Establishment of a specific cell death induction system in Bombyx mori by a transgene with the conserved apoptotic regulator, mouse Bcl-2-associated X protein (mouse Bax).

The induction of apoptosis in vivo is a useful tool for investigating the functions and importance of particular tissues. B-cell leukaemia/lymphoma 2-associated X protein (Bax) functions as a pro-apoptotic factor and induces apoptosis in several organisms. The Bax-mediated apoptotic system is widely conserved from Caenorhabditis elegans to humans. In order to establish a tissue-specific cell death system in the domestic silkworm, Bombyx mori, we constructed a transgenic silkworm that overexpressed mouse Bax (mBax) in particular tissues by the Gal4-upstream activation sequence system. We found that the expression of mBax induced specific cell death in the silk gland, fat body and sensory cells. Fragmentation of genomic DNA was observed in the fat body, which expressed mBax, thereby supporting apoptotic cell death in this tissue. Using this system, we also demonstrated that specific cell death in sensory cells attenuated the response to the sex pheromone bombykol. These results show that we successfully established a tissue-specific cell death system in vivo that enabled specific deficiencies in particular tissues. The inducible cell death system may provide useful means for industrial applications of the silkworm and possible utilization for other species.

[Complete resection of Pancoast tumor following induction chemoradiotherapy improves survival].

OBJECTIVE: Pancoast tumors are some of the most challenging thoracic malignant diseases to treat because of their proximity to vital structures at the thoracic inlet. We retrospectively analyzed 23 patients with pT3-4, N0-3 Pancoast tumors who underwent combined chest wall resection including the 1st rib, and discuss the anatomical considerations, assessment of induction therapy, and surgical approaches for these cancers. METHODS: Between 1983 and 2006, 23 patients with Pancoast tumors underwent combined resection of the 1st rib at our institute. Of those, 21 were male and 2 were female, with an average age of 58 years. There were 10 each of squamous cell carcinoma and adenocarcinoma, 2 large cell carcinoma, and 1 adenosquamous carcinoma. Over the past decade, induction chemoradiotherapy (>40Gy) was employed before surgery. RESULTS: A posterior approach was employed in 14 patients, an anterior approach in 7, and a combined anterior and posterior approach in 2. Sixteen patients underwent complete resection. One of 7 patients undergoing incomplete resection (4.3%) died on the 45th postoperative day. The 3- and 5-year survival rates were 50 and 22%, respectively, for patients with complete resection. No case survived for more than 8 months out of the 7 patients with incomplete resection. Fourteen patients with pN0 showed significantly better survival than those with pN1-3 (p = 0.0053). CONCLUSION: Recent literature and our results suggest that patients with pN0 and/or a pathological complete response (pCR) after induction chemoradiotherapy could achieve long-term survival after complete resection.

Covariation of pupillary and auditory cortical activity in rats under isoflurane anesthesia.

Very slow fluctuations of spontaneous activities significantly influence not only behavioral performance in a conscious state, but also neural activities in an unconscious state. Covariation of pupil and cortical activities may lend important insights into the state-dependent modulation of stimulus encoding, yet this phenomenon has received little attention, especially with regard to non-visual cortices. In the present study, we investigated co-fluctuation of pupil size and neural activity in the auditory cortex of rats under isoflurane anesthesia. Pupil fluctuation consisted of longitudinal irregular shifts, and 1-min cyclic modulations. Both spontaneous and auditory-evoked potentials (AEPs) covaried with the longitudinal fluctuation of pupil size, but not with the 1-min cycle. Pupil size exhibited a positive correlation with spontaneous activity and negative correlation with AEP amplitude, particularly when the pupil size was beyond the normal range. Stimulus-specific adaptation characterized using an oddball paradigm was less dependent on pupil size than AEP. In contrast to the cortical activity, heart rate covaried with pupil size with the 1-min oscillatory component, but not the non-oscillatory component. Furthermore, light exposure induced the pupil reflex through the autonomic system, but did not modify cortical activity, indicating that autonomic activity was not causing the cortical modulation. These results together suggest that cortical activities spontaneously covary with pupillary activity through central cholinergic modulation that triggers sympathetic nerve activation. Such a state-dependent property may be a confounding factor in cortical electrophysiology studies.

Dichotic listening in patients with situs inversus: brain asymmetry and situs asymmetry.

In order to investigate the relation between situs asymmetry and functional asymmetry of the human brain, a consonant-vowel syllable dichotic listening test known as the Standard Dichotic Listening Test (SDLT) was administered to nine subjects with situs inversus (SI) that ranged in age from 6 to 46 years old (mean of 21.8 years old, S.D. = 15.6); the four males and five females all exhibited strong right-handedness. The SDLT was also used to study twenty four age-matched normal subjects that were from 6 to 48 years old (mean 21.7 years old, S.D. = 15.3); the twelve males and twelve females were all strongly right-handed and served as a control group. Eight out of the nine subjects (88.9%) with SI more often reproduced the sounds from the right ear than sounds from the left ear; this is called right ear advantage (REA). The ratio of REA in the control group was almost the same, i.e., nineteen out of the twenty-four subjects (79.1%) showed REA. Results of the present study suggest that the left-right reversal in situs inversus does not involve functional asymmetry of the brain. As such, the system that produces functional asymmetry in the human brain must independently recognize laterality from situs asymmetry.

A dual-channel FM transmitter for acquisition of flight muscle activities from the freely flying hawkmoth, Agrius convolvuli.

Moths can perform various flight maneuvers by the contraction of some direct and indirect flight muscles. Multi-channel recording from these flight muscles and analysis of their interaction is very important for understanding insect flight motor system. In this study, we developed a dual-channel FM transmitter for acquisition of muscle potentials, with which a male hawkmoth (Agrius convolvuli) could fly freely and perform pheromone triggered zigzag flight in a wind tunnel. The transmitter weighs only 0.25 g including single battery, has a 5 m receivable range and works for more than 30 min. Doubling channels was achieved by providing two oscillators (the carrier frequencies were 82 and 85 MHz), and interference between them was overcome by buffer amplifiers and independent reference electrodes for each channel. With this transmitter, we could acquire muscle potentials from some direct and indirect muscles during free flight. Combined with simultaneous high-speed video analysis, we observed distinct changes of motor patterns during takeoff. Our radio-telemetric system allows acquisition of actual information from freely flying moths; such information will lead to further progress in the study of insect flight.

Descending protocerebral neurons related to the mating dance of the male silkworm moth.

Descending protocerebral neurons in the male silkworm moth brain responding to the sexual pheromone (Bombykol) were identified. The neurons responded well with a tonic type of response and the high-frequency spikes evoked continued even after the end of the stimulus. Characteristics of the dose-response curves of the neurons to the pheromone remarkably resembled those of the wing vibration which is one of the mating behavioral components of the male moths.

Long-lasting excitation of protocerebral bilateral neurons in the pheromone-processing pathways of the male moth Bombyx mori.

Intracellular recording and staining with Lucifer yellow were used to characterize the responses and structure of pheromone-processing bilateral neurons in the protocerebrum of the brain of the male silkworm moth Bombyx mori. Numerous olfactory bilateral neurons innervated a particular neuropil region lateral to the central body, the lateral accessory lobe (LAL). The LALs are linked to each other by bilateral neurons with arborizations in each LAL. The LAL appears to be important for collecting the olfactory information from both sides of the brain. Many of the bilateral neurons showed a characteristic long-lasting excitation (LLE) that outlasted the olfactory stimuli (1.5 s). In some preparations, the LLE lasted more than 20 s and the firing gradually decreased to the background level.

Localization of odor-induced oscillations in the bumblebee antennal lobe.

Odor-induced oscillatory signals have been reported in odor discrimination in various insects. To understand dynamic olfactory coding in the first order olfactory center, the antennal lobe, localization of oscillatory signals was investigated using an optical recording technique with a voltage-sensitive dye. We present here the first report of visualization of the spatial distribution pattern of odor-induced oscillations in the bumblebee antennal lobe. Analysis of the odor-induced optical responses by a maximum entropy method allowed visualization of oscillatory regions in the antennal lobe. The oscillatory signals were usually localized to regions corresponding to a single or several glomerular structures. Our results suggest that glomerular structures may be functional units of odor processing from the viewpoint of odor-induced population responses, the oscillations.

High-speed voltage-sensitive dye imaging of an in vivo insect brain.

We have developed an improved optical recording technique with high spatio-temporal resolution to investigate neural activity from an in vivo insect brain. Optical images were acquired from hawk moth antennal lobe (AL) neurons. When activity was elicited by electrical stimulation of the antennal nerve using a stereoscopic microscope, depolarization followed by hyperpolarization was consistently recorded in the AL. When gamma-aminobutyric acid (GABA)-mediated synaptic transmission was blocked by the picrotoxin, the typical hyperpolarization was greatly reduced while no changes were observed in the depolarization. This suggests that the hyperpolarization results from inhibitory postsynaptic potentials (IPSPs) originating from GABA-sensitive chloride channels on the AL neurons.

Spatio-temporal activities in the antennal lobe analyzed by an optical recording method in the male silkworm moth Bombyx mori.

Optical recordings with a voltage-sensitive dye showed that the spatio-temporal pattern of depolarizing responses evoked by electrical stimulation of antennal nerve (AN) was non-homologously distributed in the antennal lobe (AL) of the male silkworm moth, Bombyx mori. Time courses of postsynaptic activities and GABAergic inhibitory potentials of AL neurons were individually demonstrated by pharmacological experiments, i.e. Ca2+ free and bicuculline conditions. GABAergic inhibitory potentials began with a ca. 3 ms delay from the beginning of the postsynaptic activities. Intensity of the postsynaptic activities and GABAergic inhibitory potentials were non-homologously distributed in the AL. Relatively strong postsynaptic activities and GABAergic inhibitory potentials were consistently observed in some parts of the macroglomerular complex (MGC) and/or in some ordinary glomeruli (Gs) in the medial and ventral part of the AL.

Odour-tracking capability of a silkmoth driving a mobile robot with turning bias and time delay.

The reconstruction of mechanisms behind odour-tracking behaviours of animals is expected to enable the development of biomimetic robots capable of adaptive behaviour and effectively locating odour sources. However, because the behavioural mechanisms of animals have not been extensively studied, their behavioural capabilities cannot be verified. In this study, we have employed a mobile robot driven by a genuine insect (insect-controlled robot) to evaluate the behavioural capabilities of a biological system implemented in an artificial system. We used a male silkmoth as the 'driver' and investigated its behavioural capabilities to imposed perturbations during odour tracking. When we manipulated the robot to induce the turning bias, it located the odour source by compensatory turning of the on-board moth. Shifting of the orientation paths to the odour plume boundaries and decreased orientation ability caused by covering the visual field suggested that the moth steered with bilateral olfaction and vision to overcome the bias. An evaluation of the time delays of the moth and robot movements suggested an acceptable range for sensory-motor processing when the insect system was directly applied to artificial systems. Further evaluations of the insect-controlled robot will provide a 'blueprint' for biomimetic robots and strongly promote the field of biomimetics.

Tonotopic-column-dependent variability of neural encoding in the auditory cortex of rats.

Neural computation could benefit from the heterogeneity of neurons to achieve energy efficiency. Beyond a single neuron level, adaptation to biologically important signals should also make functional columns heterogeneous. In the present study, we test a hypothesis that variability of neural response depends on tonotopic columns in the primary auditory cortex (A1) of rats. Mutual information (MI) was estimated from multi-unit responses in A1 of anesthetized rats, to quantify how spike count (SC) and the first spike latency (FSL) carried information about frequency and intensity of test tones. Consequently, for both SC and FSL, we found best frequency (BF)-dependent MI distributions with wide variances in high BF regions. These MI distributions were caused by BF-dependence of the amount of information that neurons conveyed, i.e., total entropy, rather than the transmission efficiency. In addition, the relationship between the transmission efficiency and the total entropy differentiated SC encoding and FSL encoding, suggesting that SC encoding and FSL encoding are not redundant but each plays a different role in intensity encoding. These results provide compelling evidence that BF columns are heterogeneous. Such heterogeneity of columns may make the global computation in A1 more efficient. Thus, the efficient coding in the neural system could be achieved by multiple-scale heterogeneity.

Learning-stage-dependent, field-specific, map plasticity in the rat auditory cortex during appetitive operant conditioning.

Cortical reorganizations during acquisition of motor skills and experience-dependent recovery after deafferentation consist of several distinct phases, in which expansion of receptive fields is followed by the shrinkage and use-dependent refinement. In perceptual learning, however, such non-monotonic, stage-dependent plasticity remains elusive in the sensory cortex. In the present study, microelectrode mapping characterized plasticity in the rat auditory cortex, including primary, anterior, and ventral/suprarhinal auditory fields (A1, AAF, and VAF/SRAF), at the early and late stages of appetitive operant conditioning. We first demonstrate that most plasticity at the early stage was tentative, and that long-lasting plasticity after extended training was able to be categorized into either early- or late-stage-dominant plasticity. Second, training-induced plasticity occurred both locally and globally with a specific temporal order. Conditioned-stimulus (CS) frequency used in the task tended to be locally over-represented in AAF at the early stage and in VAF/SRAF at the late stage. The behavioral relevance of neural responses suggests that the local plasticity also occurred in A1 at the early stage. In parallel, the tone-responsive area globally shrank at the late stage independently of CS frequency, and this shrinkage was also correlated with the behavioral improvements. Thus, the stage-dependent plasticity may commonly underlie cortical reorganization in the perceptual learning, yet the interactions of local and global plasticity have led to more complicated reorganization than previously thought. Field-specific plasticity has important implications for how each field subserves in the learning; for example, consistent with recent notions, A1 should construct filters to better identify auditory objects at the early stage, while VAF/SRAF contribute to hierarchical computation and storage at the late stage.

Auditory cortical plasticity induced by intracortical microstimulation under pharmacological blockage of inhibitory synapses.

Electrical stimulation that can reorganize our neural system has a potential for promising neurorehabilitation. We previously demonstrated that temporally controlled intracortical microstimulation (ICMS) could induce the spike time-dependant plasticity and modify tuning properties of cortical neurons as desired. A 'pairing' ICMS following tone-induced excitatory post-synaptic potentials (EPSPs) produced potentiation in response to the paired tones, while an 'anti-pairing' ICMS preceding the tone-induced EPSPs resulted in depression. However, the conventional ICMS affected both excitatory and inhibitory synapses, and thereby could not quantify net excitatory synaptic effects. In the present work, we evaluated the ICMS effects under a pharmacological blockage of inhibitory inputs. The pharmacological blockage enhanced the ICMS effects, suggesting that inhibitory inputs determine a plastic degree of the neural system. Alternatively, the conventional ICMS had an inadequate timing to control excitatory synaptic inputs, because inhibitory synapse determined the latency of total neural inputs.

Physiology and morphology of protocerebral olfactory neurons in the male moth Manduca sexta.

1. We have used intracellular recording and staining with Lucifer Yellow, followed by reconstruction from serial sections, to characterize the responses and structure of olfactory neurons in the protocerebrum (PC) of the brain of the male sphinx moth Manduca sexta. 2. Many olfactory protocerebral neurons (PCNs) innervate a particular neuropil region lateral to the central body, the lateral accessory lobe (LAL), which appears to be important for processing olfactory information. 3. Each LAL is linked by its constituent neurons to the ipsilateral lateral PC, where projection neurons from the antennal lobe terminate, as well as to other regions of the PC. The LALs are also linked to each other by bilateral neurons with arborizations in each LAL. 4. Some PC neurons showed long-lasting excitation (LLE) that outlasted the olfactory stimuli by greater than or equal to 1 s, and as long as 30 s in some preparations. LLE was more frequently elicited by the sex-pheromone blend than by individual pheromone components. All bilateral neurons that showed LLE had arborizations in the LALs. LLE responses were also recorded in a single local neuron innervating the mushroom body. 5. In some other PC neurons, pheromonal stimuli elicited brief excitations that recovered to background firing rates less than 1 s after stimulation.

Physiology and morphology of descending neurons in pheromone-processing olfactory pathways in the male moth Manduca sexta.

1. We have characterized the responses and structure of olfactory descending neurons (DNs) that reside in the protocerebrum (PC) of the brain of male sphinx moths Manduca sexta and project toward thoracic ganglia. 2. Excitatory responses of DNs to pheromone blends were of two general types: (a) brief excitation (BE) that recovered to background in less than 1 s after the stimulus, and (b) long-lasting excitation (LLE) that outlasted the stimulus by greater than or equal to 1 s and, in many cases, as long as 30 s. Individual pheromone components were ineffective in eliciting LLE. 3. Some neurons showing LLE also exhibited state-dependent responses to pheromonal stimuli. When such neurons were in a state of low background firing, stimulation with pheromone blend elicited LLE. When they were in a state of LLE, an identical stimulus reduced firing for 5-10 s after which firing gradually increased to the initial higher level. 4. Thirteen stained DNs were reconstructed from serial sections for detailed analysis of their morphology in the brain. DNs exhibiting LLE had neurites concentrated in the lateral accessory lobes (LALs) in the protocerebrum and adjacent neuropil. Most DNs exhibiting only BE to pheromonal stimuli and other DNs showing responses only to visual or mechanosensory stimuli did not have branches in the LALs.

Physiology and morphology of projection neurons in the antennal lobe of the male moth Manduca sexta.

1. We have used intracellular recording and staining, followed by reconstruction from serial sections, to characterize the responses and structure of projection neurons (PNs) that link the antennal lobe (AL) to other regions of the brain of the male sphinx moth Manduca sexta. 2. Dendritic arborizations of the AL PNs were usually restricted either to ordinary glomeruli or to the male-specific macroglomerular complex (MGC) within the AL neuropil. Dendritic fields in the MGC appeared to belong to distinct partitions within the MGC. PNs innervating the ordinary glomeruli had arborizations in a single glomerulus (uniglomerular) or in more than one ordinary glomerulus of one AL (multiglomerular) or in one case, in single glomeruli in both ALs (bilateral-uniglomerular). One PN innervated the MGC and many or all ordinary glomeruli of the AL. 3. PNs with dendritic arborizations in the ordinary glomeruli and PNs associated with the MGC typically projected both to the calyces of the ipsilateral mushroom body and to the lateral protocerebrum, but some differences in the patterns of termination in those regions have been noted for the two classes of PNs. One PN conspicuously lacked branches in the calyces but did project to the lateral protocerebrum. The PN innervating the MGC and many ordinary glomeruli projected to the calyces of the ipsilateral mushroom body and the superior protocerebrum. 4. Crude sex-pheromone extracts excited all neurons with arborizations in the MGC, although some were inhibited by other odors. One P(MGC) was excited by crude sex-pheromone extract and by a mimic of one component of the pheromone blend but was inhibited by another component of the blend. 5. PNs with dendritic arborizations in ordinary glomeruli were excited or inhibited by certain non-pheromonal odors. Some of these PNs also responded to mechanosensory stimulation of the antennae. 6. The PN with dendritic arborizations in the MGC and many ordinary glomeruli was excited by crude sex-pheromone extracts and non-pheromonal odors and also responded to mechanosensory stimulation of the antenna.

Formation of Copper(II) Thiocyanato and Cadmium(II) Iodo Complexes in Micelles of Nonionic Surfactants with Varying Poly(ethylene oxide) Chain Lengths.

Formation of copper(II) thiocyanato and cadminum(II) iodo complexes in micelles of poly(ethylene oxide) (PEO)-type nonionic surfactants with varying PEO chain lengths of 9.5 (Triton X-100), 30 (Triton X-305), and 40 (Triton X-405) has been studied by titration spectrophotometry and calorimetry at 298 K. In a given surfactant solution, all data obtained were analyzed by assuming formation of ternary complexes MX(n)Y(m)((2-n)+) (M = Cu(II),Cd(II); X = SCN(-), I(-); Y = surfactant), and the complexes thus form in aqueous phase (m = 0) or in micelles (m = 1). In the Cu(II)-SCN(-) system, spectrophotometric data obtained by varying concentrations of the surfactant can be explained well in terms of formation of Cu(NCS)(2)Y in micelles and Cu(NCS)(+) and Cu(NCS)(2) in an aqueous phase, and it turned out that formation constant of Cu(NCS)(2)Y increases with increasing PEO chain length. In the Cd(II)-I(-) system, the formation of CdI(3)Y(-) and CdI(4)Y(2-) is concluded in micelles, and that of CdI(+), CdI(3)(-), and CdI(4)(2-) in an aqueous phase. Interestingly, formation enthalpies of CdI(3)Y(-) and CdI(4)Y(2-) become significantly less negative with increasing PEO chain length. This suggests that transfer of the complexes from aqueous solution to a hydrophobic octylphenyl (OP) moiety in micelles is significantly more exothermic than that to a hydrophilic PEO one. Thermodynamic parameters of transfer of CdI(3)(-) and CdI(4)(2-) from aqueous solution to the OP and PEO moieties of micelles have been evaluated. Copyright 2000 Academic Press.