Takayuki Yamashita

The direct reprogramming of cells has tremendous potential in in vitro neurological studies. Previous attempts to convert blood cells into induced neurons have presented several challenges, necessitating a less invasive, efficient, rapid, and convenient approach. The current study introduces an optimized method for converting somatic cells into neurons using a nonsurgical approach that employs peripheral blood cells as an alternative source to fibroblasts. We have demonstrated the efficacy of a unique combination of transcription factors, including NEUROD1, and four Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4, and c-MYC), in generating glutamatergic neurons within 3 wk. This approach, which requires only five pivotal factors (NEUROD1, OCT3/4, SOX2, KLF4, and c-MYC), has the potential to create functional neurons and circumvents the need for induced pluripotent stem cell (iPSC) intermediates, as evidenced by single-cell RNA sequencing and whole-genome bisulfite sequencing, along with lineage-tracing experiments using Cre-LoxP system. While fibroblasts have been widely used for neuronal reprogramming, our findings suggest that peripheral blood cells offer a potential alternative, particularly in contexts where minimally invasive sampling and procedures convenient for patients are emphasized. This method provides a rapid strategy for modeling neuronal diseases and contributes to advancements in drug discovery and personalized medicine.

When exposed to X-rays, scintillators emit visible luminescence. X-ray-mediated optogenetics employs scintillators for remotely activating light-sensitive proteins in biological tissue through X-ray irradiation. This approach offers advantages over traditional optogenetics, allowing for deeper tissue penetration and wireless control. Here, we assessed the short-term safety and efficacy of candidate scintillator materials for neuronal control. Our analyses revealed that lead-free halide scintillators, such as Cs3Cu2I5, exhibited significant cytotoxicity within 24 h and induced neuroinflammatory effects when injected into the mouse brain. In contrast, cerium-doped gadolinium aluminum gallium garnet (Ce:GAGG) nanoparticles showed no detectable cytotoxicity within the same period, and injection into the mouse brain did not lead to observable neuroinflammation over four weeks. Electrophysiological recordings in the cerebral cortex of awake mice showed that X-ray-induced radioluminescence from Ce:GAGG nanoparticles reliably activated 45% of the neuronal population surrounding the implanted particles, a significantly higher activation rate than europium-doped GAGG (Eu:GAGG) microparticles, which activated only 10% of neurons. Furthermore, we established the cell-type specificity of this technique by using Ce:GAGG nanoparticles to selectively stimulate midbrain dopamine neurons. This technique was applied to freely behaving mice, allowing for wireless modulation of place preference behavior mediated by midbrain dopamine neurons. These findings highlight the unique suitability of Ce:GAGG nanoparticles for X-ray-mediated optogenetics. The deep tissue penetration, short-term safety, wireless neuronal control, and cell-type specificity of this system offer exciting possibilities for diverse neuroscience applications and therapeutic interventions.

Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

Although dietary behaviors are affected by neuropsychiatric disorders, various environmental conditions can have strong effects as well. We found that mice under multiple stresses, including social isolation, intermittent high-fat diet, and physical restraint, developed feeding behavior patterns characterized by a deviated bait approach (fixated feeding). All the tested stressors affected dopamine release at the nucleus accumbens (NAcc) shell and dopamine normalization reversed the feeding defects. Moreover, inhibition of dopaminergic activity in the ventral tegmental area that projects into the NAcc shell caused similar feeding pattern aberrations. Given that the deviations were not consistently accompanied by changes in the amount consumed or metabolic factors, the alterations in feeding behaviors likely reflect perturbations to a critical stress-associated pathway in the mesolimbic dopamine system. Thus, deviations in feeding behavior patterns that reflect reward system abnormalities can be sensitive biomarkers of psychosocial and physical stress.

Body movements influence brain-wide neuronal activities. In the sensory cortex, thalamocortical bottom-up inputs and motor-sensory top-down inputs are thought to affect the dynamics of membrane potentials (V_m) of neurons and change their processing of sensory information during movements. However, direct perturbation of the axons projecting to the sensory cortex from other remote areas during movements has remained unassessed, and therefore the interareal circuits generating motor-related signals in sensory cortices remain unclear. Using a G_i-coupled opsin, eOPN3, we here inhibited interareal signals incoming to the whisker primary somatosensory barrel cortex (wS1) of awake male mice and tested their effects on whisking-related changes in neuronal activities in wS1. Spontaneous whisking in air induced the changes in spike rates of a fraction of wS1 neurons, which were accompanied by depolarization and substantial reduction of slow-wave oscillatory fluctuations of V_m. Despite an extensive innervation, inhibition of inputs from the whisker primary motor cortex (wM1) to wS1 did not alter the spike rates and V_mdynamics of wS1 neurons during whisking. In contrast, inhibition of axons from the whisker-related thalamus (wTLM) and the whisker secondary somatosensory cortex (wS2) to wS1 largely attenuated the whisking-related supra- and sub-threshold V_mdynamics of wS1 neurons. Notably, silencing inputs from wTLM markedly decreased the modulation depth of whisking phase-tuned neurons, while inhibiting wS2 inputs did not impact the whisking variable tuning of wS1 neurons. Thus, sensorimotor integration in wS1 during spontaneous whisking is predominantly facilitated by direct synaptic inputs from wTLM and wS2 rather than from wM1. Significance statementThe traditional viewpoint underscores the importance of motor-sensory projections in shaping movement-induced neuronal activity within sensory cortices. However, this study challenges such established views. We reveal that the synaptic inputs from the whisker primary motor cortex do not alter the activity patterns and membrane potential dynamics of neurons in the whisker primary somatosensory cortex (wS1) during spontaneous whisker movements. Furthermore, we make a novel observation that inhibiting inputs from the whisker secondary somatosensory cortex (wS2) substantially curtails movement-related activities in wS1, leaving the tuning to whisking variables unaffected. These findings provoke a reconsideration of the role of motor-sensory projections in sensorimotor integration and bring to light a new function for wS2-to-wS1 projections.

Photostimulation of Bar^CRH or Bar^ESR1 axons in the adult mouse spinal cord elicits excitatory or inhibitory postsynaptic responses in multiple cell types related to the autonomic nervous system including preganglionic neurons (PGNs) in the lumbosacral intermediolateral nucleus and interneurons in the lumbosacral dorsal commissure nucleus. Integration of excitatory inputs from Bar and from visceral primary afferents in PGNs may be important in the regulation of micturition behavior.

Microbial rhodopsins widely used for optogenetics are sensitive to light in the visible spectrum. As visible light is heavily scattered and absorbed by tissue, stimulating light for optogenetic control does not reach deep in the tissue irradiated from outside the subject body. Conventional optogenetics employs fiber optics inserted close to the target, which is highly invasive and poses various problems for researchers. Recent advances in material science integrated with neuroscience have enabled remote optogenetic control of neuronal activities in living animals using up- or down-conversion phosphors. The development of these methodologies has stimulated researchers to test novel strategies for less invasive, wireless control of cellular functions in the brain and other tissues. Here, we review recent reports related to these new technologies and discuss the current limitations and future perspectives toward the establishment of non-invasive optogenetics for clinical applications.

<title>Abstract</title>Scintillators emit visible luminescence when irradiated with X-rays. Given the unlimited tissue penetration of X-rays, the employment of scintillators could enable remote optogenetic control of neural functions at any depth of the brain. Here we show that a yellow-emitting inorganic scintillator, Ce-doped Gd₃(Al,Ga)₅O₁₂ (Ce:GAGG), can effectively activate red-shifted excitatory and inhibitory opsins, ChRmine and GtACR1, respectively. Using injectable Ce:GAGG microparticles, we successfully activated and inhibited midbrain dopamine neurons in freely moving mice by X-ray irradiation, producing bidirectional modulation of place preference behavior. Ce:GAGG microparticles are non-cytotoxic and biocompatible, allowing for chronic implantation. Pulsed X-ray irradiation at a clinical dose level is sufficient to elicit behavioral changes without reducing the number of radiosensitive cells in the brain and bone marrow. Thus, scintillator-mediated optogenetics enables minimally invasive, wireless control of cellular functions at any tissue depth in living animals, expanding X-ray applications to functional studies of biology and medicine.

Volatile anesthetics are widely used for surgery, but neuronal mechanisms of anesthesia remain unidentified. At the calyx of Held in brainstem slices from rats of either sex, isoflurane at clinical doses attenuated excitatory post-synaptic currents by decreasing the release probability and the number of readily releasable vesicles. In presynaptic recordings of Ca2+ currents and exocytic capacitance changes, isoflurane attenuated exocytosis by inhibiting Ca2+ currents evoked by a short presynaptic depolarization, whereas it inhibited exocytosis evoked by a prolonged depolarization via directly blocking exocytic machinery downstream of Ca2+ influx. Since the length of presynaptic depolarization can simulate the frequency of synaptic inputs, isoflurane anesthesia is likely mediated by distinct dual mechanisms, depending upon input frequencies. In simultaneous pre- and postsynaptic action potential recordings, isoflurane impaired the fidelity of repetitive spike transmission, more strongly at higher frequencies. Furthermore, in the cerebrum of adult mice, isoflurane inhibited monosynaptic cortico-cortical spike transmission, preferentially at a higher frequency. We conclude that dual presynaptic mechanisms operate for the anesthetic action of isoflurane, of which direct inhibition of exocytic machinery plays a low-pass filtering role in spike transmission at central excitatory synapses.Significance Statement:Synaptic mechanisms of general anesthesia remain unidentified. In rat brainstem slices, isoflurane inhibits excitatory transmitter release by blocking presynaptic Ca2+ channels and exocytic machinery, with the latter mechanism predominating in its inhibitory effect on high-frequency transmission. Both in slice and in vivo, isoflurane preferentially inhibits spike transmission induced by high-frequency presynaptic inputs. This low-pass filtering action of isoflurane likely plays a significant role in general anesthesia.