Modulation of deep neural circuits with sonogenetics.

Noninvasive control of neuronal activity in the deep brain can be illuminating for probing brain function and treating dysfunctions. Here, we present a sonogenetic approach for controlling distinct mouse behavior with circuit specificity and subsecond temporal resolution. Targeted neurons in subcortical regions were made to express a mutant large conductance mechanosensitive ion channel (MscL-G22S), enabling ultrasound to trigger activity in MscL-expressing neurons in the dorsal striatum and increase locomotion in freely moving mice. Ultrasound stimulation of MscL-expressing neurons in the ventral tegmental area could activate the mesolimbic pathway to trigger dopamine release in the nucleus accumbens and modulate appetitive conditioning. Moreover, sonogenetic stimulation of the subthalamic nuclei of Parkinson's disease model mice improved their motor coordination and mobile time. Neuronal responses to ultrasound pulse trains were rapid, reversible, and repeatable. We also confirmed that the MscL-G22S mutant is more effective to sensitize neurons to ultrasound compared to the wild-type MscL. Altogether, we lay out a sonogenetic approach which can selectively manipulate targeted cells to activate defined neural pathways, affect specific behaviors, and relieve symptoms of neurodegenerative disease.

The mechanosensitive ion channel Piezo1 contributes to ultrasound neuromodulation.

Transcranial low-intensity ultrasound is a promising neuromodulation modality, with the advantages of noninvasiveness, deep penetration, and high spatiotemporal accuracy. However, the underlying biological mechanism of ultrasonic neuromodulation remains unclear, hindering the development of efficacious treatments. Here, the well-known Piezo1 was studied through a conditional knockout mouse model as a major mediator for ultrasound neuromodulation ex vivo and in vivo. We showed that Piezo1 knockout (P1KO) in the right motor cortex of mice significantly reduced ultrasound-induced neuronal calcium responses, limb movement, and muscle electromyogram (EMG) responses. We also detected higher Piezo1 expression in the central amygdala (CEA), which was found to be more sensitive to ultrasound stimulation than the cortex was. Knocking out the Piezo1 in CEA neurons showed a significant reduction of response under ultrasound stimulation, while knocking out astrocytic Piezo1 showed no-obvious changes in neuronal responses. Additionally, we excluded an auditory confound by monitoring auditory cortical activation and using smooth waveform ultrasound with randomized parameters to stimulate P1KO ipsilateral and contralateral regions of the same brain and recording evoked movement in the corresponding limb. Thus, we demonstrate that Piezo1 is functionally expressed in different brain regions and that it is an important mediator of ultrasound neuromodulation in the brain, laying the ground for further mechanistic studies of ultrasound.

Photonic Nanojet-Mediated Optogenetics.

Optogenetics has become a widely used technique in neuroscience research, capable of controlling neuronal activity with high spatiotemporal precision and cell-type specificity. Expressing exogenous opsins in the selected cells can induce neuronal activation upon light irradiation, and the activation depends on the power of incident light. However, high optical power can also lead to off-target neuronal activation or even cell damage. Limiting the incident power, but enhancing power distribution to the targeted neurons, can improve optogenetic efficiency and reduce off-target effects. Here, the use of optical lenses made of polystyrene microspheres is demonstrated to achieve effective focusing of the incident light of relatively low power to neighboring neurons via photonic jets. The presence of microspheres significantly localizes and enhances the power density to the target neurons both in vitro and ex vivo, resulting in increased inward current and evoked action potentials. In vivo results show optogenetic stimulation with microspheres that can evoke significantly more motor behavior and neuronal activation at lowered power density. In all, a proof-of-concept of a strategy is demonstrated to increase the efficacy of optogenetic neuromodulation using pulses of reduced optical power.

Suppression of Pain in the Late Phase of Chronic Trigeminal Neuropathic Pain Failed to Rescue the Decision-Making Deficits in Rats.

Trigeminal neuropathic pain (TNP) led to vital cognitive functional deficits such as impaired decision-making abilities in a rat gambling task. Chronic TNP caused hypomyelination in the anterior cingulate cortex (ACC) associated with decreased synchronization between ACC spikes and basal lateral amygdala (BLA) theta oscillations. The aim of this study was to investigate the effect of pain suppression on cognitive impairment in the early or late phases of TNP. Blocking afferent signals with a tetrodotoxin (TTX)-ELVAX implanted immediately following nerve lesion suppressed the allodynia and rescued decision-making deficits. In contrast, the TTX used at a later phase could not suppress the allodynia nor rescue decision-making deficits. Intra-ACC administration of riluzole reduced the ACC neural sensitization but failed to restore ACC-BLA spike-field phase synchrony during the late stages of chronic neuropathic pain. Riluzole suppressed allodynia but failed to rescue the decision-making deficits during the late phase of TNP, suggesting that early pain relief is important for recovering from pain-related cognitive impairments. The functional disturbances in ACC neural circuitry may be relevant causes for the deficits in decision making in the chronic TNP state.

Trigeminal neuropathy causes hypomyelination in the anterior cingulate cortex, disrupts the synchrony of neural circuitry, and impairs decision-making in male rats.

Infraorbital nerve-chronic constriction injury (ION-CCI) has become the most popular chronic trigeminal neuropathic pain (TNP) injury animal model which causes prolonged mechanical allodynia. Accumulative evidence suggests that TNP interferes with cognitive functions, however the underlying mechanisms are not known. The aim of this study was to investigate decision-making performance as well as synaptic and large-scale neural synchronized alterations in the spinal trigeminal nucleus (SpV) circuitry and anterior cingulate cortex (ACC) neural circuitry in male rats with TNP. Rat gambling task showed that ION-CCI led to decrease the proportion of good decision makers and increase the proportion of poor decision makers. Electrophysiological recordings showed long-lasting synaptic potentiation of local field potential in the trigeminal ganglia-SpV caudalis (SpVc) synapses in TNP rats. In this study, TNP led to disruption of ACC spike timing and basolateral amygdala (BLA) theta oscillation associated with suppressed synchronization of theta oscillation between the BLA and ACC, indicating reduced neuronal communications. Myelination is critical for information flow between brain regions, and myelin plasticity is an important feature for learning. Neural activity in the cortical regions impacts myelination by regulating oligodendrocyte (OL) proliferation, differentiation, and myelin formation. We characterized newly formed oligodendrocyte progenitor cells, and mature OLs are reduced in TNP and are associated with reduced myelin strength in the ACC region. The functional disturbances in the BLA-ACC neural circuitry is pathologically associated with the myelin defects in the ACC region which may be relevant causes for the deficits in decision-making in chronic TNP state.

Schema-like learning and memory consolidation acting through myelination.

Memory is a dynamic brain function that is continually processed after encoding. Although psychologic concepts of mental schema are now well established, they have rarely been considered in animal studies. We used a behavior paradigm of multiple flavor-place paired associates (PAs) and showed that memory schema facilitates fast acquisition of new PAs in a single trial. The hippocampus is necessary for the encoding of new PAs and for memory retrieval within a certain time window-24 h following new PA consolidation. Whereas the anterior cingulate cortex (ACC) plays a critical role for dynamic PA learning and consolidation during training sessions, ACC is essential in schema representation and activation. New myelin generation is essential for learning. Neural activity in the cortical regions impacts myelination by regulating oligodendrocyte (OL) proliferation, differentiation, and myelin formation. Here, we show that newly formed OL progenitor cells and mature OLs are increased following repeated PA learning and that establishment of the memory schema is associated with enhanced myelin strength in the ACC region. Furthermore, to ensure that myelination is necessary for the acquisition of paired-associate learning, ACC lysolecithin-induced demyelination revealed impaired PA learning associated with decrease in ACC θ band power and reduced spike-field coherence and phase-locking in ACC.-Hasan, M., Kanna, M. S., Jun, W., Ramkrishnan, A. S., Iqbal, Z., Lee, Y., Li, Y. Schema-like learning and memory consolidation acting through myelination.

Generation of Autologous Platelet-Rich Plasma by the Ultrasonic Standing Waves.

Platelet-rich plasma (PRP) is a volume of autologous plasma that has a higher platelet concentration above baseline. It has already been approved as a new therapeutic modality and investigated in clinics, such as bone repair and regeneration, and oral surgery, with low cost-effectiveness ratio. At present, PRP is mostly prepared using a centrifuge. However, this method has several shortcomings, such as long preparation time (30 min), complexity in operation, and contamination of red blood cells (RBCs). In this paper, a new PRP preparation approach was proposed and tested. Ultrasound waves (4.5 MHz) generated from piezoelectric ceramics can establish standing waves inside a syringe filled with the whole blood. Subsequently, RBCs would accumulate at the locations of pressure nodes in response to acoustic radiation force, and the formed clusters would have a high speed of sedimentation. It is found that the PRP prepared by the proposed device can achieve higher platelet concentration and less RBCs contamination than a commercial centrifugal device, but similar growth factor (i.e., PDGF-ßß). In addition, the sedimentation process under centrifugation and sonication was simulated using the Mason-Weaver equation and compared with each other to illustrate the differences between these two technologies and to optimize the design in the future. Altogether, ultrasound method is an effective method of PRP preparation with comparable outcomes as the commercially available centrifugal products.

Transsclera Drug Delivery by Pulsed High-Intensity Focused Ultrasound (HIFU): An Ex Vivo Study.

UNLABELLED: PURPOSE/AIM OF STUDY: Drug delivery to the ocular posterior segment is of importance, but it is a challenge in the treatment of irreversible blindness disease, such as age-related macular degeneration. Although some methods (i.e. intraocular injection, sustained release by polymer and iontophoresis) have been applied, some technical drawbacks, such as slow rate and damage to the eye, need to be overcome for wide use. MATERIALS AND METHODS: In this study, the feasibility of high-intensity focused ultrasound (HIFU) to enhance the transsclera drug delivery was tested for the first time. One-hundred HIFU pulses with the driving frequency of 1.1 MHz, acoustic power of 105.6 W, pulse duration of 10-50 ms and pulse repetition frequency of 1 Hz were delivered to the fresh ex vivo porcine sclera specimen. RESULTS: In comparison to the passive diffusion (control), 50-ms HIFU can increase the penetration depth by 2.0 folds (501.7 ± 126.4 µm versus 252.4 ± 29.2 µm) using bicinchoninic acid assay and Rhodamine 6 G fluorescence intensity by 3.1 folds (22.4 ± 12.3 versus 7.1 ± 4.1) and coverage area by 2.6 folds (40.4 ± 9.1% versus 15.8 ± 2.9%). No morphological changes on the sonicated sclera samples were found using a surface electron microscope. CONCLUSIONS: In summary, pulsed-HIFU may be an effective modality in the transsclera drug delivery with a high transporting rate and depth. In vivo studies are necessary to further evaluate its performance, including the drug penetration and its possible side effects.

Effect of clot aging and cholesterol content on ultrasound-assisted thrombolysis.

Exposure to 2-MHz transcranial diagnostic ultrasound enhances the thrombolytic activity of intravenously administered tissue plasminogen activator (IV-tPA) in acute ischemic stroke (sonothrombolysis). However, rates of arterial recanalization vary widely, depending upon the clot burden, its location, and stroke subtype. We evaluated the influence of age and cholesterol level of the blood clots on sonothrombolysis in an in vitro model. To "age" the clots, serum was replaced by fresh blood periodically. We increased the cholesterol content of the clots by adding cholesterin to the blood. The clots were lysed by tPA and/or transcranial Doppler ultrasound sonication for 1 h. The extent of thrombolysis induced by various treatment protocols (controls, sonication, tPA, and sonothrombolysis) was evaluated with relative changes in the clot weights and in the clot structure by scanning electron microscopy (SEM) at end of the experiment. Sonothrombolysis induced significantly higher weight reduction in fresh clots (37.3 % in 2-h old clots versus 24.8 % in 10-h ones, p < 0.005) as well as the clots with higher cholesterol levels (41.7 versus 30.6 % in normal cholesterol clots, p < 0.005). SEM demonstrated patterns of clot dissolution among various treatment modalities. Sonothrombolysis induced better clot lysis in fresh thrombi with high cholesterol levels.