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Steering is one of the three in-equivalent forms of nonlocal correlations intermediate between Bell nonlocality and entanglement. Schrödinger-Robertson uncertainty relation (SRUR), has been widely used to detect entanglement and steering. However, the steering criterion in earlier works, based on SRUR, did not involve complete inferred-variance uncertainty relation. In this paper, by considering the local hidden state model and Reid's formalism, we derive a complete inferred-variance EPR-steering criterion based on SRUR in the bipartite scenario. Furthermore, we check the effectiveness of our steering criterion with discrete variable bipartite two-qubit and two-qutrit isotropic states., (© 2024. The Author(s).)

The cell intrinsic mechanisms directing peripheral nerve regeneration have remained largely understudied, thus limiting our understanding of these processes and constraining the advancement of novel clinical therapeutics. The use of primary adult rat dorsal root ganglion (DRG) neurons cultured in vitro is well established. Despite this, these cells can be challenging to culture and have so far not been amenable to robust transfection or live-cell imaging. The ability to transfect these cells with fluorescent plasmid constructs to label subcellular structures, combined with high resolution time-lapse imaging has the potential to provide invaluable insight into how peripheral neurons coordinate their regenerative response, and which specific cellular structures are involved in this process. Here we describe a protocol that facilitates transfection and subsequent live-imaging of adult rat DRG neurons., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

To investigate the cell behavior underlying neuronal differentiation in a physiologically relevant context, differentiating neurons must be studied in their native tissue environment. Here, we describe an accessible protocol for fluorescent live imaging of differentiating neurons within ex vivo embryonic chicken spinal cord slice cultures, which facilitates long-term observation of individual cells within developing tissue., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The initiation of nascent projections, or neurites, from the neuronal cell body is the first stage in the formation of axons and dendrites, and thus a critical step in the establishment of neuronal architecture and nervous system development. Neurite formation relies on the polarized remodelling of microtubules, which dynamically direct and reinforce cell shape, and provide tracks for cargo transport and force generation. Within neurons, microtubule behaviour and structure are tightly controlled by an array of regulatory factors. Although microtubule regulation in the later stages of axon development is relatively well understood, how microtubules are regulated during neurite initiation is rarely examined. Here, we discuss how factors that direct microtubule growth, remodelling, stability and positioning influence neurite formation. In addition, we consider microtubule organization by the centrosome and modulation by the actin and intermediate filament networks to provide an up-to-date picture of this vital stage in neuronal development., Competing Interests: None declared., (© The Author(s) 2022. Published by Oxford University Press.)

Cellular differentiation leads to the formation of specialized cell types and complex morphological variations. Often, differentiating cells transition between states by switching how they respond to the signaling environment. However, the mechanisms regulating these transitions are poorly understood. Differentiating neurons delaminate from the neuroepithelium through the regulated process of apical abscission, which mediates an acute loss of polarity and primary cilium disassembly. Using high-resolution live-cell imaging in chick neural tube, we show that these cells retain an Arl13b + particle, which elongates and initiates intraflagellar trafficking as it transits toward the cell body, indicating primary cilium remodeling. Notably, disrupting cilia during and after remodeling inhibits axon extension and leads to axon collapse, respectively. Furthermore, cilium remodeling corresponds to a switch from a canonical to noncanonical cellular response to Shh. This work transforms our understanding of how cells can rapidly reinterpret signals to produce qualitatively different responses within the same tissue context., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination. This activity is mimicked by a secreted variant of Crumbs2 (CRB2S) which is specifically expressed by dmNes+RG. In cultured MDCK cells, CRB2S associates with apical membranes and decreases cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice, and targeted reduction of Crb2/CRB2S in slice cultures reveal essential roles for transmembrane CRB2 (CRB2TM) and CRB2S on VL cells and dmNes+RG, respectively. We propose a model in which a CRB2S-CRB2TM interaction promotes the progressive attrition of the dVL without loss of overall VL integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination., Competing Interests: The authors have declared that no competing interests exist.

Detachment of newborn neurons from the neuroepithelium is required for correct neuronal architecture and functional circuitry. This process, also known as delamination, involves adherens-junction disassembly and acto-myosin-mediated abscission, during which the centrosome is retained while apical/ciliary membranes are shed. Cell-biological mechanisms mediating delamination are, however, poorly understood. Using live-tissue and super-resolution imaging, we uncover a centrosome-nucleated wheel-like microtubule configuration, aligned with the apical actin cable and adherens-junctions within chick and mouse neuroepithelial cells. These microtubules maintain adherens-junctions while actin maintains microtubules, adherens-junctions and apical end-foot dimensions. During neuronal delamination, acto-myosin constriction generates a tunnel-like actin-microtubule configuration through which the centrosome translocates. This movement requires inter-dependent actin and microtubule activity, and we identify drebrin as a potential coordinator of these cytoskeletal dynamics. Furthermore, centrosome compromise revealed that this organelle is required for delamination. These findings identify new cytoskeletal configurations and regulatory relationships that orchestrate neuronal delamination and may inform mechanisms underlying pathological epithelial cell detachment.

Here, we exploit the spatial separation of temporal events of neural differentiation in the elongating chick body axis to provide the first analysis of transcriptome change in progressively more differentiated neural cell populations in vivo. Microarray data, validated against direct RNA sequencing, identified: (1) a gene cohort characteristic of the multi-potent stem zone epiblast, which contains neuro-mesodermal progenitors that progressively generate the spinal cord; (2) a major transcriptome re-organisation as cells then adopt a neural fate; and (3) increasing diversity as neural patterning and neuron production begin. Focussing on the transition from multi-potent to neural state cells, we capture changes in major signalling pathways, uncover novel Wnt and Notch signalling dynamics, and implicate new pathways (mevalonate pathway/steroid biogenesis and TGFβ). This analysis further predicts changes in cellular processes, cell cycle, RNA-processing and protein turnover as cells acquire neural fate. We show that these changes are conserved across species and provide biological evidence for reduced proteasome efficiency and a novel lengthening of S phase. This latter step may provide time for epigenetic events to mediate large-scale transcriptome re-organisation; consistent with this, we uncover simultaneous downregulation of major chromatin modifiers as the neural programme is established. We further demonstrate that transcription of one such gene, HDAC1, is dependent on FGF signalling, making a novel link between signals that control neural differentiation and transcription of a core regulator of chromatin organisation. Our work implicates new signalling pathways and dynamics, cellular processes and epigenetic modifiers in neural differentiation in vivo, identifying multiple new potential cellular and molecular mechanisms that direct differentiation., (© 2014. Published by The Company of Biologists Ltd.)

Withdrawal of differentiating cells from proliferative tissue is critical for embryonic development and adult tissue homeostasis; however, the mechanisms that control this cell behavior are poorly understood. Using high-resolution live-cell imaging in chick neural tube, we uncover a form of cell subdivision that abscises apical cell membrane and mediates neuron detachment from the ventricle. This mechanism operates in chick and mouse, is dependent on actin-myosin contraction, and results in loss of apical cell polarity. Apical abscission also dismantles the primary cilium, known to transduce sonic-hedgehog signals, and is required for expression of cell-cycle-exit gene p27/Kip1. We further show that N-cadherin levels, regulated by neuronal-differentiation factor Neurog2, determine cilium disassembly and final abscission. This cell-biological mechanism may mediate such cell transitions in other epithelia in normal and cancerous conditions.

A shelf stable, convenience product egg yolk paneer (EYP) was developed by incorporation of optimized quantities of binders, salt, natural antioxidants and egg yolk. Dehydrated EYP was packed in metalised polyester pouches, stored at ambient temperature (27 ± 2 °C) for 6 months and sampled periodically for quality evaluation. The protein and fat content of dehydrated EYP was 26.2 ± 1.75% and 36.1 ± 2.46, respectively. The shelf stability of the product was achieved by keeping a moisture content (5.6 ± 0.50%) and water activity (0.43 ± 0.05) low. An excellent rehydration capacity (64.8 ± 5.39%) was observed in the EYP, whereas, the rehydration ratio of the product was 1:2.7. Changes in Free Fatty Acids, Thiobarbituric acid, textural profile analysis and Hunter colour units (L, a and b) during storage did not affect the quality characteristics of the product. About 38% loss in carotenoid content was recorded during storage of the product. Staphylococcus aureus, E coli, Salmonella and Shigella, however, were not detected in any sample throughout the storage period. Sensory evaluation revealed that rehydrated yolk paneer had excellent texture and was very close to fresh ones (before drying) during storage for 6 months.

Inheritance of apical membrane is proposed to maintain vertebrate neural stem cell proliferation. However, evidence for this is contradictory. Using direct clonal analysis and live imaging in chick neural tube, we show that divisions that separate apical and basal components generate an apical daughter, which becomes a neuron, and a basal daughter, which rapidly re-establishes apico-basal polarity and divides again. Using a recently described real-time reporter of Notch activity, we confirm progenitor status and demonstrate that division orientation can influence Notch signalling. In addition, we reveal loss of apical complex proteins on neuronal differentiation onset, suggesting that removal of this inherited complex is part of the neuronal differentiation mechanism. These findings reconcile contradictory data, link asymmetric division to Notch signalling dynamics and identify apical complex loss as a new step towards neuronal differentiation.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis. In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution. This assay may be modified to image a range of embryonic tissues. In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Coordination between functionally related adjacent tissues is essential during development. For example, formation of trunk neural crest cells (NCCs) is highly influenced by the adjacent mesoderm, but the molecular mechanism involved is not well understood. As part of this mechanism, fibroblast growth factor (FGF) and retinoic acid (RA) mesodermal gradients control the onset of neurogenesis in the extending neural tube. In this paper, using gain- and loss-of-function experiments, we show that caudal FGF signaling prevents premature specification of NCCs and, consequently, premature epithelial-mesenchymal transition (EMT) to allow cell emigration. In contrast, rostrally generated RA promotes EMT of NCCs at somitic levels. Furthermore, we show that FGF and RA signaling control EMT in part through the modulation of elements of the bone morphogenetic protein and Wnt signaling pathways. These data establish a clear role for opposition of FGF and RA signaling in control of the timing of NCC EMT and emigration and, consequently, coordination of the development of the central and peripheral nervous system during vertebrate trunk elongation.

Background: The vertebrate peripheral nervous system contains sensory neurons that arise from ectodermal placodes. Placodal cells ingress to move inside the head to form sensory neurons of the cranial ganglia. To date, however, the process of placodal cell ingression and underlying cellular behavior are poorly understood as studies have relied upon static analyses on fixed tissues. Visualizing placodal cell behavior requires an ability to distinguish the surface ectoderm from the underlying mesenchyme. This necessitates high resolution imaging along the z-plane which is difficult to accomplish in whole embryos. To address this issue, we have developed an imaging system using cranial slices that allows direct visualization of placode formation., Results: We demonstrate an effective imaging assay for capturing placode development at single cell resolution using chick embryonic tissue ex vivo. This provides the first time-lapse imaging of mitoses in the trigeminal placodal ectoderm, ingression, and intercellular contacts of placodal cells. Cell divisions with varied orientations were found in the placodal ectoderm all along the apical-basal axis. Placodal cells initially have short cytoplasmic processes during ingression as young neurons and mature over time to elaborate long axonal processes in the mesenchyme. Interestingly, the time-lapse imaging data reveal that these delaminating placodal neurons begin ingression early on from within the ectoderm, where they start to move and continue on to exit as individual or strings of neurons through common openings on the basal side of the epithelium. Furthermore, dynamic intercellular contacts are abundant among the delaminating placodal neurons, between these and the already delaminated cells, as well as among cells in the forming ganglion., Conclusions: This new imaging assay provides a powerful method to analyze directly development of placode-derived sensory neurons and subsequent ganglia formation for the first time in amniotes. Viewing placode development in a head cross-section provides a vantage point from which it is possible to study comprehensive events in placode formation, from differentiation, cell ingression to ganglion assembly. Understanding how placodal neurons form may reveal a new mechanism of neurogenesis distinct from that in the central nervous system and provide new insight into how cells acquire motility from a stationary epithelial cell type.

The size, composition and functioning of the spinal cord is likely to depend on appropriate numbers of progenitor and differentiated cells of a particular class, but little is known about how cell numbers are controlled in specific cell cohorts along the dorsoventral axis of the neural tube. Here, we show that FatJ cadherin, identified in a large-scale RNA interference (RNAi) screen of cadherin genes expressed in the neural tube, is localised to progenitors in intermediate regions of the neural tube. Loss of function of FatJ promotes an increase in dp4-vp1 progenitors and a concomitant increase in differentiated Lim1(+)/Lim2(+) neurons. Our studies reveal that FatJ mediates its action via the Hippo pathway mediator Yap1: loss of downstream Hippo components can rescue the defect caused by loss of FatJ. Together, our data demonstrate that RNAi screens are feasible in the chick embryonic neural tube, and show that FatJ acts through the Hippo pathway to regulate cell numbers in specific subsets of neural progenitor pools and their differentiated progeny.

Vertebrate cranial sensory ganglia, responsible for sensation of touch, taste and pain in the face and viscera, are composed of both ectodermal placode and neural crest cells. The cellular and molecular interactions allowing generation of complex ganglia remain unknown. Here, we show that proper formation of the trigeminal ganglion, the largest of the cranial ganglia, relies on reciprocal interactions between placode and neural crest cells in chick, as removal of either population resulted in severe defects. We demonstrate that ingressing placode cells express the Robo2 receptor and early migrating cranial neural crest cells express its cognate ligand Slit1. Perturbation of this receptor-ligand interaction by blocking Robo2 function or depleting either Robo2 or Slit1 using RNA interference disrupted proper ganglion formation. The resultant disorganization mimics the effects of neural crest ablation. Thus, our data reveal a novel and essential role for Robo2-Slit1 signaling in mediating neural crest-placode interactions during trigeminal gangliogenesis.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

Exposure to methylmercuric chloride (MMC) has been shown to significantly affect development of the lung and pulmonary surfactant system of the fetus. Preliminary results suggest it may also affect adult lung and associated bronchoalveolar lavage (BAL), which represents the extracellular surfactant pool. To determine if mercury exposure has the potential to alter surfactant function, adult mice were treated with MMC, 15 mg/kg by intragastric intubation on 4 successive days. BAL was collected by repeated intratracheal lavage 24 h after the last treatment. Nucleated cell numbers in lavage were determined. Tissue was prepared for scanning electron microscopy (SEM). Lavage fluid was extracted into chloroform:methanol and phospholipid concentration determined. A sample of the extract was used at a constant phospholipid concentration to measure surface activity on a bubble surfactometer. Lung weight to body weight ratio increased whereas total numbers of nucleated cells in BAL were not altered by MMC. SEM of samples from lungs of animals exposed to MMC showed normal architecture. Surface tension measurements suggest that the mean time to minimum surface tension and the minimum surface tension were greater in BAL from mice exposed to MMC for 4 days. In addition samples of BAL were prepared for Fourier-transform infrared spectrophotometry (FT-IR). Spectra showed changes in both lipid and protein components of BAL. Morphometric analyses of micrographs showed that mean alveolar diameter was reduced and wall thickness increased after mercury exposure. These results suggest that methylmercury exposure may significantly affect surface tension characteristics and composition of BAL, possibly through leakage of edematous interstitial tissue.

Surfactant obtained from bronchoalveolar lavage (BAL) can be separated into subfractions based on sedimentation characteristics. It has been suggested that the 10,000 x g, 60,000 x g and 100,000 x g subfractions isolated by this approach represent stages of surfactant extracellular processing. These three subfractions have been reported to differ in their morphology, composition and ability to lower surface tension. We wished to determine if infrared spectroscopy, which may be applied as a non-invasive technique could potentially prove useful for characterization and quantification of bronchoalveolar lavage (BAL) protein and phospholipid, and if this approach could detect differences in intermediate surfactant processing stages. Subfractions were collected from adult rabbit lungs by BAL and differential centrifugation and analyzed by Fourier transform infrared (FT-IR) spectroscopy. Biochemical assay of phospholipid and protein showed differences between subfractions that correlated well with the phospholipid/protein ratios obtained from FT-IR spectra (r = 0.939; r2 = 0.882). The subfraction sedimenting at 100,000 x g (P100) exhibited spectral shifts in the Amide I band, suggesting that the protein secondary structure was different compared to other fractions. Spectra obtained after separation of lipids and protein components showed an apparent disordering of protein secondary structure but little or no effect on the structure or mobility of phospholipids. These results support the idea that subfractions represent various processing stages of surfactant. In addition, they show that results from FT-IR analyses correlate significantly with traditional biochemical assay methods which may prove of clinical use.(ABSTRACT TRUNCATED AT 250 WORDS)

Infrared spectroscopy which has traditionally been utilized by chemists and physicists for characterization and identification of the structural properties of chemical compounds is now becoming more relevant as a biodiagnostic tool. Recent reports suggest that arthritis and Alzheimer's disease can be diagnosed by using this technique. Changes associated with these diseases diagnosable with this technique are generally overt. In this study we have used 'Fourier transform infrared spectroscopy' (FT-IR) to analyze subtle changes in composition and structure of lipids and proteins in lung tissue, bronchoalveolar lavage and purified lamellar body fraction of mice exposed to methylmercury. Infrared measurements were made in attenuated total reflection mode using the Split Pea (Harrick Scientific Corporation, USA). Mice were treated with 4 doses of methylmercuric chloride (15 mg/kg body weight/dose), and control animals received an equivalent volume of physiological saline. Comparison of the control and experimental spectra revealed alterations in the intensities and frequencies of vibrational modes of lipids following methylmercury exposure. Results indicate that FT-IR spectral analyses may be a valuable tool for detecting subtle variations in biological components associated with drug exposure to lungs and, in particular may be very useful for assessing changes in bronchoalveolar lavage.

Trichloroethylene (TCE) has previously been shown, in adult mouse lung, to alter the distribution of phospholipid associated with the pulmonary surfactant and to affect the activity of phospholipase A2, an enzyme involved in synthesis of the surfactant. However there are no data available on the effects of maternal exposure to TCE on fetal lung development. To determine if maternal TCE exposure may affect fetal pulmonary development, pregnant mice were treated with TCE (3000 mg/kg body wt.) administered intraperitoneally on day 17 of pregnancy. Fetuses were examined on the 18th and 19th gestational days and the 1st, 5th and 10th postnatal days. TCE caused increased mortality among 18- and 19-day-old fetuses and 1-day-old newborn mice. In addition mean body weight was reduced on the 1st postnatal day in animals exposed to TCE. Specific lung weights were also significantly reduced on both the 18th and 19th gestational days. More importantly, total lung phospholipid content was depressed immediately before birth; total DNA was not affected. These results suggest that prenatal maternal exposure to TCE may delay pulmonary maturation during the critical period when surfactant synthesis is initiated.

Mercury ingested from dietary sources has potent neurotoxic and teratogenic effects. Initial studies have shown that mercury may also affect fetal lung development. Since these pulmonary effects may play a role in subsequent neonatal morbidity and mortality due to compromising of the development of the lung, mercury effects in fetal and neonatal lung were investigated. Methylmercuric chloride (MMC), 1,000 ppm (15 mg/kg of body weight); was administered via an intragastric tube to timed-pregnant Swiss/Webster mice on day 9 of gestation. Lungs from fetuses on gestational day 18 and from neonates on days 1, 5, or 10 after birth were studied. Significant changes in MMC-exposed lungs compared to controls occurred at postnatal day 1. At this time, lung weight per gram body weight increased, phospholipid content per gram of lung or per microgram of DNA decreased, while DNA per gram of lung increased. Methylmercury appears to have delayed lung maturation. Cuboidal epithelial cells in alveolar tubules contained conspicuous glycogen deposits, and differentiation of alveolar type II cells was adversely affected. These results suggest that prenatal exposure to methylmercury may be detrimental to lung development, specifically to the initiation of surfactant synthesis, by delaying the normal pattern of maturation of the alveolar type II cells within the lungs.

Fetal lung fibroblasts interact with type II epithelial cells, inducing their maturation. This interaction arises by secretion of factors which alter fetal type II cell function. To analyze these factors, conditioned medium (CM) was produced by exposing serum-free minimum essential medium, with [35S]methionine (5 microCi/ml), to confluent cultures of fetal rabbit lung fibroblasts. This medium was tested for ability to stimulate [3H]choline incorporation by fetal type II cells and subsequently fractionated on molecular weight filtration columns P60 (2.5cm x 90cm; NMW cutoff, 60kd; 1M acetic acid) and A1.5m (2.5cm x 90cm; NMW cutoff, 1,500kd; Tris-buffered saline) and a hydroxyapatite column (HT) (1.5cm x 30cm; NaCl and 0.01-0.3M phosphate). Crude medium stimulated choline incorporation into phosphatidylcholine. [35S]methionine was resolved in void volume material and in material of apparent molecular weight of 6000 daltons on the P60 filtration column. Filtration on the A1.5m column showed two major fractions with radiolabel incorporation. Each of these was resolved into two subfractions on HT chromatography. The high molecular mass fraction contained material which stimulated [3H]choline incorporation by fetal type II cells. The low molecular mass fraction tended to inhibit [3H]choline incorporation. The second subfractions of both the first and second primary fractions inhibited [3H]thymidine incorporation into DNA by fetal type II cells. SDS-PAGE electrophoresis and autoradiography showed that under reducing conditions, each peak contained several proteins. However few of these displayed radioactivity. These results indicate that protein factors produced by fetal lung fibroblasts may be involved in regulating both differentiation and replication of fetal type II cells.

Twelve millimeters of the thoracolumbar spinal cord of mice has been treated with a radiofrequency heating system which has been shown previously to produce localized and controllable elevation of temperature. The severity of neurological damage was assessed by measuring the reduction in the reflex leg extension of the hind legs of the mice from video-recorded images and by scoring the performance of the mice by a negative geotaxis test. The response to treatment was rapid with maximum paralysis occurring within a few days after treatment. Only minor symptoms were observed in those animals which had not developed paralysis within 2 weeks. A 40% reduction in the reflex leg extension was chosen as an end point, and the percentage of mice having reached the end point for different thermal doses was determined in groups of nine mice. The ED50 for heating for 1 h was 43.1 degrees C and for heating at 45 degrees C was 10.8 min. An increase in temperature by 1 degree C required a decrease in time by a factor of 2.25 to produce the same effect. Thermotolerance was observed 24 h after preheating at 45 degrees C for 1.9 min with a thermotolerance ratio of 1.7. The rapid response and high sensitivity of the spinal cord will have to be taken into consideration in the clinical application of hyperthermia.

Three systems for the localized heating of the spinal cord of the mouse have been evaluated by measuring the temperatures in the spinal canal (Tsp); at a reference location dorsal to the spine (Tdo), and by numerically calculating temperature distributions throughout two-dimensional transverse cross-sections through the middle of the heated region. The systems assessed were water bath heating alone, water bath-rf combination and rf heating alone with oblique, dorsally located electrodes. It has been established that (1) for all systems delta T (where delta T = Tdo-Tsp) decreased throughout a 1 h heating period-this was attributed to changes in blood flow; (2) there existed a considerable variation in the experimental value of delta T, particularly for rf heating. The resulting error in the estimation of Tsp from a measured value of Tdo can be reduced by making use of the observed correlation between delta T and the slope of a temperature decay curve measured at the beginning of the heating period; (3) rf alone best spares adjacent visceral and superficial tissues from significant elevation of temperature.

This paper describes a quantitative and ultrastructural study of the degeneration of the intra-epithelial nerves in the epithelium of the lower respiratory tract of the cat following unilateral cervical infra-nodose vagotomy and section of the superior laryngeal nerve. A significant reduction in the number of intra-epithelial axons was found on the denervated side, degeneration being more complete at the hilus than in the trachea. Cellular inclusions resembling degenerating axons were observed especially on the denervated side, and their ultrastructural morphology is described. The functions of nerves which degenerated, and of those which remained unaffected, are discussed.

Early responses of lungs to a single radiation dose of 30 Gy were marked by an inflammatory reaction and the onset of pneumonitis within 4 weeks following hemithorax radiation in the rat. Superoxide dismutase reduced the severity of radiation lesions in lungs.

[3H]Thymidine autoradiography was used to study cell proliferation during decidualization induced by intraluminal oil in ovariectomized mice treated with oestrogen and progesterone. Development of the decidual reaction involves two distinct populations of stromal cells. Periluminal cells start to synthesize DNA 11--15 h after instillation and by 17--20 h, without dividing, differentiate into epithelioid decidual cells which continue to incorporate [3H]thymidine, presumably becoming polyploid. Cells peripheral to this zone also start to synthesize DNA between 11 and 15 h, but at 18.5 h many have divided before differentiating. None of these dividing cells had been arrested in G2. The periluminal and peripheral cells do not appear to differ in their proliferative antecedents.

The circadian rhythm of deoxyribonucleic acid (DNA) synthesis and mitosis in lung alveolar wall cells was studied in the mouse at 4 h intervals during postnatal days 4 and 5. Rhythmic variations in DNA synthesis and mitosis were observed both days, but their peak activities differed. On day 4, the greatest DNA synthesis occurred at 10 p.m. and the highest mitotic rate occurred at 2 p.m. On day 5, the greatest DNA synthesis and the highest mitotic rate both occurred at 10 p.m. On postnatal day 4, both DNA synthetic and mitotic rates showed peaks that occurred at the same time both at 2 p.m. and at 10 p.m. On postnatal day 4, both DNA synthetic and mitotic rates showed peaks that occurred at the same time both at 2 p.m. and at the 10 p.m. On postnatal day 5, 2 DNA synthesis peaks were observed, one at 10 a.m. and one at 10 p.m., with only 1 mitotic peak at 10 p.m. The length of intermitotic phases S and G2 of lung alveolar wall cells appears to be 5.5 h and 3 h, respectively.

beta-Aminopropionitrile (beta APN) was administered intraperitoneally to rats on postnatal days 1, 3, and 5. Body weight, lung volume, lung weight, number of alveoli per unit area and volume, total number of alveoli in the lung, and the total length of elastic fibers in the lung decreased, and the average alveolar volume increased in comparison with control animals similarly treated with saline. From Day 2 of age to Day 6 the total length of elastic fibers increased in control lungs but remained almost the same following beta APN treatment. Ultrastructurally, both the quality and quantity of elastin in lung alveolar wall were affected. beta APN also inhibited the synthesis of deoxyribonucleic acid in interstitial, endothelial, and Type II epithelial cells of the lung alveolar wall. The diminished number of alveoli gives support to the hypothesis that the elastin-collagen network may play a key role in postnatal alveolar multiplication.

A quantitative ultrastructural study of intra-epithelial axons in the lower respiratory tract of the cat has compared the innervation at four airway levels, two extra- and two intrapulmonary. The morphology of intra-epithelial axons has been described, and their association with different epithelial cell types recorded. Their morphology suggests that most are afferent in function.

The effect of starvation on postnatal lung growth in rats was investigated. Litters were starved twice, each time for 24 hours, on Day 1 and Day 5 after birth. One group of littermates was sacrificed on Day 7, and another group, on Day 14 of postnatal life. Intermittent starvation diminished lung growth. This was accompanied by reduced somatic growth. On postnatal Day 7, lung volume, total number of alveoli, and internal surface area of the lung were decreased in starved rats, but structurally their lungs appeared similar to control lungs. On postnatal Day 14, a striking morphologic difference was observed between the lungs of control and starved pups. Following starvation, retardation of lung growth was manifested in all the parameters studied. One week of normal uninterrupted suckling could not overcome the starvation-induced initial effects on growing lungs. It is concluded that starvation, soon after birth, affects adversely the normal development of lung.