the rehabilitation of stroke patients.

Gene therapy blockade of dorsal striatal p11 improves motor function and dyskinesia in parkinsonian mice

Complications of dopamine replacement for Parkinson's disease (PD) can limit therapeutic options, leading to interest in identifying novel pathways that can be exploited to improve treatment. p11 (S100A10) is a cellular scaffold protein that binds to and potentiates the activity of various ion channels and neurotransmitter receptors. We have previously reported that p11 can influence ventral striatal function in models of depression and drug addiction, and thus we hypothesized that dorsal striatal p11 might mediate motor function and drug responses in parkinsonian mice. To focally inhibit p11 expression in the dorsal striatum, we injected an adeno-associated virus (AAV) vector producing a short hairpin RNA (AAV.sh.p11). This intervention reduced the impairment in motor function on forced tasks, such as rotarod and treadmill tests, caused by substantia nigra lesioning in mice. Measures of spontaneous movement and gait in an open-field test declined as expected in control lesioned mice, whereas AAV.sh.p11 mice remained at or near normal baseline. Mice with unilateral lesions were then challenged with l-dopa (levodopa) and various dopamine receptor agonists, and resulting rotational behaviors were significantly reduced after ipsilateral inhibition of dorsal striatal p11 expression. Finally, p11 knockdown in the dorsal striatum dramatically reduced l-dopa-induced abnormal involuntary movements compared with control mice. These data indicate that focal inhibition of p11 action in the dorsal striatum could be a promising PD therapeutic target to improve motor function while reducing l-dopa-induced dyskinesias.

Intracortical inhibition is modulated by phase of prosthetic rehabilitation in transtibial amputees.

Reorganization of primary motor cortex (M1) is well-described in long-term lower limb amputees. In contrast cortical reorganization during the rehabilitation period after amputation is poorly understood. Thirteen transtibial amputees and 13 gender matched control participants of similar age were recruited. Transcranial magnetic stimulation was used to assess corticomotor and intracortical excitability of M1 bilaterally. Neurophysiological assessments were conducted at admission, prosthetic casting, first walk and discharge. Gait variability at discharge was assessed as a functional measure. Compared to controls, amputees had reduced short-latency intracortical inhibition (SICI) for the ipsilateral M1 at admission (p = 0.01). Analysis across rehabilitation revealed SICI was reduced for the contralateral M1 at first walk compared to discharge (p = 0.003). For the ipsilateral M1 both short and long-latency intracortical inhibition were reduced at admission (p < 0.05) and prosthetic casting (p < 0.02). Analysis of the neurophysiology and gait function revealed several interesting relationships. For the contralateral M1, reduced inhibition at admission (p = 0.04) and first walk (p = 0.05) was associated with better gait function. For the ipsilateral M1, reduced inhibition at discharge (p = 0.05) was associated with poor gait function. This study characterized intracortical excitability in rehabilitating amputees. A dichotomous relationship between reduced intracortical inhibition for each M1 and gait function was observed at different times. Intracortical inhibition may be an appropriate cortical biomarker of gait function in lower limb amputees during rehabilitation, but requires further investigation. Understanding M1 intracortical excitability of amputees undertaking prosthetic rehabilitation provides insight into brain reorganization in the sub-acute post-amputation period and may guide future studies seeking to improve rehabilitation outcomes.

Normalized activation in the somatosensory cortex 30 years following nerve repair in children: an fMRI study.

The clinical outcome following a peripheral nerve injury in the upper extremity is generally better in young children than in teenagers and in adults, but the mechanism behind this difference is unknown. In 28 patients with a complete median nerve injury sustained at the ages of 1-13 years (n = 13) and 14-20 years (n = 15), the cortical activation during tactile finger stimulation of the injured and healthy hands was monitored at a median time since injury of 28 years using functional magnetic resonance imaging (fMRI) at 3 Tesla. The results from the fMRI were compared with the clinical outcome and electroneurography. The cortical activation pattern following sensory stimulation of the median nerve-innervated fingers was dependent on the patient's age at injury. Those injured at a young age (1-13 years) had an activation pattern similar to that of healthy controls. Furthermore, they showed a clinical outcome significantly superior (P = 0.001) to the outcome in subjects injured at a later age; however, electroneurographical parameters did not differ between the groups. In subjects injured at age 14-20 years, a more extended activation of the contralateral hemisphere was seen in general. Interestingly, these patients also displayed changes in the ipsilateral hemisphere where a reduced inhibition of somatosensory areas was seen. This loss of ipsilateral inhibition correlated to increasing age at injury as well as to poor recovery of sensory functions in the hand. In conclusion, cerebral changes in both brain hemispheres may explain differences in clinical outcome following a median nerve injury in childhood or adolescence.

Mirror illusion reduces ipsilateral motor cortical inhibition in healthy young adults: implications for rehabilitation

Mirror illusion reduces ipsilateral motor cortical inhibition in healthy young adults: implications for rehabilitation

High-Resolution fMRI Reveals Laminar Differences in Neurovascular Coupling between Positive and Negative BOLD Responses

The six cortical layers have distinct anatomical andphysiological properties, like different energy use and different feedforward and feedback connectivity. It is not known if and how layer-specific neural processes are reflected in the fMRI signal. To address this question we used high-resolution fMRI to measure BOLD, CBV, and CBF responses to stimuli that elicit positive and negative BOLD signals in macaque primary visual cortex. We found that regions with positive BOLD responses had parallel increases in CBV and CBF, whereas areas with negative BOLD responses showed a decrease in CBF but an increase in CBV. For positive BOLD responses, CBF and CBV increased in the center of the cortex, but for negative BOLD responses, CBF decreased superficially while CBV increased in the center. Our findings suggest different mechanisms for neurovascular coupling for BOLD increases and decreases, aswell as laminar differences in neurovascular coupling.

Discharge patterns in the lateral superior olive of decerebrate cats

Anatomical and pharmacological studies have shown that the lateral superior olive (LSO) receives inputs from a number of sources and that LSO cells can alter the balance of their own excitatory and inhibitory drive. It is thus likely that the ongoing sound-evoked responses of LSO cells reflect a complex interplay of excitatory and inhibitory events, which may be affected by anesthesia. The goal of this study was to characterize the temporal discharge patterns of single units in the LSO of unanesthetized, decerebrate cats in response to long-duration ipsilateral best-frequency tone bursts. A decision tree is presented to partition LSO units on the basis of poststimulus time histogram shape, adaptation of instantaneous firing rate as a function of time, and sustained discharge rate. The results suggest that LSO discharge patterns form a continuum with four archetypes: sustained choppers that show two or more peaks of activity at stimulus onset and little adaptation of rate throughout the response, transient choppers that undergo a decrease in rate that eventually stabilizes with time, primary-like units that display an initial peak of activity followed by a monotonic decline in rate to a steady-state value, and onset-sustained units that exhibit an initial peak of activity at stimulus onset followed by a low sustained activity. Compared with the chopper units, the nonchopper units tend to show longer first-spike latencies, lower peak firing rates, and more irregular sustained discharge patterns. Modeling studies show that the full range of LSO response types can be obtained from an underlying sustained chopper by varying the strength and latency of a sound-driven ipsilateral inhibition relative to that of excitation. Together, these results suggest that inhibition plays a major role in shaping the temporal discharge patterns of units in unanesthetized preparations.

Functional studies (NeuroSPECT) of the human auditory pathway after stimulating binaurally with pure tones

Conclusions: Our observations support the concept of bilateral cortical activation with monaural and binaural auditory stimulation. The observation that most of the significantly activated areas were the same with monaural or binaural stimulation suggests that the differences in auditory perception with binaural stimulation are not due to the involvement of significantly different processing centers but, more likely, to the type of information that reaches these centers for processing. The observation that the degree of stimulation was less intense in binaural than in monaural stimulation supports the concept that a richer binaural auditory stimulation compared with monaural stimulation does not mean summation of stimuli but integration and better processing of the information. For normal bilateral hearing subjects, a monaural stimulus is an uncommon event and may thus explain the more intense response. The repeatability of the results for monaural and binaural stimulation with pure tones in the same subjects confirms the consistency of the testing method. Objectives: (1) To determine which areas of the cerebral cortex and basal ganglia are activated by binaural stimulation with pure tones (left and right ear simultaneously) and what type of response occurs (e.g. excitatory or inhibitory) in these different areas. (2) To determine the degree of ipsilateral and/or contralateral cortical activation and/or inhibition. (3) To compare the data with our previous reports of monaural stimulation using the same technique and the same subjects. (4) To evaluate the consistency of our testing method. Methods: Brain perfusion single photon emission computed tomography (SPECT) evaluation was conducted using auditory binaural stimulation with pure tones in six normal volunteers. Both ears were stimulated simultaneously. Tc99m HMPAO was injected while pure tones were delivered and the SPECT imaging was done 1 h later. Results: After delivering pure tones there was bilateral activation in Brodmann areas 7 (somatosensory association cortex), 9 and 10 (executive frontal areas), 17, 18, and 19 (associative visual cortex). There was also activation in temporal areas 21, 22 (auditory association areas), and parietal areas 39 and 40 (Wernicke). There was also marked activation in both thalami. These activated areas were the same as those in our previous reports with monaural stimulation in the same subjects. However, except for areas 17, 18, 23, 31, and 32 (which remained over 4 SD above the normal maximum), the degree of activation was less intense in binaural compared with monaural stimulation. Inhibition was also less intense in binaural stimulation.

Motor inhibition and response expectancy: A Laplacian ERP study

In between-hand choice reaction time tasks, the motor cortex involved in the required response (contralateral) has been shown to be activated while the motor cortex involved in the non-required response (ipsilateral) has been shown to be inhibited. The aim of the present study was to test the hypothesis that ipsilateral inhibition reflects an active mechanism aimed at preventing errors. To this end, the risk of committing errors in between-hand choice reaction time tasks was manipulated by introducing a response probability bias. The surface Laplacian transforms of electroencephalographic waves recorded over the motor cortices, contralateral and ipsilateral to the responding hand were compared. Results showed that contralateral activation was not modulated by the risk of committing errors while ipsilateral inhibition was sensitive to this risk in a gradual manner: the higher the risk, the stronger the inhibition.

Synaptic patterning of left-right alternation in a computational model of the rodent hindlimb central pattern generator

Establishing, maintaining, and modifying the phase relationships between extensor and flexor muscle groups is essential for central pattern generators in the spinal cord to coordinate the hindlimbs well enough to produce the basic walking rhythm. This paper investigates a simplified computational model for the spinal hindlimb central pattern generator (CPG) that is abstracted from experimental data from the rodent spinal cord. This model produces locomotor-like activity with appropriate phase relationships in which right and left muscle groups alternate while extensor and flexor muscle groups alternate. Convergence to this locomotor pattern is slow, however, and the range of parameter values for which the model produces appropriate output is relatively narrow. We examine these aspects of the model's coordination of left-right activity through investigation of successively more complicated subnetworks, focusing on the role of the synaptic architecture in shaping motoneuron phasing. We find unexpected sensitivity in the phase response properties of individual neurons in response to stimulation and a need for high levels of both inhibition and excitation to achieve the walking rhythm. In the absence of cross-cord excitation, equal levels of ipsilateral and contralateral inhibition result in a strong preference for hopping over walking. Inhibition alone can produce the walking rhythm, but contralateral inhibition must be much stronger than ipsilateral inhibition. Cross-cord excitatory connections significantly enhance convergence to the walking rhythm, which is achieved most rapidly with strong crossed excitation and greater contralateral than ipsilateral inhibition. We discuss the implications of these results for CPG architectures based on unit burst generators.

Influence of inhibitory synaptic kinetics on the interaural time difference sensitivity in a linear model of binaural coincidence detection

Temporal correlations between the sound waves arriving at the two ears are used to extract the azimuthal position of sound sources. Nerve cells in the mammalian medial superior olive (MSO) that extract these binaural correlations are sensitive to interaural time differences (ITDs) in the range of about 10 micros. These neurons receive inputs from the two ears via four pathways, two excitatory and two inhibitory ones. In this paper, a simple linear model is fitted to the frequency dependence of ITD sensitivity of MSO neurons, which is quantified by the two parameters, characteristic phase and characteristic delay. The fit parameters are the relative delays and the relative strengths of the two inhibitory pathways and thus specify the underlying ITD-detecting circuit assuming a non-Jeffress-like situation, i.e., no excitatory delay lines but phase-locked inhibition. The fitting procedure finds the parameters of these inhibitory pathways such that they account for a desired frequency dependence of ITD sensitivity. It is found that positive characteristic delays require a finite amount of ipsilateral inhibition that arrives at roughly the same time as ipsilateral excitation as well as contralateral inhibition that lags contralateral excitation so much that it effectively leads excitation of the next cycle.

6. Effect of muscle fatigue due to grip task on ipsilateral intracortical inhibition

6. Effect of muscle fatigue due to grip task on ipsilateral intracortical inhibition

Response Properties of Single Units in the Dorsal Nucleus of the Lateral Lemniscus of Decerebrate Cats

The dorsal nucleus of the lateral lemniscus (DNLL) receives afferent inputs from many brain stem nuclei and, in turn, is a major source of inhibitory inputs to the inferior colliculus (IC). The goal of this study was to characterize the monaural and binaural response properties of neurons in the DNLL of unanesthetized decerebrate cat. Monaural responses were classified according to the patterns of excitation and inhibition observed in contralateral and ipsilateral frequency response maps. Binaural classification was based on unit sensitivity to interaural level differences. The results show that units in the DNLL can be grouped into three distinct types. Type v units produce contralateral response maps that show a wide V-shaped excitatory area and no inhibition. These units receive ipsilateral excitation and exhibit binaural facilitation. The contralateral maps of type i units show a more restricted I-shaped region of excitation that is flanked by inhibition. Type o maps display an O-shaped island of excitation at low stimulus levels that is bounded by inhibition at higher levels. Both type i and type o units receive ipsilateral inhibition and exhibit binaural inhibition. Units that produce type v maps have a low best frequency (BF), whereas type i and type o units have high BFs. Type v and type i units give monotonic rate-level responses for both BF tones and broadband noise. Type o units are inhibited by tones at high levels, but are excited by high-level noise. These results show that the DNLL can exert strong, differential effects in the IC.

Neural Pathways for Vertical Saccades: From the Superior Colliculus to Trochlear Motoneurons

The pathways from the superior colliculus (SC) to vertical ocular motoneurons were analyzed in anesthetized cats using intracellular recording from trochlear motoneurons. Electrical stimulation of the ipsilateral or contralateral SC evoked disynaptic excitation and inhibition in trochlear motoneurons. Stimulation of the ipsilateral Forel's field H (FFH) and the contralateral interstitial nucleus of Cajal (INC) evoked monosynaptic excitation and inhibition in trochlear motoneurons, respectively. Preconditioning stimulation of the SC facilitated FFH-evoked monosynaptic excitation. The results showed that trochlear motoneurons receive disynaptic excitation via the FFH from the ipsilateral SC and disynaptic inhibition via the INC from the contralateral SC.