Randomized trial of granulocyte colony-stimulating factor for spinal cord injury.

Attenuation of the secondary injury of spinal cord injury (SCI) can suppress the spread of spinal cord tissue damage, possibly resulting in spinal cord sparing that can improve functional prognoses. Granulocyte colony-stimulating factor (G-CSF) is a haematological cytokine commonly used to treat neutropenia. Previous reports have shown that G-CSF promotes functional recovery in rodent models of SCI. Based on preclinical results, we conducted early phase clinical trials, showing safety/feasibility and suggestive efficacy. These lines of evidence demonstrate that G-CSF might have therapeutic benefits for acute SCI in humans. To confirm this efficacy and to obtain strong evidence for pharmaceutical approval of G-CSF therapy for SCI, we conducted a phase 3 clinical trial designed as a prospective, randomized, double-blinded and placebo-controlled comparative trial. The current trial included cervical SCI [severity of American Spinal Injury Association (ASIA) Impairment Scale (AIS) B or C] within 48 h after injury. Patients are randomly assigned to G-CSF and placebo groups. The G-CSF group was administered 400 µg/m2/day × 5 days of G-CSF in normal saline via intravenous infusion for five consecutive days. The placebo group was similarly administered a placebo. Allocation was concealed between blinded evaluators of efficacy/safety and those for laboratory data, as G-CSF markedly increases white blood cell counts that can reveal patient treatment. Efficacy and safety were evaluated by blinded observer. Our primary end point was changes in ASIA motor scores from baseline to 3 months after drug administration. Each group includes 44 patients (88 total patients). Our protocol was approved by the Pharmaceuticals and Medical Device Agency in Japan and this trial is funded by the Center for Clinical Trials, Japan Medical Association. There was no significant difference in the primary end point between the G-CSF and the placebo control groups. In contrast, one of the secondary end points showed that the ASIA motor score 6 months (P = 0.062) and 1 year (P = 0.073) after drug administration tend to be higher in the G-CSF group compared with the placebo control group. Moreover, in patients aged over 65 years old, motor recovery 6 months after drug administration showed a strong trend towards a better recovery in the G-CSF treated group (P = 0.056) compared with the control group. The present trial failed to show a significant effect of G-CSF in primary end point although the subanalyses of the present trial suggested potential G-CSF benefits for specific population.

Spontaneous network activity visualized by ultrasensitive Ca(2+) indicators, yellow Cameleon-Nano.

We report ultrasensitive Ca(2+) indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca(2+)-sensing domain of a genetically encoded Ca(2+) indicator, YC2.60 or YC3.60. Their high Ca(2+) affinities (K(d) = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca(2+) transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.

Characteristics of Postoperative C5 Palsy Following Anterior Decompression and Fusion Surgery for Cervical Degenerative Disorders: Trends Associated with Advancements in Surgical Technique.

OBJECTIVE: To investigate trends in the characteristics of postoperative C5 palsy following anterior decompression and fusion associated with advancements in this surgical procedure to treat cervical degenerative disorders. METHODS: We included 801 consecutive patients who underwent anterior decompression and fusion for cervical degenerative disorders from 2006 to 2019 and investigated the incidence, onset, and prognosis of C5 palsy. In addition, we compared the incidence of C5 palsy with that found in our previous investigation. RESULTS: The cases of 42 (5.2%) patients were complicated by C5 palsy. For patients with ossification of the longitudinal ligament (OPLL), 22 (12.4%) of 177 were complicated with C5 palsy, and the incidence was significantly higher than that in patients without OPLL (20 [3.2%] of 624, P < 0.01). The incidence of C5 palsy in patients without OPLL was significantly lower than that found in our previous investigation (P < 0.01). The incidence of C5 palsy in patients that required contiguous multilevel corpectomy was significantly higher in patients that required within a single corpectomy (P < 0.01). At 1-year follow-up, muscle strength in 3 (6.1%) of 49 limbs had not improved sufficiently. CONCLUSIONS: With advancements in surgical techniques which allowed necessary and sufficient spinal cord decompression and avoided unnecessary corpectomy, the incidence of C5 palsy in patients without OPLL was decreased significantly. By contrast, for patients with OPLL, the incidence of C5 palsy was similar to the incidence found previously, perhaps because a broad and contiguous multilevel corpectomy was usually needed to decompress the spinal cord sufficiently.

Ptch1-mediated dosage-dependent action of Shh signaling regulates neural progenitor development at late gestational stages.

Sonic hedgehog (Shh) signaling regulates cell differentiation and proliferation during brain development. However, the role of Shh in neurogenesis during late gestation (embryonic day 13.5-18.5) remains unclear. Herein, we used a genetic approach and in utero electroporation to investigate the role of mouse Shh and patched homolog 1 (Ptch1), the putative receptor for Shh. Proliferating cortical intermediate (basal) progenitor cells (IPCs) were severely reduced in Shh mutant mice, suggesting that endogenous Shh signaling could play an essential role in cortical IPC development. During cortical neurogenesis, strong upregulation of Shh signaling enhanced the transition from ventricular zone (VZ) progenitors to ventralized IPCs, while low levels of signaling enhanced the generation and proliferation of cortical IPCs in the subventricular zone. The effects of Shh upregulation in this study were consistent with a phenotype of conditional loss of function of Ptch1, and the phenotype of a hypomorphic allele of Ptch1, respectively. These data indicated that endogenous Ptch1 mediates the broad effects of Shh on the transition from VZ progenitors to IPCs and activation of proliferation of the IPCs in the cortex during late gestational stages.

Development and evolution of cerebellar neural circuits.

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.

Neuroprotective therapy using granulocyte colony-stimulating factor for patients with worsening symptoms of compression myelopathy, Part 1: a phase I and IIa clinical trial.

OBJECTIVE: Based on the neuroprotective effects of granulocyte colony-stimulating factor (G-CSF) on experimental spinal cord injury, we initiated a clinical trial that evaluated the safety and efficacy of neuroprotective therapy using G-CSF for patients with worsening symptoms of compression myelopathy. METHODS: We obtained informed consent from 15 patients, in whom the Japanese Orthopaedic Association (JOA) score for cervical myelopathy decreased two points or more during a recent 1-month period. G-CSF (5 or 10 µg/kg/day) was intravenously administered for five consecutive days. We evaluated motor and sensory functions of the patients and the presence of adverse events related to G-CSF therapy. RESULTS: G-CSF administration suppressed the progression of myelopathy in all 15 patients. Neurological improvements in motor and sensory functions were obtained in all patients after the administration, although the degree of improvement differed among the patients. Nine patients in the 10-µg group (n=10) underwent surgical treatment at 1 month or later after G-CSF administration. In the 10-µg group, the mean JOA recovery rates 1 and 6 months after administration were 49.9±15.1 and 59.1±16.3%, respectively. On the day following the start of G-CSF therapy, the white blood cell count increased to more than 22,700 cells/mm3. It varied from 12,000 to 50,000 and returned to preadministration levels 3 days after completing G-CSF treatment. No serious adverse events occurred during or after treatment. CONCLUSION: The results indicate that G-CSF administration at 10 µg/kg/day is safe for patients with worsening symptoms of compression myelopathy and may be effective for their neurological improvement.

Neuroprotective therapy using granulocyte colony-stimulating factor for acute spinal cord injury: a phase I/IIa clinical trial.

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) is a cytokine that is clinically used to treat neutropenia. G-CSF also has non-hematopoietic functions and could potentially be used to treat neuronal injury. To confirm the safety and feasibility of G-CSF administration for acute spinal cord injury (SCI), we have initiated a phase I/IIa clinical trial of neuroprotective therapy using G-CSF. METHODS: The trial included a total of 16 SCI patients within 48 h of onset. In the first step, G-CSF (5 µg/kg/day) was intravenously administered for 5 consecutive days to 5 patients. In the second step, G-CSF (10 µg/kg/day) was similarly administered to 11 patients. We evaluated motor and sensory functions of patients using the American Spinal Cord Injury Association (ASIA) score and ASIA impairment scale (AIS) grade. RESULTS: In all 16 patients, neurological improvement was obtained after G-CSF administration. AIS grade increased by one step in 9 of 16 patients. A significant increase in ASIA motor scores was detected 1 day after injection (P < 0.01), and both light touch and pin prick scores improved 2 days after injection (P < 0.05) in the 10 µg group. No severe adverse effects were observed after G-CSF injection. CONCLUSION: These results indicate that intravenous administration of G-CSF (10 µg/kg/day) for 5 days is essentially safe, and suggest that some neurological recovery may occur in most patients. We suggest that G-CSF administration could be therapeutic for patients with acute SCI.

C5 palsy following anterior decompression and spinal fusion for cervical degenerative diseases.

Postoperative C5 palsy is a common complication after cervical spine decompression surgery. However, the incidence, prognosis, and etiology of C5 palsy after anterior decompression with spinal fusion (ASF) have not yet been fully established. In the present study, we analyzed the clinical and radiological characteristics of patients who developed C5 palsy after ASF for cervical degenerative diseases. The cases of 199 consecutive patients who underwent ASF were analyzed to clarify the incidence of postoperative C5 palsy. We also evaluated the onset and prognosis of C5 palsy. The presence of high signal changes (HSCs) in the spinal cord was analyzed using T2-weighted magnetic resonance images. C5 palsy occurred in 17 patients (8.5%), and in 15 of them, the palsy developed after ASF of 3 or more levels. Among ten patients who had a manual muscle test (MMT) grade ≤2 at the onset, five patients showed incomplete or no recovery. Sixteen of the 17 C5 palsy patients presented neck and shoulder pain prior to the onset of muscle weakness. In the ten patients with a MMT grade ≤2 at the onset, nine patients showed HSCs at the C3-C4 and C4-C5 levels. The present findings demonstrate that, in most patients with severe C5 palsy after ASF, pre-existing asymptomatic damage of the anterior horn cells at C3-C4 and C4-C5 levels may participate in the development of motor weakness in combination with the nerve root lesions that occur subsequent to ASF. Thus, when patients with spinal cord lesions at C3-C4 and C4-C5 levels undergo multilevel ASF, we should be alert to the possible occurrence of postoperative C5 palsy.

Segmental motion at the peak of the ossification foci is independent risk factor except for mal-alignment and thick ossification foci for poor outcome after laminoplasty for cervical ossification of the posterior longitudinal ligament: analyses in patients with positive K-line, lordotic alignment, and lower canal occupying ratio.

PURPOSE: To elucidate the independent preoperative factors that have a significant impact on poor surgical outcome after laminoplasty for K-line (+) ossification of the posterior longitudinal ligament (OPLL). Analyses in K-line (+) patient population can exclude the influence by mal-alignment and thick OPLL, both of which are well known two major factors that have significant impact on clinical outcome. METHODS: The present study included 72 patients (50 male and 22 female) who underwent laminoplasty for K-line (+) cervical OPLL and were followed-up for at least 1 year. Recovery of Japanese Orthopedic Association score (JOA score) for cervical myelopathy was used as the measure of clinical outcome. For radiographic assessment, the type of OPLL, the maximum OPLL occupation ratio, the C2-C7 angle, and the segmental range of motion at the peak of OPLL (segmental ROM) were assessed. To elucidate the factors that are significantly associated with a poor clinical outcome after laminoplasty for K-line (+) OPLL, statistical analyses were conducted. RESULTS: The mean preoperative JOA score was 8.9 points and improved to 12.8 points after surgery. The recovery of JOA score was 47 ± 35%. Stepwise logistic regression following univariate analyses revealed that preoperative segmental ROM at the peak of OPLL is an independent factor associated with a poor outcome (p = 0.04, odds ratio = 1.15). CONCLUSIONS: Large preoperative segmental ROM at the peak of the OPLL is an independent factor that has significant impact on poor surgical outcome after laminoplasty for K-line (+) OPLL.

Cervical myelopathy in patients with ossification of the posterior longitudinal ligament.

OBJECT: The authors assessed the clinical course in patients with a narrowed cervical spinal canal caused by ossification of the posterior longitudinal ligament (OPLL), but who have no or only mild myelopathy. Additionally, the authors analyzed the factors contributing to the development and aggravation of myelopathy in patients with OPLLinduced spinal canal stenosis. METHODS: Between 1997 and 2004, the authors selected treatments for patients with cervical OPLL in whom the residual space available for the spinal cord was < or = 12 mm. Treatment decisions were based on the severity of myelopathy at presentation. Twenty-one patients with no or mild myelopathy (defined as a Japanese Orthopaedic Association [JOA] scale score > or = 14 points) received conservative treatment, with a mean follow-up period of 4.5 years. In 20 patients with moderate or severe myelopathy (JOA scale score < 14 points), the authors performed surgery via an anterior approach. The clinical course in these patients was assessed with the JOA scale and the OPLL types were classified. The authors evaluated the range of motion between C-1 and C-7, the developmental segmental sagittal diameter, the percentage of spinal canal diameter occupied by the OPLL (% ratio), and the residual space available for the spinal cord on cervical radiographs; T2-weighted MR images were examined for high signal changes (HSCs). RESULTS: In the conservative treatment group, 8 patients showed improvement, 12 remained unchanged, and 1 patient's condition became slightly worse during the observation period. Fifteen patients in this group had mixedtype, 3 had continuous-type, 2 had localized-type, and 1 had a segmental-type OPLL. In the surgically treated group, there were 12 patients with segmental-type, 10 patients with mixed-type, and 1 with localized-type OPLL. The mean range of motion at C1-7 was 36.4 degrees in the conservatively treated group and 46.5 degrees in the surgical group (p < 0.05). No significant difference was seen between the groups in terms of developmental segmental sagittal diameter, % ratio, or residual space available for the cord. No HSCs were noted in the conservative group, while 17 patients in the surgical group had HSCs (p < 0.05). CONCLUSIONS: In the present study, the authors demonstrate that the mobility of the cervical spine and the type of OPLL are important factors contributing to the development and aggravation of myelopathy in patients with OPLLinduced spinal canal stenosis. The authors advocate conservative treatment in most patients with OPLLs who have no or only mild myelopathy, even in the presence of spinal canal narrowing.

Optical intrinsic signal imaging with optogenetics reveals functional cortico-cortical connectivity at the columnar level in living macaques.

Despite extensive research on primate cognitive function, understanding how anatomical connectivity at a neural circuit level relates to information transformation across different cortical areas remains primitive. New technology is needed to visualize inter-areal anatomical connectivity in living monkeys and to tie this directly to neurophysiological function. Here, we developed a novel method to investigate this structure-function relationship, by combining optical intrinsic signal imaging (OISI) with optogenetic stimulation in living monkeys (opto-OISI). The method involves expressing channelrhodophsin-2 in one area (source) followed by optical imaging of optogenetic activations in the other area (target). We successfully demonstrated the potential of the method with interhemispheric columnar projection patterns between V1/V2 border regions. Unlike the combination of optogenetics and functional magnetic resonance imaging (opto-fMRI), opto-OISI has the advantage of enabling us to detect responses of small clusters of neurons, even if the clusters are sparsely distributed. We suggest that opto-OISI can be a powerful approach to understanding cognitive function at the neural circuit level, directly linking inter-areal circuitry to fine-scale structure and function.

Anatomical Evidence for a Direct Projection from Purkinje Cells in the Mouse Cerebellar Vermis to Medial Parabrachial Nucleus.

Cerebellar malformations cause changes to the sleep-wake cycle, resulting in sleep disturbance. However, it is unclear how the cerebellum contributes to the sleep-wake cycle. To examine the neural connections between the cerebellum and the nuclei involved in the sleep-wake cycle, we investigated the axonal projections of Purkinje cells in the mouse posterior vermis by using an adeno-associated virus (AAV) vector (serotype rh10) as an anterograde tracer. When an AAV vector expressing humanized renilla green fluorescent protein was injected into the cerebellar lobule IX, hrGFP and synaptophysin double-positive axonal terminals were observed in the region of medial parabrachial nucleus (MPB). The MPB is involved in the phase transition from rapid eye movement (REM) sleep to Non-REM sleep and vice versa, and the cardiovascular and respiratory responses. The hrGFP-positive axons from lobule IX went through the ventral spinocerebellar tract and finally reached the MPB. By contrast, when the AAV vector was injected into cerebellar lobule VI, no hrGFP-positive axons were observed in the MPB. To examine neurons projecting to the MPB, we unilaterally injected Fast Blue and AAV vector (retrograde serotype, rAAV2-retro) as retrograde tracers into the MPB. The cerebellar Purkinje cells in lobules VIII-X on the ipsilateral side of the Fast Blue-injected MPB were retrogradely labeled by Fast Blue and AAV vector (retrograde serotype), but no retrograde-labeled Purkinje cells were observed in lobules VI-VII and the cerebellar hemispheres. These results indicated that Purkinje cells in lobules VIII-X directly project their axons to the ipsilateral MPB but not lobules VI-VII. The direct connection between lobules VIII-X and the MPB suggests that the cerebellum participates in the neural network controlling the sleep-wake cycle, and cardiovascular and respiratory responses, by modulating the physiological function of the MPB.

Study protocol for the G-SPIRIT trial: a randomised, placebo-controlled, double-blinded phase III trial of granulocyte colony-stimulating factor-mediated neuroprotection for acute spinal cord injury.

INTRODUCTION: Granulocyte colony-stimulating factor (G-CSF) is generally used for neutropaenia. Previous experimental studies revealed that G-CSF promoted neurological recovery after spinal cord injury (SCI). Next, we moved to early phase of clinical trials. In a phase I/IIa trial, no adverse events were observed. Next, we conducted a non-randomised, non-blinded, comparative trial, which suggested the efficacy of G-CSF for promoting neurological recovery. Based on those results, we are now performing a phase III trial. METHODS AND ANALYSIS: The objective of this study is to evaluate the efficacy of G-CSF for acute SCI. The study design is a prospective, multicentre, randomised, double-blinded, placebo-controlled comparative study. The current trial includes cervical SCI (severity of American Spinal Injury Association (ASIA) Impairment Scale B/C) within 48 hours after injury. Patients are randomly assigned to G-CSF and placebo groups. The G-CSF group is administered 400 µg/m2/day×5 days of G-CSF in normal saline via intravenous infusion for 5 consecutive days. The placebo group is similarly administered a placebo. Our primary endpoint is changes in ASIA motor scores from baseline to 3 months. Each group includes 44 patients (88 total patients). ETHICS AND DISSEMINATION: The study will be conducted according to the principles of the World Medical Association Declaration of Helsinki and in accordance with the Japanese Medical Research Involving Human Subjects Act and other guidelines, regulations and Acts. Results of the clinical study will be submitted to the head of the respective clinical study site as a report after conclusion of the clinical study by the sponsor-investigator. Even if the results are not favourable despite conducting the clinical study properly, the data will be published as a paper. TRIAL REGISTRATION NUMBER: UMIN000018752.

Target regulation of V2R expression and functional maturation in vomeronasal sensory neurons in vitro.

Vomeronasal receptors from the V1R and V2R gene families mediate the detection of chemical stimuli such as pheromones via the vomeronasal organ (VNO). The differential expression of vomeronasal receptors might contribute in part to a variety of pheromonal effects, which are different sexually, developmentally and even individually. However, little is known about the mechanisms controlling vomeronasal receptor expression. Cultured vomeronasal sensory neurons (VSNs) bear phenotypic resemblance to the intact VNO but they remain immature. Because indices of VSN maturation are increased by coculture with the target cells for VSNs, accessory olfactory bulb (AOB) neurons, AOB neurons may regulate vomeronasal receptor expression and functional maturation in VSNs. To test this hypothesis, we examined the expression of V2R-type vomeronasal receptors (VR1 and VR4) and chemosensory responsiveness in VNOs cocultured with AOB neurons. Immunoblot and immunocytochemical analysis revealed that the coculture of VNOs with AOB neurons resulted in a greater expression of VR1 and VR4 after 10 days than VNOs cultured alone. Moreover, calcium imaging analysis showed that cocultured VNOs responded to urine components applied iontophoretically into their cavities with a time course similar to the V2R expression, in contrast to singly cultured VNOs that displayed no response. These results demonstrate that AOB neurons induce the expression of vomeronasal receptors in VSNs, allowing them to function.

Perioperative Complications in 155 Patients Who Underwent Oblique Lateral Interbody Fusion Surgery: Perspectives and Indications From a Retrospective, Multicenter Survey.

STUDY DESIGN: A retrospective multicenter survey. OBJECTIVE: To investigate the perioperative complications of oblique lateral interbody fusion (OLIF) surgery. SUMMARY OF BACKGROUND DATA: OLIF has been widely performed to achieve minimally invasive, rigid lumbar lateral interbody fusion. The associated perioperative complications are not yet well described. METHODS: The participants were patients who underwent OLIF surgery under the diagnosis of degenerative lumbar diseases between April 2013 and May 2015 at 11 affiliated medical institutions. The collected data were classified into intraoperative and early-stage postoperative (≤1 mo) complications. The intraoperative complications were then subcategorized into organ damage (neural, vertebral, vascular, and others) and other complications, mainly related to instrumental failure. The collected data were also divided and analyzed based on whether the surgeon was certified to perform the surgery and the incidence of complications in the early (April 2013-March 2014) and late stages (April 2014-May 2015) of OLIF introduction. RESULTS: In the 155 included patients, 75 complications were reported (incidence rate, 48.3%). The most common complication was endplate fracture/subsidence (18.7%), followed by transient psoas weakness and thigh numbness (13.5%) and segmental artery injury (2.6%). Almost all these complications were transient, except for three patients who had permanent damage: one had ureteral injury and two had neurological injury. Postoperative complications included surgical site infection (1.9%) and reoperation (1.9%). Whether the primary operator was experienced did not affect the incidence of complications. Regarding the introductory stage, the incidence of complications was 50% in the early stage and 38% in the late stage. CONCLUSION: The overall incidence of perioperative complications of OLIF surgery reached 48.3%, of which only 1.9% resulted in permanent damage. Our analysis based on surgeon experience indicated that the OLIF procedure could be performed without increasing incidence of complications, under the guidance of experienced supervisors. LEVEL OF EVIDENCE: 3.

Cell-cycle-independent transitions in temporal identity of mammalian neural progenitor cells.

During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode.

Neuronal birthdate-specific gene transfer with adenoviral vectors.

The multilayered structure of the cerebral cortex has been studied in detail. Early-born neurons migrate into the inner layer and late-born neurons migrate into more superficial layers, thus establishing an inside-out gradient. The progenitor cells appear to acquire layer-specific properties at the time of neuronal birth; however, the molecular mechanisms of cell-fate acquisition are still unclear, because it has been difficult to identify a cohort of birthdate-related progenitor cells. Using replication-defective adenoviral vectors, we successfully performed "pulse gene transfer" into progenitor cells in a neuronal birthdate-specific manner. When adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic day 10.5 (E10.5) and E14.5, the adenoviral vectors introduced a foreign gene into a specific cohort of birthdate-related progenitor cells. The virally infected cohorts developed normally into cortical neurons and formed the canonical cortical layers in an inside-out manner. This technique allows us to distinguish a cohort of birthdate-related progenitor cells from other progenitor cells with different birthdates and to introduce a foreign gene into specific subsets of cortical layers by performing adenoviral injection at specific times. This adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birthdate.

Mediolateral compartmentalization of the cerebellum is determined on the "birth date" of Purkinje cells.

The adult cerebellum is functionally compartmentalized into clusters along the mediolateral axis (M-L clusters), and a variety of molecular makers are expressed in specific subsets of M-L clusters. These M-L clusters appear to be the basic structure in which cerebellar functions are performed, but the mechanisms by which cerebellar mediolateral compartmentalization is established are still unclear. To address these questions, we examined the development of M-L clusters using replication-defective adenoviral vectors. The adenoviral vectors effectively introduced foreign genes into the neuronal progenitor cells of the cerebellum in a birth date-specific manner, allowing us to observe the native behavior of each cohort of birth date-related progenitor cells. When the adenoviral vectors were injected into the midbrain ventricle of mouse embryos on embryonic days 10.5 (E10.5), E11.5, and E12.5, the virally infected cerebellar progenitor cells developed into Purkinje cells. Notably, the Purkinje cells that shared the same birth date formed specific subsets of M-L clusters in the cerebellum. Each subset of M-L clusters displayed nested and, in part, mutually complementary patterns, and these patterns were unchanged from the late embryonic stage to adulthood, suggesting that Purkinje cell progenitors are fated to form specific subsets of M-L clusters after their birth between E10.5 and E12.5. This study represents the first such direct observation of Purkinje cell development. Moreover, we also show that there is a correlation between the M-L clusters established by the birth date-related Purkinje cells and the domains of engrailed-2, Wnt-7B, L7/pcp2, and EphA4 receptor tyrosine kinase expression.

Programmable wireless light-emitting diode stimulator for chronic stimulation of optogenetic molecules in freely moving mice.

We produced a miniaturized, multicode, multiband, and programmable light-emitting diode (LED) stimulator for wireless control of optogenetic experiments. The LED stimulator is capable of driving three independent LEDs upon reception of an infrared (IR) signal generated by a custom-made IR transmitter. Individual LED photopulse patterns are assigned to different codes of the IR signals (up to 256 codes). The photopulse patterns can be programmed in the on-board microcontroller by specifying the parameters of duration ([Formula: see text]), frequency ([Formula: see text]), and pulse width ([Formula: see text]). The IR signals were modulated at multiple carrier frequencies to establish multiband IR transmission. Using these devices, we could remotely control the moving direction of a Thy1-ChR2-YFP transgenic mouse by transcranially illuminating the corresponding hemisphere of the primary motor cortex. IR transmitter and LED stimulator will be particularly useful in experiments where free movement or patterned concurrent stimulation is desired, such as testing social communication of rodents.

Expression of human solute carrier family transporters in skin: possible contributor to drug-induced skin disorders.

Solute carrier (SLC) transporters play important roles in absorption and disposition of drugs in cells; however, the expression pattern of human SLC transporters in the skin has not been determined. In the present study, the expression patterns of 28 human SLC transporters were determined in the human skin. Most of the SLC transporter family members were either highly or moderately expressed in the liver, while their expression was limited in the skin and small intestine. Treatment of human keratinocytes with a reactive metabolite of ibuprofen significantly reduced cell viability. Expression array analysis revealed that S100 calcium binding protein A7A (S100A7A) was induced nearly 50-fold in dermal cells treated with ibuprofen acyl-glucuronide. Determination of the expression of drug-metabolizing enzymes as well as drug transporters prior to the administration of drugs would make it possible to avoid the development of idiosyncratic skin diseases in individuals.