INTRODUCTION:Transcranial ultrasound stimulation (TUS) shows great promise for inducing neuroplastic changes that persist long after stimulation. Evidence of stimulation-locked (online) neural changes would enable the development of closed-loop application of TUS. However, such responses have been difficult to distinguish from coincident neural activity caused by auditory and somatosensory effects of TUS. METHODS:To dissociate genuine online neuromodulatory effects from peripheral confounds, we leveraged the contralateral retinotopic organization of the early visual cortex in nineteen subjects. Using a hemifield visual stimulation paradigm combined with high-precision, functional MRI-guided TUS, we applied TUS to the left early visual cortex while participants viewed checkerboards presented in the left or right visual hemifield. Randomized delivery of TUS on half of the trials enabled within-subject comparisons of hemisphere-specific, pattern-locked visual evoked potentials (VEPs) across stimulated and unstimulated hemispheres, as well as across visual-stimulus and no-stimulus conditions. RESULTS:TUS to the left visual cortex reduced mean VEP amplitude over the stimulated (left) hemisphere for right-hemifield (contralateral) stimuli. No such reduction appeared at the symmetric right-hemisphere sites for left-hemifield stimuli. The degree of online suppression correlated positively with target engagement, estimated by modelling the TUS field that accounted for inter-individual heterogeneity in skull transmission and its overlap with the fMRI-defined target. This relationship suggests that greater target engagement is reliably associated with stronger TUS-induced neural modulation. CONCLUSIONS:These findings provide clear evidence of online, spatially specific TUS-induced neural modulation, dissociated from peripheral confounds. This approach establishes a robust framework for future studies aiming to map the TUS parameter space in real time by leveraging topographic organization to control for peripheral confounds, and supports the development of closed-loop neuromodulation protocols.

Sleep is essentially contributing to human health and well-being through multiple biological functions, including restoration and biosynthesis, brain clearance, energy metabolism, immunological and endocrine processing, synaptic plasticity, memory consolidation, and regulation of cognitive and emotional processes. Sleep disturbances are highly prevalent and are both a symptom and a contributing risk factor for psychiatric, neurological, and somatic disorders. Given the limitations of pharmacological interventions, noninvasive neuromodulation techniques ranging from noninvasive transcranial [transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS), transcranial alternating current stimulation (tACS), transcranial random noise stimulation (tRNS), temporal interference stimulation (tTIS), and transcranial ultrasound stimulation (TUS)] to peripheral sensory (auditory, olfactory, visual, tactile, vestibular) and electrical nerve (galvanic vestibular, transcutaneous vagus nerve, and median nerve) stimulation have gained increasing attention as potential tools to modulate sleep physiology. These techniques offer promising avenues for both therapeutic applications and fundamental research into sleep-dependent neuroplasticity, interregional communication, and oscillatory activity. However, sleep is not a uniform state but a highly complex and dynamic phenomenon, with intricate macrostructural [e.g., non-rapid eye movement (NREM)-rapid eye movement (REM) sleep balance, sleep efficiency] and microstructural (e.g., hierarchically nested slow waves and spindles) characteristics that contribute to a variety of functions. This complexity necessitates precise targeting strategies, often employing real-time brain state-dependent stimulation, to modulate specific sleep-related processes effectively. In this review, we summarize the functions of sleep and the available noninvasive tools for its modulation, addressing key methodological challenges and providing recommendations for best practices in sleep neuromodulation.

Low-intensity Transcranial Ultrasonic Stimulation (TUS) is a non-invasive brain stimulation technique enabling cortical and deep brain targeting with unprecedented spatial accuracy. Given the high rate of adoption by new users with varying levels of expertise and interdisciplinary backgrounds, practical guidelines are needed to ensure state-of-the-art TUS application and reproducible outcomes. Therefore, the International Transcranial Ultrasonic Stimulation Safety and Standards (ITRUSST) consortium has formed a subcommittee, endorsed by the International Federation of Clinical Neurophysiology (IFCN), to develop recommendations for best practice in TUS applications in humans. The practical guide presented here provides a brief introduction into ultrasound physics and sonication parameters. It explains the requirements of TUS lab equipment and transducer selection and discusses experimental design and procedures alongside potential confounds and control conditions. Finally, the guide elaborates on essential steps of application planning for stimulation safety and efficacy, as well as considerations when combining TUS with neuroimaging, electrophysiology, or other brain stimulation techniques. We hope that this practical guide to TUS will assist both novice and experienced users in planning and conducting high-quality studies and provide a solid foundation for further advancements in this promising field.

Focused ultrasound can non-invasively modulate neural activity, but whether effective stimulation parameters generalize across brain regions and cell types remains unknown. We used focused ultrasound coupled with fiber photometry to identify optimal neuromodulation parameters for four different arousal centers of the brain in an effort to yield overt changes in behavior. Applying coordinate descent, we found that optimal parameters for excitation or inhibition are highly distinct, the effects of which are generally conserved across brain regions and cell types. Optimized stimulations induced clear, target-specific behavioral effects, whereas non-optimized protocols of equivalent energy resulted in substantially less or no change in behavior. These outcomes were independent of auditory confounds and, contrary to expectation, accompanied by a cyclooxygenase-dependent and prolonged reduction in local blood flow and temperature with brain-region-specific scaling. These findings demonstrate that carefully tuned and targeted ultrasound can exhibit powerful effects on complex behavior and physiology.

IntroductionOpioid use disorder (OUD) is a serious and persistent problem in the United States with limited non-pharmacological treatment options, especially for the concomitant sleep disorders experienced by most individuals with addiction. While new, non-invasive interventions such as low-intensity focused ultrasound (LIFU) have shown promise in targeting the brain regions impacted throughout addiction and recovery, the devices used are not amenable to outpatient treatment in their current form factor and cannot be used at night during sleep. To bridge this gap and provide a much-needed treatment option for repeated, at-home use, we developed a wearable LIFU device out-of-clinic use.MethodsThis study evaluated the feasibility and acceptability of the portable treatment device among individuals recovering from OUD in an unsupervised, at-home setting. 31 subjects were recruited from a Baltimore, Maryland (USA) outpatient treatment facility and, along with a separate group of 14 healthy controls (HC), were asked to wear a prototype EEG-only (non-LIFU) device for 7 consecutive nights to assess their willingness and adherence to nightly use. Participants used a smartphone application, TrialKit (ePRO), to self-report nightly sleep data (e.g. duration, quality, possible disturbances, and device comfort).ResultsOf the 31 OUD participants recruited, 30 (97%) successfully completed the at-home study, and the majority responded that they would participate in future studies using the head wearable device (OUD, 87%; HC, 71%). OUD participants were statistically more likely than HCs to respond that they would consider using the device in the future to help them sleep (OUD, 70%; HC, 29%). Despite some participants facing technological issues (e.g. lack of reliable phone access or cellular data plans), the OUD group demonstrated high study compliance on par with the healthy control group.DiscussionParticipant’s daily ePRO and exit interview results established that at-home use of advanced treatment technology is feasible in a population group challenged with recovering from OUD. Even more so, numerous participants noted strong willingness to participate in future LIFU-enabled intervention studies to address their persistent sleep issues during recovery.

Sleep and feeding are vital homeostatic behaviors, and disruptions in either can result in substantial metabolic consequences. Distinct neuronal manipulations in Drosophila can dissociate sleep loss from subsequent homeostatic rebound, offering an optimal platform to examine the precise interplay between these fundamental behaviors. Here, we investigate concomitant changes in sleep and food intake in individual animals, as well as respiratory metabolic expenditure, that accompany behavioral and genetic manipulations that induce sleep loss in Drosophila melanogaster. We find that sleep disruptions resulting in energy deficit through increased metabolic expenditure and manifested as increased food intake were consistently followed by rebound sleep. In contrast, "soft" sleep loss, which does not induce rebound sleep, is not accompanied by increased metabolism and food intake. Our results demonstrate that homeostatic sleep rebound is linked to energy deficit accrued during sleep loss. Collectively, these findings support the notion that sleep functions to conserve energy and highlight the need to examine the effects of metabolic therapeutics on sleep.

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.

Our understanding of brain circuit operations and disorders has rapidly outpaced our ability to intervene and restore them. Developing technologies that can precisely interface with any brain region and circuit may combine diagnostics with therapeutic intervention, expediting personalised brain medicine. Transcranial ultrasound stimulation (TUS) is a promising noninvasive solution to this challenge, offering focal precision and scalability. By exploiting the biomechanics of pressure waves on brain tissue, TUS enables multi-site targeted neuromodulation across distributed circuits in the cortex and deeper areas alike. In this Essay, we explore the emergent evidence that TUS can functionally test and modify dysfunctional regions, effectively serving as a search and rescue tool for the brain. We define the challenges and opportunities faced by TUS as it moves towards greater target precision and integration with advanced brain monitoring and interventional technology. Finally, we propose a roadmap for the evolution of TUS as it progresses from a research tool to a clinically validated therapeutic for brain disorders.

Endotherms can survive low temperatures and food shortage by actively entering a hypometabolic state known as torpor. Although the decrease in metabolic rate and body temperature during torpor is controlled by the brain, the underlying neural circuits remain largely unknown. In this study, we identify the neural circuits involved in torpor regulation by combining whole-brain mapping of torpor-activated neurons, cell type-specific manipulation of neural activity, and viral tracing-based circuit mapping. We found that Trpm2-positive neurons in the preoptic area and Vgat-positive neurons in the dorsal medial hypothalamus are activated during torpor. Genetic silencing shows the activity of either cell type is necessary to enter the torpor state. Finally, we show that these cells receive projections from the arcuate and suprachiasmatic nucleus and send projections to brain regions involved in thermoregulation. Our results demonstrate an essential role of hypothalamic neurons in the regulation of body temperature and metabolic rate during torpor and identify critical nodes of the torpor-regulatory network.

Neuropeptide S (NPS) is a highly conserved peptide found in all tetrapods that functions in the brain to promote heightened arousal; however, the subpopulations mediating these phenomena remain unknown. We generated mice expressing Cre recombinase from the Nps gene locus (NpsCre) and examined populations of NPS+ neurons in the lateral parabrachial area (LPBA), the peri-locus coeruleus (peri-LC) region of the pons, and the dorsomedial thalamus (DMT). We performed brain-wide mapping of input and output regions of NPS+ clusters and characterized expression patterns of the NPS receptor 1 (NPSR1). While the activity of all three NPS+ subpopulations tracked with vigilance state, only NPS+ neurons of the LPBA exhibited both increased activity prior to wakefulness and decreased activity during REM sleep, similar to the behavioral phenotype observed upon NPSR1 activation. Accordingly, we found that activation of the LPBA but not the peri-LC NPS+ neurons increased wake and reduced REM sleep. Furthermore, given the extended role of the LPBA in respiration and the link between behavioral arousal and breathing rate, we demonstrated that the LPBA but not the peri-LC NPS+ neuronal activation increased respiratory rate. Together, our data suggest that NPS+ neurons of the LPBA represent an unexplored subpopulation regulating breathing, and they are sufficient to recapitulate the sleep/wake phenotypes observed with broad NPS system activation.

Transcranial ultrasound neuromodulation is a promising potential therapeutic tool for the noninvasive treatment of neuropsychiatric disorders. However, the expansive parameter space and difficulties in controlling for peripheral auditory effects make it challenging to identify ultrasound sequences and brain targets that may provide therapeutic efficacy. Careful preclinical investigations in clinically relevant behavioral models are critically needed to identify suitable brain targets and acoustic parameters. However, there is a lack of ultrasound devices allowing for multi-target experimental investigations in awake and unrestrained rodents. We developed a miniaturized 64-element ultrasound array that enables neurointerventional investigations with within-trial active control targets in freely behaving rats. We first characterized the acoustic field with measurements in free water and with transcranial propagation. We then confirmed in vivo that the array can target multiple brain regions via electronic steering, and verified that wearing the device does not cause significant impairments to animal motility. Finally, we demonstrated the performance of our system in a high-throughput neuromodulation experiment, where we found that ultrasound stimulation of the rat central medial thalamus, but not an active control target, promotes arousal and increases locomotor activity.

Neuropeptide S (NPS) is a highly conserved peptide found in all tetrapods that functions in the brain to promote heightened arousal; however, the subpopulations mediating these phenomena remain unknown. We generated mice expressing Cre recombinase from the Nps gene locus (NpsCre) and examined populations of NPS+ neurons in the lateral parabrachial area (LPBA), the peri-locus coeruleus (peri-LC) region of the pons, and the dorsomedial thalamus (DMT). We performed brain-wide mapping of input and output regions of NPS+ clusters and characterized expression patterns of the NPS receptor 1 (NPSR1). While the activity of all three NPS+ subpopulations tracked with vigilance state, only NPS+ neurons of the LPBA exhibited both increased activity prior to wakefulness and decreased activity during REM sleep, similar to the behavioral phenotype observed upon NPSR1 activation. Accordingly, we found that activation of the LPBA but not the peri-LC NPS+ neurons increased wake and reduced REM sleep. Furthermore, given the extended role of the LPBA in respiration and the link between behavioral arousal and breathing rate, we demonstrated that the LPBA but not the peri-LC NPS+ neuronal activation increased respiratory rate. Together, our data suggest that NPS+ neurons of the LPBA represent an unexplored subpopulation regulating breathing, and they are sufficient to recapitulate the sleep/wake phenotypes observed with broad NPS system activation.

Endotherms can survive low temperatures and food shortage by actively entering a hypometabolic state known as torpor. Although the decrease in metabolic rate and body temperature (Tb) during torpor is controlled by the brain, the specific neural circuits underlying these processes have not been comprehensively elucidated. In this study, we identify the neural circuits involved in torpor regulation by combining whole-brain mapping of torpor-activated neurons, cell-type-specific manipulation of neural activity, and viral tracing-based circuit mapping. We find that Trpm2-positive neurons in the preoptic area and Vgat-positive neurons in the dorsal medial hypothalamus are activated during torpor. Genetic silencing shows that the activity of either cell type is necessary to enter the torpor state. Finally, we show that these cells receive projections from the arcuate and suprachiasmatic nucleus and send projections to brain regions involved in thermoregulation. Our results demonstrate an essential role of hypothalamic neurons in the regulation of Tb and metabolic rate during torpor and identify critical nodes of the torpor regulatory network.

Transcranial ultrasound stimulation (TUS) holds great potential as a tool to alter neural circuits non-invasively in both animals and humans. In contrast to established non-invasive brain stimulation methods, ultrasonic waves can be focused on both cortical and deep brain targets with the unprecedented spatial resolution as small as a few cubic millimeters. This focusing allows exclusive targeting of small subcortical structures, previously accessible only by invasive deep brain stimulation devices. The neuromodulatory effects of TUS are likely derived from the kinetic interaction of the ultrasound waves with neuronal membranes and their constitutive mechanosensitive ion channels, to produce short term and long-lasting changes in neuronal excitability and spontaneous firing rate. After decades of mechanistic and safety investigation, the technique has finally come of age, and an increasing number of human TUS studies are expected. Given its excellent compatibility with non-invasive brain mapping techniques, such as electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), as well as neuromodulatory techniques, such as transcranial magnetic stimulation (TMS), systemic TUS effects can readily be assessed in both basic and clinical research. In this review, we present the fundamentals of TUS for a broader audience. We provide up-to-date information on the physical and neurophysiological mechanisms of TUS, available readouts for its neural and behavioral effects, insights gained from animal models and human studies, potential clinical applications, and safety considerations. Moreover, we discuss the indirect effects of TUS on the nervous system through peripheral co-stimulation and how these confounding factors can be mitigated by proper control conditions.

Focused ultrasound (FUS) is a powerful tool for noninvasive modulation of deep brain activity with promising therapeutic potential for refractory epilepsy; however, tools for examining FUS effects on specific cell types within the deep brain do not yet exist. Consequently, how cell types within heterogeneous networks can be modulated and whether parameters can be identified to bias these networks in the context of complex behaviors remains unknown. To address this, we developed a fiber Photometry Coupled focused Ultrasound System (PhoCUS) for simultaneously monitoring FUS effects on neural activity of subcortical genetically targeted cell types in freely behaving animals. We identified a parameter set that selectively increases activity of parvalbumin interneurons while suppressing excitatory neurons in the hippocampus. A net inhibitory effect localized to the hippocampus was further confirmed through whole brain metabolic imaging. Finally, these inhibitory selective parameters achieved significant spike suppression in the kainate model of chronic temporal lobe epilepsy, opening the door for future noninvasive therapies.