Change in EEG Activity is Associated with a Decrease in Tinnitus Awareness after rTMS.

OBJECTIVE: To examine how 1Hz and 10Hz rTMS temporarily influence ratings of tinnitus loudness, annoyance, and awareness. The thalamocortical dysrhythmia (TCD) model of tinnitus was tested by examining changes in spectral power and coherence of resting state EEGs from baseline to each phase of treatment and correlating these data with change in tinnitus. METHODS: Nineteen participants completed a double-blind, placebo (sham rTMS) controlled, within-subjects study with crossover between the two active rTMS treatment conditions. An imposed order effect, sham rTMS first, eliminated drift of active treatment into the placebo condition. The primary outcome measures were analogue ratings of tinnitus loudness, annoyance, and awareness, assessed repeatedly at baseline and during treatment, and 64 channel, resting state EEGs collected at baseline and the end of each treatment phase. Active rTMS consisted of 1800 pulses at 110% of motor threshold over temporal cortex delivered at 1Hz and 10Hz over four days. The research design also examined the effect of rTMS immediately following stimulation, regression to the mean in tinnitus ratings made over multiple days, and differences between treatment responders and non-responders. RESULTS: There was no immediate effect of rTMS on tinnitus during a single rTMS session. Regression to the mean in tinnitus ratings occurred over three days of baseline and four days of treatment (both sham and active rTMS). After accounting for regression to the mean in the statistical model, 1Hz rTMS led to a significant decrease in tinnitus awareness from baseline and 10Hz rTMS trended in the same direction, whereas sham rTMS showed little change from baseline other than regression to the mean. Changes from baseline in spectral power of the resting state EEG provided partial support for predictions based on TCD model of tinnitus for active 1 and 10Hz rTMS but not sham rTMS. However, only an increase in beta coherence correlated significantly with a decrease in tinnitus awareness. Changes in the EEG were robust in treatment responders but absent among non-responders and during sham rTMS. CONCLUSIONS: A positive response to rTMS for tinnitus is associated with an rTMS-induced change in beta coherence of the EEG. Increased beta coherence may be a biomarker of the rTMS effect; a "top-down" modulation of the EEG that promotes habituation to tinnitus. Participants whose tinnitus did not improve after rTMS did not show any changes in the EEG.

Focus on the pedunculopontine nucleus. Consensus review from the May 2018 brainstem society meeting in Washington, DC, USA.

The pedunculopontine nucleus (PPN) is located in the mesopontine tegmentum and is best delimited by a group of large cholinergic neurons adjacent to the decussation of the superior cerebellar peduncle. This part of the brain, populated by many other neuronal groups, is a crossroads for many important functions. Good evidence relates the PPN to control of reflex reactions, sleep-wake cycles, posture and gait. However, the precise role of the PPN in all these functions has been controversial and there still are uncertainties in the functional anatomy and physiology of the nucleus. It is difficult to grasp the extent of the influence of the PPN, not only because of its varied functions and projections, but also because of the controversies arising from them. One controversy is its relationship to the mesencephalic locomotor region (MLR). In this regard, the PPN has become a new target for deep brain stimulation (DBS) for the treatment of parkinsonian gait disorders, including freezing of gait. This review is intended to indicate what is currently known, shed some light on the controversies that have arisen, and to provide a framework for future research.

Bottom-up gamma maintenance in various disorders.

Maintained gamma band activity is a key element of higher brain function, participating in perception, executive function, and memory. The pedunculopontine nucleus (PPN), as part of the reticular activating system (RAS), is a major source of the "bottom-up" flow of gamma activity to higher regions. However, interruption of gamma band activity is associated with a number of neurological and psychiatric disorders. This review will focus on the role of the PPN in activating higher regions to induce arousal and descending pathways to modulate posture and locomotion. As such, PPN deep brain stimulation (DBS) can not only help regulate arousal and stepping, but continuous application may help maintain necessary levels of gamma band activity for a host of other brain processes. We will explore the potential future applications of PPN DBS for a number of disorders that are characterized by disturbances in gamma band maintenance.

The pedunculopontine nucleus: From posture and locomotion to neuroepigenetics.

In this review, we discuss first an example of one of the symptoms of PD, freezing of gait (FOG), then we will turn to the use of deep brain stimulation (DBS) of the pedunculopontine nucleus (PPN) to treat PD, and the original studies that led to identification of the PPN as one source of locomotor control and why stimulation frequency is critical, and then describe the intrinsic properties of PPN neurons that require beta/gamma stimulation in order to fully activate all types of PPN neurons. Finally, we will describe recent findings on the proteomic and molecular consequences of gamma band activity in PPN neurons, with emphasis on the potential neuroepigenetic sequelae. These considerations will provide essential information for the appropriate refining and testing of PPN DBS as a potential therapy for PD, as well as alternative options.

Bottom-up gamma and stages of waking.

Gamma activity has been proposed to promote the feed forward or "bottom-up" flow of information from lower to higher regions of the brain during perception. The pedunculopontine nucleus (PPN) modulates waking and REM sleep, and is part of the reticular activating system (RAS). The properties of PPN cells are unique in that all PPN neurons fire maximally at gamma band frequency regardless of electrophysiological or transmitter type, thus proposed as one origin of "bottom-up" gamma. This property is based on the presence of intrinsic membrane oscillations subserved by high threshold, voltage-dependent calcium channels. Moreover, some PPN cells are electrically coupled. Assuming that the population of PPN neurons has the capacity to fire at ∼40Hz coherently, then the population as a whole can be expected to generate a stable gamma band signal. But what if not all the neurons are firing at the peaks of the oscillations? That means that some cells may fire only at the peaks of every second oscillation. Therefore, the population as a whole can be expected to be firing at a net ∼20Hz. If some cells are firing at the peaks of every fourth oscillation, then the PPN as a whole would be firing at ∼10Hz. Firing at rates below 10Hz would imply that the system is seldom firing at the peaks of any oscillation, basically asleep, in slow wave sleep, thus the activation of the RAS is insufficient to promote waking. This hypothesis carries certain implications, one of which is that we awaken in stages as more and more cells are recruited to fire at the peaks of more and more oscillations. For this system, it would imply that, as we awaken, we step from ∼10Hz to ∼20Hz to ∼30Hz to ∼40Hz, that is, in stages and presumably at different levels of awareness. A similar process can be expected to take place as we fall asleep. Awakening can then be considered to be stepwise, not linear. That is, the implication is that the process of waking is a stepwise event, not a gradual increase, suggesting that the brain can spend time at each of these different stages of arousal.

The 10 Hz Frequency: A Fulcrum For Transitional Brain States.

A 10 Hz rhythm is present in the occipital cortex when the eyes are closed (alpha waves), in the precentral cortex at rest (mu rhythm), in the superior and middle temporal lobe (tau rhythm), in the inferior olive (projection to cerebellar cortex), and in physiological tremor (underlying all voluntary movement). These are all considered resting rhythms in the waking brain which are "replaced" by higher frequency activity with sensorimotor stimulation. That is, the 10 Hz frequency fulcrum is replaced on the one hand by lower frequencies during sleep, or on the other hand by higher frequencies during volition and cognition. The 10 Hz frequency fulcrum is proposed as the natural frequency of the brain during quiet waking, but is replaced by higher frequencies capable of permitting more complex functions, or by lower frequencies during sleep and inactivity. At the center of the transition shifts to and from the resting rhythm is the reticular activating system, a phylogenetically preserved area of the brain essential for preconscious awareness.

Implications of gamma band activity in the pedunculopontine nucleus.

The fact that the pedunculopontine nucleus (PPN) is part of the reticular activating system places it in a unique position to modulate sensory input and fight-or-flight responses. Arousing stimuli simultaneously activate ascending projections of the PPN to the intralaminar thalamus to trigger cortical high-frequency activity and arousal, as well as descending projections to reticulospinal systems to alter posture and locomotion. As such, the PPN has become a target for deep brain stimulation for the treatment of Parkinson's disease, modulating gait, posture, and higher functions. This article describes the latest discoveries on PPN physiology and the role of the PPN in a number of disorders. It has now been determined that high-frequency activity during waking and REM sleep is controlled by two different intracellular pathways and two calcium channels in PPN cells. Moreover, there are three different PPN cell types that have one or both calcium channels and may be active during waking only, REM sleep only, or both. Based on the new discoveries, novel mechanisms are proposed for insomnia as a waking disorder. In addition, neuronal calcium sensor protein-1 (NCS-1), which is over expressed in schizophrenia and bipolar disorder, may be responsible for the dysregulation in gamma band activity in at least some patients with these diseases. Recent results suggest that NCS-1 modulates PPN gamma band activity and that lithium acts to reduce the effects of over expressed NCS-1, accounting for its effectiveness in bipolar disorder.

Arousal and the control of perception and movement.

Recent discoveries on the nature of the activity generated by the reticular activating system (RAS) suggest that arousal is much more involved in perception and movement than previously thought. The RAS is not simply an amorphous, unspecific region but rather a distinct group of nuclei with specific cell and transmitter types that control waking and modulate such processes as perception and movement. Thus, disturbances in the RAS will affect a number of neurological disorders. The discovery of gamma band activity in the RAS determined that high threshold calcium channels are responsible for generating gamma band activity in the RAS. Results showing that waking is mediated by CaMKII modulation of P/Q-type channels and REM sleep is modulated by cAMP/PK modulation of N-type channels points to different intracellular pathways influencing each state. Few studies address these important breakthroughs. Novel findings also show that the same primate RAS neurons exhibiting activity in relation to arousal are also involved in locomotion. Moreover, deep brain stimulation of this region, specifically the pedunculopontine nucleus (PPN DBS), in Parkinson's disease has salutary effects on movement, sleep, and cognition. Gamma oscillations appear to participate in sensory perception, problem solving, and memory, and coherence at these frequencies may occur at cortical or thalamocortical levels. However, rather than participating in the temporal binding of sensory events, gamma band activity generated in the RAS may help stabilize coherence related to arousal, providing a stable activation state during waking, and relay such activation to the cortex. Continuous sensory input will thus induce gamma band activity in the RAS to participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our perceptions and actions. Such a role has received little attention but promises to help understand and treat a number of neurological disorders.

Bottom-up Gamma: the Pedunculopontine Nucleus and Reticular Activating System.

Gamma rhythms have been proposed to promote the feed forward or "bottom-up" flow of information from lower to higher regions in the brain during perception. On the other hand, beta rhythms have been proposed to represent feed back or "top-down" influence from higher regions to lower. The pedunculopontine nucleus (PPN) has been implicated in sleep-wake control and arousal, and is part of the reticular activating system (RAS). This review describes the properties of the cells in this nucleus. These properties are unique, and perhaps it is the particular characteristics of these cells that allow the PPN to be involved in a host of functions and disorders. The fact that all PPN neurons fire maximally at gamma band frequency regardless of electrophysiological or transmitter type, make this an unusual cell group. In other regions, for example in the cortex, cells with such a property represent only a sub-population. More importantly, the fact that this cell group's functions are related to the capacity to generate coherent activity at a preferred natural frequency, gamma band, speaks volumes about how the PPN functions. We propose that "bottom-up" gamma band influence arises in the RAS and contributes to the build-up of the background of activity necessary for preconscious awareness and gamma activity at cortical levels.

Arousal, motor control, and parkinson's disease.

This review highlights the most important discovery in the reticular activating system (RAS) in the last 10 years, the manifestation of gamma (γ) band activity in cells of the RAS, especially in the pedunculopontine nucleus (PPN), which is in charge of the high frequency states of waking and rapid eye movement sleep. This discovery is critical to understanding the modulation of movement by the RAS and how it sets the background over which we generate voluntary and triggered movements. The presence of γ band activity in the RAS is proposed to participate in the process of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. Early findings using stimulation of this region to induce arousal, and also to elicit stepping, are placed in this context. This finding also helps explain the novel use of PPN deep brain stimulation for the treatment of Parkinson's disease, although considerable work remains to be done.

The physiology of the pedunculopontine nucleus: implications for deep brain stimulation.

This brief review resolves a number of persistent conflicts regarding the location and characteristics of the mesencephalic locomotor region, which has in the past been described as not locomotion-specific and is more likely the pedunculopontine nucleus (PPN). The parameters of stimulation used to elicit changes in posture and locomotion we now know are ideally suited to match the intrinsic membrane properties of PPN neurons. The physiology of these cells is important not only because it is a major element of the reticular activating system, but also because it is a novel target for the treatment of gait and postural deficits in Parkinson's disease (PD). The discussion explains many of the effects reported following deep brain stimulation (DBS) of the PPN by different groups and provides guidelines for the determination of long-term assessment and effects of PPN DBS. A greater understanding of the physiology of the target nuclei within the brainstem and basal ganglia, amassed over the past decades, has enabled increasingly better patient outcomes from DBS for movement disorders. Despite these improvements, there remains a great opportunity for further understanding of the mechanisms through which DBS has its effects and for further development of appropriate technology to effect these treatments. We review the scientific basis for one of the newest targets, the PPN, in the treatment of PD and other movement disorders, and address the needs for further investigation.

Gamma band activity in the RAS-intracellular mechanisms.

Gamma band activity participates in sensory perception, problem solving, and memory. This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit gamma band activity, and describes the intrinsic membrane properties behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus, intralaminar parafascicular nucleus, and pontine SubCoeruleus nucleus dorsalis all fire in the gamma band range when maximally activated, but no higher. The mechanisms involve high-threshold, voltage-dependent P/Q-type calcium channels, or sodium-dependent subthreshold oscillations. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness and provide the essential stream of information for the formulation of many of our actions. We address three necessary next steps resulting from these discoveries: an intracellular mechanism responsible for maintaining gamma band activity based on persistent G-protein activation, separate intracellular pathways that differentiate between gamma band activity during waking versus during REM sleep, and an intracellular mechanism responsible for the dysregulation in gamma band activity in schizophrenia. These findings open several promising research avenues that have not been thoroughly explored. What are the effects of sleep or REM sleep deprivation on these RAS mechanisms? Are these mechanisms involved in memory processing during waking and/or during REM sleep? Does gamma band processing differ during waking versus REM sleep after sleep or REM sleep deprivation?

Mechanism behind gamma band activity in the pedunculopontine nucleus.

The pedunculopontine nucleus (PPN), part of the reticular activating system, modulates waking and paradoxical sleep. During waking and paradoxical sleep, EEG responses are characterized by low-amplitude, high-frequency oscillatory activity in the beta-gamma band range (~20-80 Hz). We have previously reported that gamma band activity may be intrinsically generated by the membrane electroresponsiveness of PPN neurons, and that the neuronal ensemble generates different patterns of gamma activity in response to specific transmitters. This study attempted to identify the voltage-gated calcium and potassium channels involved in the rising and falling phases of gamma oscillations in PPN neurons. We found that all rat (8-14 day) PPN cell types showed gamma oscillations in the presence of TTX and synaptic blockers when membrane potential was depolarized using current ramps. PPN neurons showed gamma oscillations when voltage-clamped at holding potentials above -30 mV, suggesting that their origin may be spatially located beyond voltage-clamp control. The average frequency for all PPN cell types was 23 ± 1 Hz and this increased under carbachol (47 ± 2 Hz; anova df = 64, t = 12.5, P < 0.001). The N-type calcium channel blocker ω-conotoxin-GVIA partially reduced gamma oscillations, while the P/Q-type blocker ω-agatoxin-IVA abolished them. Both ω-CgTX and ω-Aga blocked voltage-dependent calcium currents, by 56 and 52% respectively. The delayed rectifier-like potassium channel blocker α-dendrotoxin also abolished gamma oscillations. In carbachol-induced PPN population responses, ω-agatoxin-IVA reduced higher, and ω-CgTx mostly lower, frequencies. These results suggest that voltage-dependent P/Q- and, to a lesser extent, N-type calcium channels mediate gamma oscillations in PPN.

Cholinergic and glutamatergic agonists induce gamma frequency activity in dorsal subcoeruleus nucleus neurons.

The dorsal subcoeruleus nucleus (SubCD) is involved in generating two signs of rapid eye movement (REM) sleep: muscle atonia and ponto-geniculo-occipital (PGO) waves. We tested the hypothesis that single cell and/or population responses of SubCD neurons are capable of generating gamma frequency activity in response to intracellular stimulation or receptor agonist activation. Whole cell patch clamp recordings (immersion chamber) and population responses (interface chamber) were conducted on 9- to 20-day-old rat brain stem slices. All SubCD neurons (n = 103) fired at gamma frequency when subjected to depolarizing steps. Two statistically distinct populations of neurons were observed, which were distinguished by their high (>80 Hz, n = 24) versus low (35-80 Hz, n = 16) initial firing frequencies. Both cell types exhibited subthreshold oscillations in the gamma range (n = 43), which may underlie the gamma band firing properties of these neurons. The subthreshold oscillations were blocked by the sodium channel blockers tetrodotoxin (TTX, n = 21) extracellularly and N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314) intracellularly (n = 5), indicating they were sodium channel dependent. Gamma frequency subthreshold oscillations were observed in response to the nonspecific cholinergic receptor agonist carbachol (CAR, n = 11, d = 1.08) and the glutamate receptor agonists N-methyl-d-aspartic acid (NMDA, n = 12, d = 1.09) and kainic acid (KA, n = 13, d = 0.96), indicating that cholinergic and glutamatergic inputs may be involved in the activation of these subthreshold currents. Gamma band activity also was observed in population responses following application of CAR (n = 4, P < 0.05), NMDA (n = 4, P < 0.05) and KA (n = 4, P < 0.05). Voltage-sensitive, sodium channel-dependent gamma band activity appears to be a part of the intrinsic membrane properties of SubCD neurons.

Chapter 11--novel mechanism for hyperreflexia and spasticity.

We established that hyperreflexia is delayed after spinal transection in the adult rat and that passive exercise could normalize low frequency-dependent depression of the H-reflex. We were also able to show that such passive exercise will normalize hyperreflexia in patients with spinal cord injury (SCI). Recent results demonstrate that spinal transection results in changes in the neuronal gap junction protein connexin 36 below the level of the lesion. Moreover, a drug known to increase electrical coupling was found to normalize hyperreflexia in the absence of passive exercise, suggesting that changes in electrical coupling may be involved in hyperreflexia. We also present results showing that a measure of spasticity, the stretch reflex, is rendered abnormal by transection and normalized by the same drug. These data suggest that electrical coupling may be dysregulated in SCI, leading to some of the symptoms observed. A novel therapy for hyperreflexia and spasticity may require modulation of electrical coupling.

Commentary: the pedunculopontine nucleus: clinical experience, basic questions and future directions.

This issue is dedicated to a potential new target for the treatment of movement disorders, the pedunculopontine tegmental nucleus (PPTg), or, more simply, the pedunculopontine nucleus, that some authors abbreviate as PPN. We provide an overview of the field as an introduction to the general reader, beginning with the clinical experience to date of Mazzone and co-workers in Rome, some basic questions that need to be addressed, and potential future directions required in order to ensure that the potential benefits of this work are realized.

Gamma band unit activity and population responses in the pedunculopontine nucleus.

The pedunculopontine nucleus (PPN) is involved in the activated states of waking and paradoxical sleep, forming part of the reticular activating system (RAS). The studies described tested the hypothesis that single unit and/or population responses of PPN neurons are capable of generating gamma band frequency activity. Whole cell patch clamp recordings (immersion chamber) and population responses (interface chamber) were conducted on 9- to 20-day-old rat brain stem slices. Regardless of cell type (I, II, or III) or type of response to the nonselective cholinergic receptor agonist carbachol (excitation, inhibition, biphasic), almost all PPN neurons fired at gamma band frequency, but no higher, when subjected to depolarizing steps (50 +/- 2 Hz, mean +/- SE). Nonaccommodating neurons fired at 18-100 Hz throughout depolarizing steps, while most accommodating neurons exhibited gamma band frequency of action potentials followed by gamma band membrane oscillations. These oscillations were blocked by the sodium channel blocker tetrodotoxin (TTX), suggesting that at least some are mediated by sodium currents. Population responses in the PPN showed that carbachol induced peaks of activation in the theta and gamma range, while glutamatergic receptor agonists induced overall increases in activity at theta and gamma frequencies, although in differing patterns. Gamma band activity appears to be a part of the intrinsic membrane properties of PPN neurons, and the population as a whole generates different patterns of gamma band activity under the influence of specific transmitters. Given sufficient excitation, the PPN may impart gamma band activation on its targets.

AROUSAL FROM SLICES TO HUMANS: Translational studies on sleep-wake control.

Most psychiatric and neurological disorders exhibit sleep disorders, and in some cases presage the disease. Study of the control of sleep and waking has the potential for making a major impact on a number of disorders, making translational neuroscience research on this area critical. One element of the reticular activating system (RAS) is the pedunculopontine nucleus (PPN), which is the cholinergic arm of the RAS, and projects to the thalamus to trigger thalamocortical rhythms and to the brainstem to modulate muscle tone and locomotion. We developed a research program using brainstem slices containing the PPN to tell us about the cellular and molecular organization of this region. In addition, we developed the P13 midlatency auditory evoked potential, which is generated by PPN outputs, preparation in freely moving rats. This allows the study of PPN cellular and molecular mechanisms at the level of the whole animal. We also study the P50 midlatency auditory evoked potential, which is the human equivalent of the rodent P13 potential, allowing us to study processes detected in vitro, confirmed in the whole animal, and tested in humans. This translational research program led to the discovery of a novel mechanism of sleep-wake control, pointing the way to a number of new clinical applications in the development of novel stimulants and anesthetics.

Modafinil normalized hyperreflexia after spinal transection in adult rats.

STUDY DESIGN: Hyperreflexia occurs after spinal cord injury and can be assessed by measuring low frequency-dependent depression of the H-reflex in the anesthetized animal. OBJECTIVE: To determine the effects of Modafinil (MOD), given orally, following a complete SCI compared with animals receiving MBET and transected untreated animals and examine if changes exist in Connexin 36 (Cx-36) protein levels in the lumbar enlargement of animals for the groups described. SETTING: Center for Translational Neuroscience, Little Rock, AR, USA. METHODS: Adult female rats underwent complete transection (Tx) at T10 level. H-reflex testing was performed 30 days following Tx in one group, and after initiation of treatment with MOD in another group, and after MBET training in the third group. The Lumbar enlargement tissue was harvested and western blots were performed after immunoprecipitation techniques to compare Cx-36 protein levels. RESULTS: Statistically significant decreases in low frequency-dependent depression of the H-reflex were observed in animals that received MOD and those that were treated with MBET compared with the Tx, untreated group. Statistically significant changes in Cx-36 protein levels were not observed in animals treated with MOD compared with Tx, untreated animals. CONCLUSION: Normalization of the loss of low frequency -dependent depression of the H-reflex was demonstrated in the group receiving MOD and the group receiving MBET compared with the Tx, untreated group. Further work is needed to examine if Cx-36 protein changes occur in specific subregions of the spinal cord.

The onset of hyperreflexia in the rat following complete spinal cord transection.

STUDY DESIGN: Hyperreflexia occurs after spinal cord injury (SCI) and can be assessed by measuring low frequency-dependent depression of the H-reflex. Previous studies showed the time course for the onset of hyperreflexia to occur between 6-28 days in the contusion model of SCI. OBJECTIVE: To determine the time course of the onset of hyperreflexia in the transection model of SCI and examine changes in Connexin-36 (Cx-36) protein levels in the lumbar enlargement of animals. SETTING: Spinal Cord Injury Mobilization Program of the Center for Translational Neuroscience, the research arm of the Jackson T. Stephens Neuroscience Institute, Little Rock, AR, USA. METHODS: Adult female rats underwent transection at T10 level. Low frequency-dependent depression of the H-reflex was tested at 7, 14 and 30 days post-transection. Lumbar enlargement tissue was harvested following reflex testing and western blots were performed after immunoprecipitation to compare Cx-36 protein levels. RESULTS: Significant decreases in low frequency-dependent depression of the H-reflex were observed in animals tested 14 and 30 days post-transection compared with control animals, but it was not different from control animals at 7 days. Significant decreases in Cx-36 protein levels were observed in animals 7 days post-transection compared with controls. CONCLUSION: Rats transition to a state of hyperreflexia between 7 and 14 days post-transection. Cx-36 protein levels decreased at 7 days post-transection and gradually returned to control levels by 30 days post-transection. These data suggest there may be a relationship between changes in neuronal gap junction protein levels and the delayed onset of hyperreflexia.