Improved trafficking and expression of luminopsins for more efficient optical and pharmacological control of neuronal activity

Luminopsins (LMOs) are chimeric proteins consisting of a luciferase fused to an opsin that provide control of neuronal activity, allowing for less cumbersome and less invasive optogenetic manipulation. It was previously shown that both an external light source and the luciferase substrate, coelenterazine (CTZ), could modulate activity of LMO-expressing neurons, although the magnitudes of the photoresponses remained subpar. In this study, we created an enhanced iteration of the excitatory luminopsin LMO3, termed eLMO3, that has improved membrane targeting due to the insertion of a Golgi trafficking signal sequence. In cortical neurons in culture, the expression of eLMO3 resulted in significant reductions in the formation of intracellular aggregates, as well as in a significant increase in total photocurrents. Furthermore, we corroborated the findings with injections of adeno-associated viral vectors into the deep layers of the somatosensory cortex (the barrel cortex) of male mice. We observed greatly reduced numbers of intracellular puncta in eLMO3-expressing cortical neurons compared to those expressing the original LMO3. Finally, we quantified CTZ-driven behavior, namely whisker-touching behavior, in male mice with LMO3 expression in the barrel cortex. After CTZ administration, mice with eLMO3 displayed significantly longer whisker responses than mice with LMO3. In summary, we have engineered the superior LMO by resolving membrane trafficking defects, and we demonstrated improved membrane targeting, greater photocurrents, and greater functional responses to stimulate with CTZ.     

Revealing the Pb Whisker Growth Mechanism from Al-Alloy Surface and Morphological Dependency on Material Stress and Growth Environment

Spontaneous metallic Pb whisker formation from Pb and Bi containing Al-alloy’s surfaces is a newly discovered phenomenon. The whiskers display unique formations, growth and morphology, which give the opportunity to be applied for specialized sensor and electronics applications. Within this work, the impact of environmental conditions (gas composition and moisture) is investigated and correlated with the modification of whisker evolution and growth dynamics. Furthermore, the residual stress state of the aluminum matrix using deep cryogenic treatment is modified and used to further increase whisker nucleation and growth by up to three- and seven-fold, respectively, supported by quantitative results. The results of this paper indicate the possibility to manipulate the whisker not only in terms of their kinetics but also their morphology (optimal conditions are 20% O₂ and 35% humidity). Such features allow the tailoring of the whisker structure and surface to volume ratio, which can be optimized for different applications. Finally, this research provides new insight into the growth dynamics of the whiskers through in situ and ex situ measurements, providing further evidence of the complex nucleation and growth mechanisms that dictate the spontaneous growth of Pb whiskers from Al-alloy 6026 surfaces with growth velocities up to 1.15 µm/s.     

Use of a Simplified Method of Optical Recording to Identify Foci of Maximal Neuron Activity in the Somatosensory Cortex of White Rats

Eight mongrel white male rats were studied under urethane anesthesia, and neuron activity evoked by mechanical and/or electrical stimulation of the contralateral whiskers was recorded in the primary somatosensory cortex. Recordings were made using a digital USB chamber attached to the printer port of a Pentium 200MMX computer running standard programs. Optical images were obtained in the barrel-field zone using a differential signal, i.e., the difference signal for cortex images in control and experimental animals. The results obtained here showed that subtraction of averaged sequences of frames yielded images consisting of spots reflecting the probable position of activated groups of neurons. The most effective stimulation consisted of natural low-frequency stimulation of the whiskers. The method can be used for preliminary mapping of cortical zones, as it provides for rapid and reproducible testing of the activity of neuron ensembles over large areas of the cortex.     

The rat's postero-orbital sinus hair: I. Brainstem projections and the effect of infraorbital nerve section at different ages

The central terminations, in the trigeminal nucleus, of afferents from the rat's postero-orbital (PO) sinus hair have been investigated with transganglionic transport of horseradish peroxidase (HRP) and succinic dehydrogenase (SDH) histochemistry. The normal pattern of terminations has been compared with that found after section of an adjacent nerve, the infraorbital (IO) nerve, at three ages: neonatal, 1 week old, and adult. The PO afferent fibres have three separate representations in the brainstem-in trigeminal sensory nucleus principalis (Vp) and rostral subnucleus oralis (Vo), in trigeminal subnucleus interpolaris (Vi), and in caudal trigeminal subnucleus caudalis (Vc) and C₁ dorsal horn. In coronal sections the areas of terminations were seen as oval patches lying ventrolaterally in Vp, Vo, and Vi and ventromedially in Vc and C₁. Following neonatal IO nerve section the terminal areas were approximately doubled in Vp, Vo, and Vi but were unchanged in Vc and C₁. IO nerve section at day 7 also caused a significant, though smaller (1.4× compared with 2.0×), increase in the terminal areas in the rostral three nuclei, without changing Vc and C₁. However, no significant change in area occurred after adult IO nerve section. SDH histochemistry at 3 to 4 weeks of age showed patches of terminals on both normal and lesioned sides consistent with those seen after HRP. Previous studies have reported increased functional representation of surrounding intact skin regions, including the PO sinus hairs, after neonatal but not adult, IO nerve section. The present results show that there are concomitant anatomical changes. Like the functional results, the extent of the anatomical changes are dependent on the maturity of the rat when lesioned.     

Functional mapping of rat barrel activation following whisker stimulation using activity-induced manganese-dependent contrast

BACKGROUND: Cortical whisker barrels in the primary somatosensory cortex are a well-known example of brain function in rodents. The well-defined relationship between barrels and whiskers makes this system a unique model to study neuronal function and plasticity. In this study, we sought to establish a feasible working protocol of applying manganese-enhanced MRI (MEMRI) to map the cortical barrels following whisker stimulation. METHODS: The protocol was based on the principle of activity-induced manganese-dependent (AIM) contrast. Rats were prepared by sodium pentobarbital anesthetization, intraperitoneal manganese-chloride injection, right common carotid mannitol injection and temperature maintenance. Left whiskers were connected to a speaker through a cotton thread and were stimulated by a series of rectangular pulses. MEMRI was acquired with a 3T scanner 3 h after whisker stimulation. Before MR scanning, Wistar rats were euthanized to avoid motion artifacts. To improve the signal-to-noise ratio (SNR) and detection sensitivity, image coregistration, pixel intensity normalization, statistical mapping, group averaging and subtraction were performed. The AIM enhancement of the cortical barrels was quantified using volume of interest analysis on the acquired T1WI and R1 mapping. RESULTS: Both experimental and control groups showed greater enhancement in the right hemisphere, same side as mannitol injection. In the experimental group, however, activity-induced enhancement was more localized in the right barrel fields, whereas in the control group, the enhancement was uniform throughout the right cortex. In the right cortical barrels, the enhancement ratios and R1 values in the experimental group were significantly higher than those in the control group. In the left cortical barrels, there was no significant difference between the two groups. Subtracted images and voxel-based statistical t-value mapping between experimental and control groups showed additional enhancement concentrated in the right cortical barrels. CONCLUSIONS: We have mapped rat whisker barrels using the AIM method and have shown a clear relationship between manganese-enhanced cortical regions and whisker tactile-sense-evoked activity. It is possible that, with sufficient SNR, the AIM method may reach whisker barrel discrimination, potentially useful to study plasticity in surgically or genetically manipulated rat brains.     

热处理时间对钛酸钾晶须增强牙用复合树脂的抗弯强度的影响

目的：研究不同热处理时间对钛酸钾晶须增强的复合树脂抗弯强度的影响。方法：将3%硅烷偶联剂处理过的钛酸钾晶须按照60%质量分数的填充量与树脂基质手工搅拌混合后制备复合树脂三点弯曲测试标准试件四组,每组6个。四组标准试件分别经120℃热处理30min、45min、1h、2h后按照ISO-10477的标准进行三点弯曲测试。结果：热处理时间1h的复合树脂抗弯强度（123.90±15.90）MPa明显高于热处理时间为30min时的抗弯强度（98.82±15.84）MPa。结论：钛酸钾晶须增强复合树脂经120℃热处理30min-1h时,随着热处理时间的增加,抗弯强度逐渐增大。     

羟基磷灰石晶须/纳米氧化锌复合抗菌人工骨体外极限降解实验

目的 将纳米氧化锌对羟基磷灰石晶须进行改性,制备合成一种抗菌植骨复合材料,研究其在极限环境中的降解和物质变化.方法 通过溶胶-凝胶法制备纳米氧化锌,将制备所得纳米氧化锌改性羟基磷灰石晶须,得到羟基磷灰石晶须/纳米氧化锌复合材料.依据IS0 10993-14标准,采用柠檬酸缓冲溶液浸泡测试失重率,并对材料表面的形态变化作扫描电镜观察和X射线衍射分析.结果 HAPw/n-ZnO、HAPw降解率分别为：25.44％、18.59％,浸泡后通过扫描电镜可以明显观察到断裂的羟基磷灰石晶须,其表面的纳米氧化锌颗粒大量减少.XRD分析羟基磷灰石的三强峰降低.结论 HAPw/n-ZnO属于低可溶性材料.     

Development of Innervation to Maxillary Whiskers in Mice

The maxillary vibrissal pad is a unique, richly innervated sensory apparatus. It is highly evolved in the rodent that it constitutes a major source of sensory information to the somatosensory cortex. In this report, indocarbocyanine tracing and immunofluorescence were used to study the embryonic and early neonatal development of innervation to maxillary vibrissal follicles in mice. The first sign of vibrissal follicle innervation occurred at embryonic day 12 (E12), when the lateral nasal and maxillary processes were penetrated by nerve branches with small terminal plexuses assuming the positions of vibrissal follicle primordia. Between E13 and E15, the nerve plexuses at the presumptive follicles grew in size and became more numerous with no signs of specific receptor subtype formation. By E17, the nerve plexuses had grown further in size and the region‐specific receptor subtype specification developed. At birth (P0), the superficial vibrissal nerves began to innervate the apical part of the inner conical body, whereas the deep vibrissal nerve gave off the recurrent cavernous branches. At P3, all of the different sets of receptor subtypes had regional distributions, densities and morphologies comparable to those described in adult mice. A 3‐day old mouse had all complements of sensory receptors necessary for somatosensory transduction as revealed not only by neuroanatomic tracing but also with immunofluorescence for several markers of neurosensory differentiation. Our data reveal a hitherto unknown time table for the development of peripheral sensory receptors in the vibrissal follicles. This time table parallels that of their central targets in the somatosensory barrel cortex, which develops at P4. Anat Rec 293:1553–1567, 2010. © 2010 Wiley‐Liss, Inc.     

四针状氧化锌晶须对复合树脂抗菌性能的影响

目的 研究四针状氧化锌晶须(tetrapod-like zinc oxide whiskers,T-ZnOw)抗菌剂对复合树脂抗菌性能的影响,为减少树脂修复后继发龋提供新方法.方法 液体稀释法测试T-ZnOw和无机载银抗菌剂对变形链球菌的最小抑菌浓度(minimal inhibitory concentration,MIC)及最小杀菌浓度(minimal bactericidal concentration,MBC).复合树脂粉剂中分别加入质量分数为3%、5%、10%的T-ZnOw和质量分数分别为l%、3%、5%的无机载银抗菌剂,直接接触法测定抗菌率,比较树脂样本加速老化处理3个月后的抗菌率.结果 T-ZnOw和无机载银抗菌剂的MIC分别为0.15625、0.15625 g/L,MBC分别为0.3125、0.15625 g/L.T-ZnOw质量分数为3%、5%、10%时,树脂对变形链球菌的抗菌率分别为(84.85±5.16)%、(94.22±3.73)%、(99.43±0.48)%.无机载银抗菌剂质量分数为1%、3%、5%时,复合树脂的抗菌率分别为(71.01±7.67)%、(90.76±5.91)%、(97.19±4.10)%.老化后添加5%T-ZnOw和5%无机载银抗菌剂的树脂抗菌率分别为(89.89±5.55)%和(78.79±7.81)%.结论 添加T-ZnOw可赋予复合树脂抗菌性能,其质量分数达5%时即可取得较好的抗菌效果,且抗菌长效性优于无机载银抗菌剂.     

Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage-sensitive dyes

We have identified large-scale patterns of electrical activity in cortical circuits that occur in response to stimulation of peripheral receptors. Our focus was on primary (S1) vibrissal cortex of anesthetized rat, and we used optical techniques in conjunction with voltage-sensitive dyes to measure depolarization of the upper layers of cortex. Displacement of one vibrissa produced a field of activity that extends over very many cortical columns in S1. There are multiple, focal maxima within this field. A global maximum is located near the center of the field of activity, and, as determined electrically and histologically, this site maps to the cortical column appropriate for the deflected vibrissa. The amplitude of this component attains a steady-state value under continuous stimulation. Additional temporal characteristics are revealed by the response to a single displacement; the signal was triphasic and began with a prompt depolarization that was followed by a transient phase of inhibition and a final phase of long-lasting depolarization. The somatotopy of the other, satellite maxima in the field of activity were established through the reconstruction of the fields of activity produced by individual stimulation of other vibrissae. Local maxima for one vibrissa were seen to overlie the global maximum found for stimulation of nearest- and next-nearest-neighbor vibrissae. In contrast to the amplitude of the global maxima, the amplitude associated with the local maxima was not maintained with either continuous or infrequent but repetitive stimulation. Finally, the field of activity induced by alternate deflection of two neighboring vibrissae was suppressed in amplitude in comparison to the summed amplitudes of the signals elicited by deflection of each vibrissa alone. We suggest that these patterns of activity are a manifestation of the dynamic interaction among neighboring cortical columns. © 1996 Wiley-Liss, Inc.