The Integration of Top-down and Bottom-up Inputs to the Striatal Cholinergic Interneurons.

BACKGROUND: Cholinergic interneurons (ChIs) are important for learning and memory. They exhibit a multiphasic excitation-pause-rebound response to reward or sensory cues indicating a reward, believed to gate dopamine-dependent learning. Although ChIs receive extensive top-down inputs from the cortex and bottom-up inputs from the thalamus and midbrain, it is unclear which inputs are involved in the development of ChI multiphasic activity. METHODS: We used a single-unit recording of putative ChIs (pChIs) in response to cortical and visual stimulation to investigate how top-down and bottom-up inputs regulate the firing pattern of ChIs. RESULTS: We demonstrated that cortical stimulation strongly regulates pChIs, with the maximum firing rate occurring at the peak of the inverted local field potential (iLFP), reflecting maximum cortical stimulation. Pauses in pChIs occurred during the descending phase of iLFP, indicating withdrawal of excitatory cortical input. Visual stimulation induced long pauses in pChIs, but it is unlikely that bottom- up inputs alone induce pauses in behaving animals. Also, the firing pattern of ChIs triggered by visual stimulation did not correlate with the iLFP as it did after cortical stimulation. Top-down and bottom-up inputs independently regulate the firing pattern of ChIs with similar efficacy but notably produce a well-defined pause in ChI firing. CONCLUSION: This study provides in vivo evidence that the multiphasic ChI response may require both top-down and bottom-up inputs. The findings suggest that the firing pattern of ChIs correlated to the iLFP might be a useful tool for estimating the degree of contribution of top-down and bottom-up inputs in regulating the firing activity of ChIs.

Sensory Reinforced Corticostriatal Plasticity.

BACKGROUND: Regional changes in corticostriatal transmission induced by phasic dopaminergic signals are an essential feature of the neural network responsible for instrumental reinforcement during discovery of an action. However, the timing of signals that are thought to contribute to the induction of corticostriatal plasticity is difficult to reconcile within the framework of behavioural reinforcement learning, because the reinforcer is normally delayed relative to the selection and execution of causally-related actions. OBJECTIVE: While recent studies have started to address the relevance of delayed reinforcement signals and their impact on corticostriatal processing, our objective was to establish a model in which a sensory reinforcer triggers appropriately delayed reinforcement signals relayed to the striatum via intact neuronal pathways and to investigate the effects on corticostriatal plasticity. METHODS: We measured corticostriatal plasticity with electrophysiological recordings using a light flash as a natural sensory reinforcer, and pharmacological manipulations were applied in an in vivo anesthetized rat model preparation. RESULTS: We demonstrate that the spiking of striatal neurons evoked by single-pulse stimulation of the motor cortex can be potentiated by a natural sensory reinforcer, operating through intact afferent pathways, with signal timing approximating that required for behavioural reinforcement. The pharmacological blockade of dopamine receptors attenuated the observed potentiation of corticostriatal neurotransmission. CONCLUSION: This novel in vivo model of corticostriatal plasticity offers a behaviourally relevant framework to address the physiological, anatomical, cellular, and molecular bases of instrumental reinforcement learning.

High-Definition Transcranial Infraslow Pink-Noise Stimulation Can Influence Functional and Effective Cortical Connectivity in Individuals With Chronic Low Back Pain: A Pilot Randomized Placebo-Controlled Study.

INTRODUCTION: Pain can be regarded as an emergent property of multiple interacting, dynamically changing brain networks and thus needs a targeted treatment approach. A novel high-definition transcranial infraslow pink-noise stimulation (HD-tIPNS) technique was developed to modulate the key hubs of the three main nociceptive pathways simultaneously, ie, the pregenual anterior cingulate cortex (pgACC) (descending inhibitory pathway), the dorsal anterior cingulate cortex (dACC) (medial nociceptive pathway), and both somatosensory cortices (S1) (lateral nociceptive pathway). This study aimed to evaluate safety and verify whether a single session of HD-tIPNS may disrupt functional and effective connectivity between targeted cortical regions. MATERIALS AND METHODS: A pilot double-blind randomized two-arm placebo-controlled parallel trial was conducted. Participants (N = 30) with chronic low back pain were equally randomized to receive a single session of either sham stimulation or HD-tIPNS (targeting the pgACC, dACC, and bilateral S1). Primary outcomes included safety and electroencephalographic measures, and secondary outcomes included pain measures, collected after treatment. A Mann-Whitney U test was used to compare between-group differences in percentage changes with baseline for each outcome measures. A Wilcoxon signed-rank test was used to identify difference in effective connectivity measure before and after HD-tIPNS. RESULTS: No serious adverse events were reported. A significant decrease in instantaneous functional connectivity was noted between the pgACC and dACC (U = 47.0, Z = -2.72, p = 0.007) and the pgACC and left S1 (U = 41.0, Z = -2.97, p = 0.003) in the infraslow band after HD-tIPNS when compared with sham stimulation. A significant decrease in instantaneous effective connectivity was noted in the direction of the dACC to the pgACC (Z = -2.10, p = 0.035), in the infraslow band after HD-tIPNS when compared with baseline. No changes in clinical pain measures were detected. CONCLUSIONS: HD-tIPNS can safely modulate the functional and effective connectivity between targeted pain-related cortical hubs. Further studies are warranted to evaluate whether repeated exposures to HD-tIPNS can incur clinical benefits through inducing changes in functional and effective connectivity at targeted cortical regions. CLINICAL TRIAL REGISTRATION: The Clinicaltrials.gov registration number for the study is ACTRN12621001438842.

Harnessing Ultrasound for Targeting Drug Delivery to the Brain and Breaching the Blood-Brain Tumour Barrier.

Despite significant advances in developing drugs to treat brain tumours, achieving therapeutic concentrations of the drug at the tumour site remains a major challenge due to the presence of the blood-brain barrier (BBB). Several strategies have evolved to enhance brain delivery of chemotherapeutic agents to treat tumours; however, most approaches have several limitations which hinder their clinical utility. Promising studies indicate that ultrasound can penetrate the skull to target specific brain regions and transiently open the BBB, safely and reversibly, with a high degree of spatial and temporal specificity. In this review, we initially describe the basics of therapeutic ultrasound, then detail ultrasound-based drug delivery strategies to the brain and the mechanisms by which ultrasound can improve brain tumour therapy. We review pre-clinical and clinical findings from ultrasound-mediated BBB opening and drug delivery studies and outline current therapeutic ultrasound devices and technologies designed for this purpose.

The Sheep as a Large Animal Model for the Investigation and Treatment of Human Disorders.

An essential aim of biomedical research is to translate basic science information obtained from preclinical research using small and large animal models into clinical practice for the benefit of humans. Research on rodent models has enhanced our understanding of complex pathophysiology, thus providing potential translational pathways. However, the success of translating drugs from pre-clinical to clinical therapy has been poor, partly due to the choice of experimental model. The sheep model, in particular, is being increasingly applied to the field of biomedical research and is arguably one of the most influential models of human organ systems. It has provided essential tools and insights into cardiovascular disorder, orthopaedic examination, reproduction, gene therapy, and new insights into neurodegenerative research. Unlike the widely adopted rodent model, the use of the sheep model has an advantage over improving neuroscientific translation, in particular due to its large body size, gyrencephalic brain, long lifespan, more extended gestation period, and similarities in neuroanatomical structures to humans. This review aims to summarise the current status of sheep to model various human diseases and enable researchers to make informed decisions when considering sheep as a human biomedical model.

Mapping sheep to human brain: The need for a sheep brain atlas.

A brain atlas is essential for understanding the anatomical relationship between neuroanatomical structures. Standard stereotaxic coordinates and reference systems have been developed for humans, non-human primates and small laboratory animals to contribute to translational neuroscience research. Despite similar neuroanatomical and neurofunctional features between the sheep and human brain, little is known of the sheep brain stereotaxy, and a detailed sheep atlas is scarce. Here, we briefly discuss the value of using sheep in neurological research and the paucity of literature concerning the coordinates system during neurosurgical approaches. Recent advancements such as computerized tomography, positron emission tomography, magnetic resonance imaging, functional magnetic resonance imaging and diffusion tensor imaging are used for targeting and localizing the coordinates and brain areas in humans. Still, their application in sheep is rare due to the lack of a 3D stereotaxic sheep atlas by which to map sheep brain structures to its human counterparts. More recently, a T1- and T2-weighted high-resolution MRI 3D stereotaxic atlas of the sheep brain has been generated, however, the journey to create a sheep brain atlas by which to map directly to the human brain is still uncharted. Therefore, developing a detailed sheep brain atlas is valuable for the future to facilitate the use of sheep as a large animal experimental non-primate model for translational neurological research.

High-definition transcranial infraslow pink noise stimulation for chronic low back pain: protocol for a pilot, safety and feasibility randomised placebo-controlled trial.

INTRODUCTION: Chronic low back pain (CLBP) is a common disabling health condition. Current treatments demonstrate modest effects, warranting newer therapies. Brain imaging demonstrates altered electrical activities in cortical areas responsible for pain modulation, emotional and sensory components of pain experience. Treatments targeting to change electrical activities of these key brain regions may produce clinical benefits. This pilot study aims to (1) evaluate feasibility, safety and acceptability of a novel neuromodulation technique, high-definition transcranial infraslow pink noise stimulation (HD-tIPNS), in people with CLBP, (2) explore the trend of effect of HD-tIPNS on pain and function, and (3) derive treatment estimates to support sample size calculation for a fully powered trial should trends of effectiveness be present. METHODS AND ANALYSIS: A pilot, triple-blinded randomised two-arm placebo-controlled parallel trial. Participants (n=40) with CLBP will be randomised to either sham stimulation or HD-tIPNS (targeting somatosensory cortex and dorsal and pregenual anterior cingulate cortex). Primary outcomes include feasibility and safety measures, and clinical outcomes of pain (Brief Pain Inventory) and disability (Roland-Morris disability questionnaire). Secondary measures include clinical, psychological, quantitative sensory testing and electroencephalography collected at baseline, immediately postintervention, and at 1-week, 1-month and 3 months postintervention. All data will be analysed descriptively. A nested qualitative study will assess participants perceptions about acceptability of intervention and analysed thematically. ETHICS AND DISSEMINATION: Ethical approval has been obtained from Health and Disability Ethics Committee (Ref:20/NTB/67). Findings will be reported to regulatory and funding bodies, presented at conferences, and published in a scientific journal. TRIAL REGISTRATION NUMBER: ACTRN12620000505909p.

Ultrasound-Mediated Blood-Brain Barrier Disruption for Drug Delivery: A Systematic Review of Protocols, Efficacy, and Safety Outcomes from Preclinical and Clinical Studies.

Ultrasound-mediated blood-brain barrier (BBB) disruption has garnered focus as a method of delivering normally impenetrable drugs into the brain. Numerous studies have investigated this approach, and a diverse set of ultrasound parameters appear to influence the efficacy and safety of this approach. An understanding of these findings is essential for safe and reproducible BBB disruption, as well as in identifying the limitations and gaps for further advancement of this drug delivery approach. We aimed to collate and summarise protocols and parameters for achieving ultrasound-mediated BBB disruption in animal and clinical studies, as well as the efficacy and safety methods and outcomes associated with each. A systematic search of electronic databases helped in identifying relevant, included studies. Reference lists of included studies were further screened to identify supplemental studies for inclusion. In total, 107 articles were included in this review, and the following parameters were identified as influencing efficacy and safety outcomes: microbubbles, transducer frequency, peak-negative pressure, pulse characteristics, and the dosing of ultrasound applications. Current protocols and parameters achieving ultrasound-mediated BBB disruption, as well as their associated efficacy and safety outcomes, are identified and summarised. Greater standardisation of protocols and parameters in future preclinical and clinical studies is required to inform robust clinical translation.

Coincidence of cholinergic pauses, dopaminergic activation and depolarisation of spiny projection neurons drives synaptic plasticity in the striatum.

Dopamine-dependent long-term plasticity is believed to be a cellular mechanism underlying reinforcement learning. In response to reward and reward-predicting cues, phasic dopamine activity potentiates the efficacy of corticostriatal synapses on spiny projection neurons (SPNs). Since phasic dopamine activity also encodes other behavioural variables, it is unclear how postsynaptic neurons identify which dopamine event is to induce long-term plasticity. Additionally, it is unknown how phasic dopamine released from arborised axons can potentiate targeted striatal synapses through volume transmission. To examine these questions we manipulated striatal cholinergic interneurons (ChIs) and dopamine neurons independently in two distinct in vivo paradigms. We report that long-term potentiation (LTP) at corticostriatal synapses with SPNs is dependent on the coincidence of pauses in ChIs and phasic dopamine activation, critically accompanied by SPN depolarisation. Thus, the ChI pause defines the time window for phasic dopamine to induce plasticity, while depolarisation of SPNs constrains the synapses eligible for plasticity.

Correction: Gandhi et al. Ultrasound-Mediated Blood-Brain Barrier Disruption for Drug Delivery: A Systematic Review of Protocols, Efficacy, and Safety Outcomes from Preclinical and Clinical Studies. Pharmaceutics 2022, 14, 833.

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Throwing open the doors of perception: The role of dopamine in visual processing.

Animals form associations between visual cues and behaviours. Although dopamine is known to be critical in many areas of the brain to bind sensory information with appropriate responses, dopamine's role in the visual system is less well understood. Visual signals, which indicate the likely occurrence of a rewarding or aversive stimulus or indicate the context within which such stimuli may arrive, modulate activity in the superior colliculus and alter behaviour. However, such signals primarily originate in cortical and basal ganglia circuits, and evidence of direct signalling from midbrain dopamine neurons to superior colliculus is lacking. Instead, hypothalamic A13 dopamine neurons innervate the superior colliculus, and dopamine receptors are differentially expressed in the superior colliculus, with D1 receptors in superficial layers and D2 receptors in deep layers. However, it remains unknown if A13 dopamine neurons control behaviours through their effect on afferents within the superior colliculus. We propose that A13 dopamine neurons may play a critical role in processing information in the superior colliculus, modifying behavioural responses to visual cues, and propose some testable hypotheses regarding dopamine's effect on visual perception.

Steric stabilisers govern the colloidal and chemical stability but not in vitro cellular toxicity of linoleoylethanolamide cubosomes.

Linoleoylethanolamide (LEA) is an endogenous lipid with remarkable neuromodulatory properties. However, its therapeutic potential is limited by rapid clearance in vivo, targetability and solubility. This study aimed to formulate LEA into liquid crystalline nanoparticles (cubosomes) as a strategy to address the aforementioned challenges. The influence of three different steric stabilisers: Tween 80 and Pluronic F68, both of which have the potential to interact with receptors expressed at the blood-brain barrier and Pluronic F127 as a control, on colloidal stability, internal structure, chemical stability and cytotoxicity of the dispersions were investigated. We found that for effective stabilization of LEA dispersions, a higher concentration of Tween 80 was required compared to Pluronics. Freshly prepared dispersions showed mean particle size of <250 nm and low PDIs (<0.2), with an Im3m type cubic structure but with different lattice parameters. Upon storage at ambient temperature for a week, increased mean particle size and PDI, with a significant reduction in the concentration of LEA was observed in Tween 80-stabilised dispersions. Greater than 80% cell viability was observed at concentrations of up to 20 µg/mL LEA in the presence of all three stabilisers. Collectively, our results suggest that the stabiliser type influences colloidal and chemical stability but not cytotoxicity of LEA cubosomes. This study highlights the potential of endogenous bioactive lipids to be utilized as core cubosome forming lipids with the view to improving their solubility, rapid clearance and targetability to enable delivery of these bioactive molecules to the brain.

Low-intensity contralesional electrical theta burst stimulation modulates ipsilesional excitability and enhances stroke recovery.

Targeting interhemispheric inhibition using brain stimulation has shown potential for enhancing stroke recovery. Following stroke, increased inhibition originating from the contralesional hemisphere impairs motor activation in ipsilesional areas. We have previously reported that low-intensity electrical theta burst stimulation (TBS) applied to an implanted electrode in the contralesional rat motor cortex reduces interhemispheric inhibition, and improves functional recovery when commenced three days after cortical injury. Here we apply this approach at more clinically relevant later time points and measure recovery from photothrombotic stroke, following three weeks of low-intensity intermittent TBS (iTBS), continuous TBS (cTBS) or sham stimulation applied to the contralesional motor cortex. Interhemispheric inhibition and cellular excitability were measured in the same rats from single pyramidal neurons in the peri-infarct area, using in vivo intracellular recording. A minimal dose of iTBS did not enhance motor function when applied beginning one month after stroke. However both a high and a low dose of iTBS improved recovery to a similar degree when applied 10 days after stroke, with the degree of recovery positively correlated with ipsilesional excitability. The final level of interhemispheric inhibition was negatively correlated with excitability, but did not independently correlate with functional recovery. In contrast, contralesional cTBS left recovery unaltered, but decreased ipsilesional excitability. These data support focal contralesional iTBS and not cTBS as an intervention for enhancing stroke recovery and suggest that there is a complex relationship between functional recovery and interhemispheric inhibition, with both independently associated with ipsilesional excitability.

Pauses in Cholinergic Interneuron Activity Are Driven by Excitatory Input and Delayed Rectification, with Dopamine Modulation.

Cholinergic interneurons (ChIs) of the striatum pause their firing in response to salient stimuli and conditioned stimuli after learning. Several different mechanisms for pause generation have been proposed, but a unifying basis has not previously emerged. Here, using in vivo and ex vivo recordings in rat and mouse brain and a computational model, we show that ChI pauses are driven by withdrawal of excitatory inputs to striatum and result from a delayed rectifier potassium current (IKr) in concert with local neuromodulation. The IKr is sensitive to Kv7.2/7.3 blocker XE-991 and enables ChIs to report changes in input, to pause on excitatory input recession, and to scale pauses with input strength, in keeping with pause acquisition during learning. We also show that although dopamine can hyperpolarize ChIs directly, its augmentation of pauses is best explained by strengthening excitatory inputs. These findings provide a basis to understand pause generation in striatal ChIs. VIDEO ABSTRACT.

A real-time in vitro assay to evaluate the release of macromolecules from liposomes.

The availability of a real-time assay to experimentally investigate the release of encapsulated proteins would be beneficial given the interest in the use of liposomes as a drug delivery vehicle. Although simple assays for small molecular weight substances exist, assays to evaluate macromolecules do not. Here we describe a method that detects the release of model macromolecules from liposomes in real time. The assay employs the intermolecular distance-dependent phenomenon of fluorescence resonance energy transfer (FRET) between the fluorophore donor, fluorescein (FITC), and fluorescent quencher, QSY® 9. The macromolecular species were conjugated to the markers fluorescein (44kDa dextran) and QSY® 9 (67 kDa bovine serum albumin, BSA). Following confirmation of quenching between FITC-Dex and QSY® 9-BSA, liposomes were loaded with the macromolecular markers and subjected to various treatments (high-pressure extrusion and Triton X solubilisation) to cause release from liposomes. An increase in FITC fluorescence was observed when liposomes were subjected to extrusion cycles. Surprisingly, the addition of Triton X did not cause an increase in fluorescence probably because the FRET pair became associated with mixed micelles. This assay method should be useful in studies to investigate the mechanisms by which macromolecules are released from liposomes, particularly when liposomes are exposed to release-triggers (eg, temperature change, pH change, ultrasound). Such understanding will underpin the formulation of triggered liposomal delivery systems for macromolecules.

Reinforcement determines the timing dependence of corticostriatal synaptic plasticity in vivo.

Plasticity at synapses between the cortex and striatum is considered critical for learning novel actions. However, investigations of spike-timing-dependent plasticity (STDP) at these synapses have been performed largely in brain slice preparations, without consideration of physiological reinforcement signals. This has led to conflicting findings, and hampered the ability to relate neural plasticity to behavior. Using intracellular striatal recordings in intact rats, we show here that pairing presynaptic and postsynaptic activity induces robust Hebbian bidirectional plasticity, dependent on dopamine and adenosine signaling. Such plasticity, however, requires the arrival of a reward-conditioned sensory reinforcement signal within 2 s of the STDP pairing, thus revealing a timing-dependent eligibility trace on which reinforcement operates. These observations are validated with both computational modeling and behavioral testing. Our results indicate that Hebbian corticostriatal plasticity can be induced by classical reinforcement learning mechanisms, and might be central to the acquisition of novel actions.Spike timing dependent plasticity (STDP) has been studied extensively in slices but whether such pairings can induce plasticity in vivo is not known. Here the authors report an experimental paradigm that achieves bidirectional corticostriatal STDP in vivo through modulation by behaviourally relevant reinforcement signals, mediated by dopamine and adenosine signaling.

Differences in Motor Evoked Potentials Induced in Rats by Transcranial Magnetic Stimulation under Two Separate Anesthetics: Implications for Plasticity Studies.

Repetitive transcranial magnetic stimulation (rTMS) is primarily used in humans to change the state of corticospinal excitability. To assess the efficacy of different rTMS stimulation protocols, motor evoked potentials (MEPs) are used as a readout due to their non-invasive nature. Stimulation of the motor cortex produces a response in a targeted muscle, and the amplitude of this twitch provides an indirect measure of the current state of the cortex. When applied to the motor cortex, rTMS can alter MEP amplitude, however, results are variable between participants and across studies. In addition, the mechanisms underlying any change and its locus are poorly understood. In order to better understand these effects, MEPs have been investigated in vivo in animal models, primarily in rats. One major difference in protocols between rats and humans is the use of general anesthesia in animal experiments. Anesthetics are known to affect plasticity-like mechanisms and so may contaminate the effects of an rTMS protocol. In the present study, we explored the effect of anesthetic on MEP amplitude, recorded before and after intermittent theta burst stimulation (iTBS), a patterned rTMS protocol with reported facilitatory effects. MEPs were assessed in the brachioradialis muscle of the upper forelimb under two anesthetics: a xylazine/zoletil combination and urethane. We found MEPs could be induced under both anesthetics, with no differences in the resting motor threshold or the average baseline amplitudes. However, MEPs were highly variable between animals under both anesthetics, with the xylazine/zoletil combination showing higher variability and most prominently a rise in amplitude across the baseline recording period. Interestingly, application of iTBS did not facilitate MEP amplitude under either anesthetic condition. Although it is important to underpin human application of TMS with mechanistic examination of effects in animals, caution must be taken when selecting an anesthetic and in interpreting results during prolonged TMS recording.

Action Experience and Action Discovery in Medicated Individuals with Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder that markedly affects voluntary action. While regular dopamine treatment can help restore motor function, dopamine also influences cognitive portions of the action system. Previous studies have demonstrated that dopamine medication boosts action-effect associations, which are crucial for the discovery of new voluntary actions. In the present study, we investigated whether neural processes involved in the discovery of new actions are altered in PD participants on regular dopamine treatment, compared to healthy age-matched controls. We recorded brain electroencephalography (EEG) activity while PD patients and age-matched controls performed action discovery (AD) and action control tasks. We found that the novelty P3, a component normally present when there is uncertainty about the occurrence of the sensory effect, was enhanced in PD patients. However, AD was maintained in PD patients, and the novelty P3 demonstrated normal learning-related reductions. Crucially, we found that in PD patients the causal association between an action and its resulting sensory outcome did not modulate the amplitude of the feedback correct-related positivity (fCRP), an EEG component sensitive to the association between an action and its resulting effect. Collectively, these preliminary results suggest that the formation of long-term action-outcome representations may be maintained in PD patients on regular dopamine treatment, but the initial experience of action-effect association may be affected.

Low-intensity repetitive magnetic stimulation lowers action potential threshold and increases spike firing in layer 5 pyramidal neurons in vitro.

Repetitive transcranial magnetic stimulation (rTMS) has become a popular method of modulating neural plasticity in humans. Clinically, rTMS is delivered at high intensities to modulate neuronal excitability. While the high-intensity magnetic field can be targeted to stimulate specific cortical regions, areas adjacent to the targeted area receive stimulation at a lower intensity and may contribute to the overall plasticity induced by rTMS. We have previously shown that low-intensity rTMS induces molecular and structural plasticity in vivo, but the effects on membrane properties and neural excitability have not been investigated. Here we investigated the acute effect of low-intensity repetitive magnetic stimulation (LI-rMS) on neuronal excitability and potential changes on the passive and active electrophysiological properties of layer 5 pyramidal neurons in vitro. Whole-cell current clamp recordings were made at baseline prior to subthreshold LI-rMS (600 pulses of iTBS, n=9 cells from 7 animals) or sham (n=10 cells from 9 animals), immediately after stimulation, as well as 10 and 20min post-stimulation. Our results show that LI-rMS does not alter passive membrane properties (resting membrane potential and input resistance) but hyperpolarises action potential threshold and increases evoked spike-firing frequency. Increases in spike firing frequency were present throughout the 20min post-stimulation whereas action potential (AP) threshold hyperpolarization was present immediately after stimulation and at 20min post-stimulation. These results provide evidence that LI-rMS alters neuronal excitability of excitatory neurons. We suggest that regions outside the targeted region of high-intensity rTMS are susceptible to neuromodulation and may contribute to rTMS-induced plasticity.