Summary Video-enhanced differential interference contrast microscopy with background subtraction has made visible many structures and processes in living cells. In video-enhanced differential interference contrast, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved video-enhanced differential interference contrast by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12-bit background image from the incoming 12-bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion-enhanced differential interference contrast. By carrying out background subtraction with 12-bit images, the number of grey levels in the moving vesicles can be maximized and a single look-up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background and motion-enhanced differential interference contrast images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron.

Motor proteins convert chemical energy into work, thereby generating persistent motion of cellular and subcellular objects. The velocities of motor proteins as a function of opposing loads have been previously determined in vitro for single motors. These single molecule "force-velocity curves" have been useful for elucidating motor kinetics and for estimating motor performance under physiological loads due to, for example, the cytoplasmic drag force on transported organelles. Here we report force-velocity curves for single and multiple motors measured in vivo. Using motion enhanced differential interference contrast (MEDIC) movies of living NT2 (neuron-committed teratocarcinoma) cells at 37 degrees C, three parameters were measured-velocity (v), radius (a), and effective cytoplasmic viscosity (eta')-as they applied to moving vesicles. These parameters were combined in Stokes' equation, F = 6piaeta'v, to determine the force, F, required to transport a single intracellular particle at velocity, v. In addition, the number of active motors was inferred from the multimodal pattern seen in a normalized velocity histogram. Using this inference, the resulting in vivo force-velocity curve for a single motor agrees with previously reported in vitro single motor force-velocity curves. Interestingly, however, the curves for two and three motors lie significantly higher in both measured velocity and computed force, which suggests that motors can work cooperatively to attain higher transport forces and velocities.

Gliding assays of motor proteins such as kinesin, dynein and myosin are commonly carried out with fluorescently labeled microtubules or filamentous actin. In this paper, we show that speckled microtubules (MTs), prepared by copolymerizing 98% unlabeled tubulin with 2% rhodamine-labeled tubulin, can be localized to +/-7.4 nm (24 measurements) in images acquired every 125 ms. If the speckled MTs move at about 800 nm s(-1), ten images are sufficient to determine their velocity to a precision of +/-6.8 nm s(-1)(6 microtubules, 24 measurements). This velocity precision is four-fold better than manual methods for measuring the gliding velocity of uniformly labeled MTs by end-point localization. The improved velocity precision will permit the determination of velocity-force curves when one, two and three kinesin motors pull a single load in vitro.

The dependence of developing spinal motoneuron survival on a soluble factor(s) from their target, muscle tissue is well established both in vivo and in vitro. Considering this apparent dependence, we examined whether a specific component of the stress response mediates motoneuron survival in trophic factor-deprived environments. We demonstrate that, although endogenous expression of heat shock protein 70 (HSP70) did not change during trophic factor deprivation, application of e-rhHsp70 (exogenous recombinant human Hsp70) promoted motoneuron survival. Conversely, depletion of HSP70 from chick muscle extract (MEx) potently reduces the survival-promoting activity of MEx. Additionally, exogenous treatment with or spinal cord overexpression of Hsp70 enhances motoneuron survival in vivo during the period of naturally occurring cell death [programmed cell death (PCD)]. Hindlimb muscle cells and lumbar spinal astrocytes readily secrete HSP70 in vitro, suggesting potential physiological sources of extracellular Hsp70 for motoneurons. However, in contrast to exogenous treatment with or overexpression of Hsp70 in vivo, muscle-targeted injections of this factor in an ex vivo preparation fail to attenuate motoneuron PCD. These data (1) suggest that motoneuron survival requirements may extend beyond classical trophic factors to include HSP70,(2) indicate that the source of this factor is instrumental in determining its trophic function, and (3) may therefore influence therapeutic strategies designed to increase motoneuron Hsp70 signaling during disease or injury.

Strategies to improve function after CNS injuries must contend with the failure of axons to regrow after transection in adult mammals. In this issue of Neuron, Smith et al. provide an important advance by demonstrating that SOCS3 acts as a key negative regulator of adult optic nerve regeneration.

The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages:(1) creation of a Lab-on-Bead library, a one-bead, one-sequence library that, in turn, displays a library of candidate molecules,(2) fluorescence microscopy-aided identification of single target-bound beads and the extraction - wet or dry - of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target-binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target-binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead-based design allows for easy screening over a variety of prepared surfaces that can extend this technique's application to the discovery of diagnostic reagents and disease markers. Copyright (c) 2009 John Wiley & Sons, Ltd.

Primary neuron cultures are widely used in research due to the ease and usefulness of observing individual cells. Therefore, it is vital to understand how variations in culture conditions may affect neuron physiology. One potential variation for cultured neurons is a change in intracellular transport. As transport is necessary for the normal delivery of organelles, proteins, nucleic acids, and lipids, it is a logical indicator of a cell's physiology. We test the hypothesis that organelle transport may change with varying in vitro population densities, thus indicating a change in cellular physiology. Using a novel background subtraction imaging method we show that, at 5 days in vitro (DIV), transport of vesicular organelles in embryonic rat spinal cord neurons is positively correlated with cell density. When density increased 6.5-fold, the number of transported organelles increased 2.2+/-0.3-fold. Intriguingly, this effect was not observable at 3-4 DIV. These results show a significant change in cellular physiology with a relatively small change in plating procedure; this indicates that cells appearing to be morphologically similar, and at the same DIV, may still suffer from a great degree of variability.

STUDY OBJECTIVE: We compare the pain of intravenous (IV) cannulation in pediatric emergency department (ED) patients after applying a topical lidocaine/tetracaine patch versus placebo. We hypothesized that application of the active patch would reduce the pain of IV cannulation by at least 15 mm. METHODS: We conducted a randomized, double-blind, placebo-controlled trial in a suburban academic ED. Patients aged 3 to 17 years who required nonemergency IV cannulation were eligible for enrollment. At triage, a nurse placed a commercially available topical lidocaine/tetracaine patch or an identical-looking placebo patch over the antecubital or hand vein in patients for whom an IV catheter was anticipated. After IV cannulation by the treating nurse, the pain of cannulation was measured on a validated 100-mm visual analogue scale or Wong Baker scale. Outcomes were compared between groups with Mann-Whitney U, Student t, and chi(2) tests. A sample of 40 patients had 80% power to detect a 13-mm difference in pain scores. RESULTS: Forty-five patients were randomized to lidocaine/tetracaine patch (22) or placebo (23), and IV cannulation was attempted in 40 of these patients. Mean age was 10 years (SD=4.3), 35% were female patients. The median pain of IV cannulation in the active treatment group (18 mm [interquartile range (IQR) 1 to 40 mm]) was significantly lower than in the placebo group (35 mm [IQR 20 to 59 mm]; P=.04). Adequate pain relief was more common in the active treatment group (75%[95% confidence interval (CI) 53% to 89%] versus 35%[95% CI 18% to 57%]; difference 40%[95% CI 6% to 64%]). The number of successful IV cannulations after the first attempt was similar in both the lidocaine/tetracaine and the placebo groups (90%[95% CI 70% to 97%] versus 85%[95% CI 64% to 95%]; difference 5%(95% CI -21% to 30%). CONCLUSION: Application of a topical lidocaine/tetracaine patch resulted in a modest reduction in the pain of IV cannulation in pediatric ED patients and did not alter the rate of successful cannulations.

This protocol details methods to isolate and purify astrocytes and motoneurons (MNs) from the chick lumbar spinal cord. In addition, an approach to study the influences of astrocyte secreted factors on MNs is provided. Astrocytes are isolated between embryonic days 10 and 12 (E10-12), propagated in serum (2-3 h) and differentiated in chemically defined medium (3-4 h). When prepared according to this protocol, astrocyte cultures are more than 98% pure when assessed using the astrocyte-specific markers glial fibrillary acidic protein (GFAP) and S100beta. MNs are isolated between E5.5 and 6.0 (3-4 h) using a procedure that takes selective advantage of the large size of these cells. These cultures can be maintained using individual trophic factors, target-derived factors or astrocyte-derived factors, the preparation of which is also described (5-6 h). All or part of these techniques can be used to investigate a variety of processes that occur during nervous system development and disease or after injury.

During development, motoneurons (MNs) undergo a highly stereotyped, temporally and spatially defined period of programmed cell death (PCD), the result of which is the loss of 40-50% of the original neuronal population. Those MNs that survive are thought to reflect the successful acquisition of limiting amounts of trophic factors from the target. In contrast, maturation of MNs limits the need for target-derived trophic factors, because axotomy of these neurons in adulthood results in minimal neuronal loss. It is unclear whether MNs lose their need for trophic factors altogether or whether, instead, they come to rely on other cell types for nourishment. Astrocytes are known to supply trophic factors to a variety of neuronal populations and thus may nourish MNs in the absence of target-derived factors. We investigated the survival-promoting activities of muscle- and astrocyte-derived secreted factors and found that astrocyte-conditioned media (ACM) was able to save substantially more motoneurons in vitro than muscle-conditioned media (MCM). Our results indicate that both ACM and MCM are significant sources of MN trophic support in vitro and in ovo, but only ACM can rescue MNs after unilateral limb bud removal. Furthermore, we provide evidence suggesting that MCM facilitates the death of a subpopulation of MNs in a p75(NTR)- and caspase-dependent manner; however, maturation in ACM results in MN trophic independence and reduced vulnerability to this negative, pro-apoptotic influence from the target.

The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell death-independent role for Bax in facilitating mutant SOD1-mediated motor denervation.

DNA polymerases are protein machines that processively incorporate complimentary nucleotides into a growing double-stranded DNA (ds-DNA). Single-base nucleotide incorporation rates have been determined by stalling and restarting various polymerases, but intrinsic processive rates have been difficult to obtain, particularly for polymerases with low processivity, such as the human immunodeficiency virus type 1 reverse transcriptase (HIV RT) polymerase. Here we find, using a new fluorescence-based single-molecule polymerization assay, that the intrinsic processive DNA-dependent polymerization of HIV RT is approximately Poissionian (i.e. each nucleotide is added sequentially) with a rate of about 100 bases per second at 21 degrees C. From the same experiments, based on the stepping statistics of polymerization, we also estimate the rates for HIV RT early termination and final release of completely replicated primer-template DNA. In addition, by measuring the rate of polymerization as a function of temperature, we have estimated the activation energy for processive nucleotide incorporation.

The autonomous motion of vesicle is observed in a simple chemical system. A vesicle composed of didodecyldimethylammonium bromide DDAB breaks down by ion exchange from Br(-) to I(-). When an electrolyte is supplied to vesicles, some of them begin to move after an induction period. They continue to move, leaving behind the reaction products on the trail. The ion exchange decreases the vesicle size, and smaller vesicles remain after the motion. We examine the characteristics of this motion. The surface tension of the DDAB-containing aqueous phase depends on the KI concentration. Considering this result carefully, we conclude that vesicles can move when the ion exchange from Br(-) to I(-) proceeds irreversibly. Then, inhomogeneity in the vesicle membrane develops because of the coagulating nature of the product, didodecyldimethylammonium iodide (DDAI), which is sparingly soluble in water. Inhomogeneous properties of vesicle membranes are then generated, which induce surface transport of the reaction product and flow in the water pool. As a result, a couple of convection rolls appear in the water pool of the vesicle. The convection rolls drive vesicle motion. A simple model for the semiquantitative description is proposed.

A transition rate model of cargo transport by N molecular motors is proposed. Under the assumption of steady state, the force-velocity curve of multimotor system can be derived from the force-velocity curve of a single motor. Our work shows, in the case of low load, that the velocity of multimotor system can decrease or increase with increasing motor number, which is dependent on the single motor force-velocity curve; and most commonly, the velocity decreases. This gives a possible explanation to some recent experimental observations.

These studies have shown that the bones of guinea pigs given daily injections of parathormone from the age of 2 to 7 days to the age of 110 to 120 days, show relatively very little effect after receiving 20 units daily during the last 65 to 87 days of treatment. But it is probable that their bones underwent decalcification early in the treatment and that subsequently the parathormone, continued at the same dosage, did not maintain the effects on the bones. Healing finally occurred despite it. The bones of guinea pigs treated with intermittent injections of large doses of parathormone from the time they were 1 week old to the age of 95 to 145 days also showed relatively few changes at the end of the treatment. The injections were given at intervals of 7 to 11 days, and were stepped up from 60 units to 140 units. From our previous experience (1) we infer that the earlier injections of parathormone produced very extensive bone changes which healed in the intervals between the injections. As the guinea pigs became older the injections of parathormone did not produce as severe effects. We have found in our studies of experimental hyperparathyroidism that the bone changes after a single large dose of parathormone in young guinea pigs are soon healed. The study of a series of animals shows that healing begins at about the 48th hour after injection, and proceeds rapidly. Between the 8th and 14 days, callus may be observed at the costochondral junctions, where fractures had occurred. Now the endosteum may be lined by osteoblasts and the vessel canals by new formed bone. In adult guinea pigs extremely large single doses had little effect on the bones in 48 hours, even though the dose killed the animal. It was only when three doses pyramided over a period of 48 hours and totaling 2580 units of parathormone were given, that moderately severe bone resorption could be demonstrated in the adult. The elevation of serum calcium may be considered as one of the indices of calcium mobilization in experimental hyperparathyroidism. When the rate of calcium excretion exceeds the rate of its mobilization, or when the animal is on a low calcium diet, hypercalcemia may be absent. It is possible to raise the serum calcium of adult guinea pigs by large single doses of parathormone, but the resulting rise is not as great as in the young (2). This is confirmatory evidence of the fact that calcium is mobilized much less rapidly from the bones of old animals than from those of young ones. Collip pointed out that young normal dogs are more susceptible to parathormone (6). This observation was corroborated by Morgan and Garrison (7). We found that the same difference held also in experimental hyperparathyroidism produced in dogs by repeated doses of parathormone (8). In man, clinical experience likewise indicates the necessity of using relatively large doses of parathormone to raise the serum calcium of adults. The serum calcium of middle-aged or old adults does not rise significantly unless as much as 100 units or more of parathormone are given daily for a number of days. Charts VI and VII, in a recent paper by Merritt and Bauer (9), support our findings of the relative difficulty of obtaining a significant elevation of serum calcium in adults. If adult guinea pigs are given daily injections of parathormone which are rapidly stepped up, the animals may be killed by the ensuing acute hyperparathyroidism, only slight bone changes being produced. However, a careful avoidance of the induction of acute hyperparathyroidism by gradual stepping up of the parathormone dose permits the employment of doses continued over a long period of time that could not possibly have been tolerated otherwise. Furthermore, healing of the lesions thus produced may occur, in spite of the continuance of parathormone at this level. It seems likely that the difference in response of young and old guinea pigs to single doses of parathormone, as indicated by the bone changes, as well as by the serum calcium and phosphorus, is related to the more rapid rate of mineral metabolism in the young, actively growing animals. The calcium mobilizing effect of parathormone is most prominent in actively growing young animals, the calcium being withdrawn from the most readily available stores-the regions of most active new bone formation and most active bone reconstruction (10). In the adult animal the calcium reserves (in the formed bone) are less susceptible to the calcium mobilizing effect of parathormone. The adult guinea pig will show relatively slight bone changes even as a result of extremely large, fatal doses of parathormone. Repeated doses, as is well known, will produce, by pyramiding, greater effects than the entire amount administered at one time. In this type of experiment the young again show greater susceptibility of the bone than the adult. In time, however, some compensation takes place, and the effects of the same doses are decreased until finally healing may occur in spite of the continued treatment. Increase of the dose, however, again elicits the parathormone effects upon the bone, as well as upon the serum calcium and phosphorus, without toxic changes (1, 8). It would seem that some compensation sets in which may be overcome by increasing the dose. This compensation is especially evident in the experiments in which the parathormone doses were stepped up gradually from small amounts. In addition to the compensation observed in young and adult animals as a result of repeated injections of parathormone, we must also consider the possibility that there is a compensating mechanism in adult animals more effective than in the young. That compensation occurs is unquestionable but its nature is not clear. Apparently it is less effective during pregnancy, doses of parathormone which produce only slight bone changes in ordinary adults causing very severe lesions in advanced pregnancy (11). Parathormone has been shown to produce only one primary effect on bone, and that is decalcification. This may come about as the result of a change in the circulating tissue fluids, the salts being dissolved out of the organic matrix, and the latter disappearing secondarily. The process is most rapid in the vicinity of most active bone formation. The osteoblasts disappear from the surfaces of bone where dissolution is occurring, and at the same time the marrow connective tissue proliferates. Fusion of cells produces osteoclasts (12), which then proceed to remove the decalcified organic matrix, with the production of the deep lacunae of Howship. Frequently leucocytes are also observed actively phagocyting the decalcified organic matrix, and often leucocytes are observed within the osteoclasts (12). Healing is associated with the complete reversal of the process. The osteoclasts disappear, the connective tissue diminishes, osteoblasts reappear, and bone formation is resumed. As we have previously stated (13), parathormone produces a more continuous effect than experimental acidosis and greater changes than are seen in experimental osteoporosis. A pronounced decalcification results from it which, with its sequelae, simulates von Recklinghausen's disease. The emphasis which the older pathologists laid on osteoclasts as a special feature of ostitis fibrosa cystica is justified, for in the experimental condition the appearance of great numbers of osteoclasts is a constant feature, whenever decalcification occurs (13). There seems to be no doubt that the giant cell tumors found in ostitis fibrosa cystica are expressions of the same pathological response. The other features of the bone changes of hyperparathyroidism-marrow hemorrhage, cysts, fractures, and osteoid proliferation-are secondary to the primary decalcification. Progress of the pathological changes leads to circulatory stasis and cyst formation. Stresses and strains exerted on the progressively weakening bone may result in microscopical or gross fractures. Osteoid tissue is, as we have previously pointed out (13), merely reparative in nature, being laid down as support to the weakened or fractured bone, or as a part of healing. Osteoid borders appear on bone surfaces 48 hours after one large dose of parathormone. The mosaic picture which we have observed in the bones of some of our animals is produced by short and irregularly disposed cement lines resulting from rapid bone transformation. Schmorl (14) recently emphasized the mosaic-like appearance of the newly formed lamellar bone in Paget's disease (ostitis fibrosa deformans). The mosaic-like appearance of bone has also been described in local bone conditions, as e.g. syphilitic periostitis, and in bone in the vicinity of cysts and giant cell tumors in von Recklinghausen's disease (ostitis fibrosa cystica). However, Schmorl claims that in no disease is the mosaic appearance so constant and the arrangement of the cement lines so irregular as in Paget's disease. In chronic experimental hyperparathyroidism (von Recklinghausen's disease), the mosaic structure is not a prominent feature because of the progressive decalcification. But the bones of our young guinea pigs which received intermittent injections showed a mosaic-like appearance indicative of the periodic decalcifications and restorations which they had undergone.

Before proceeding to a discussion of the experiments upon cold-blooded animals, it is necessary to review briefly some of the work recently done with the bacillus of leprosy. The appearance of the bacillus in man and its behavior under artificial cultivation, and in the tissues of lower animals, should be considered in order that comparisons may be drawn. In their studies with the organism under cultivation, Duval and Gurd pointed out that the long, slender, and beaded appearance of the leprosy bacillus described by Hansen, in 1872, is lost when removed for several generations from the parent stem, and under artificial cultivation the organism becomes unbeaded, short, and coccoid. Duval also noted that these changes in morphology were always followed by rapid multiplication of the organism. Duval argues, a priori, that the bacillus is not in a favorable environment in the human tissues. If these deductions are correct, the morphology of the leprosy bacillus should vary according to the resistance offered by the tissues of different animals. The resistance of the human host to the leprosy bacillus becomes more evident in the light of the clinical aspect of the disease. The long period of incubation, the duration of the disease, and the disappearance of the bacilli preceding the healing of the infected foci show that the resistance offered to the bacillus by the human tissues is not to be overestimated. This opinion is confirmed when the behavior of the leprosy bacillus under cultivation and in the tissues of various mammals is compared. When cats, rabbits, bats, guinea pigs, and rats are inoculated either below the skin or into the peritoneal cavity with large quantities of Bacillus leprae, a slight local reaction follows within twenty-four to forty-eight hours, but no definite lesions are produced and the bacilli soon disappear. The resistance of some animals to Bacillus leprae is well illustrated by two cats which were inoculated subcutaneously and intraperitoneally with a heavy suspension of Bacillus leprae. These animals were killed and examined three days later, but the bacilli were not demonstrable from the regions about the sites of inoculation. Pigeons are likewise refractory. It is impossible to cause a local reaction in these birds, and the injected bacilli disappear rapidly. Hence, probably no multiplication takes place in them. Goats, young pigs, and white and dancing mice are in a degree susceptible to injections, and though undoubted lesions are produced, and multiplication of the bacilli occurs, the lesions and bacilli disappear after a limited time. Acid-fast bacilli which are recovered from the lesions are long, slim, and beaded, though the organisms used in the inoculations were short, unbeaded, and coccoid. Monkeys inoculated with cultures of the short unbeaded forms react promptly. The lesions resulting, though confined in most instances to the site of inoculation, occasionally appear at distant points. The number of bacilli present in the nodules and their arrangement within typical lepra cells show that multiplication has taken place. The organism has, however, changed from the short coccoid form to the long, slender, beaded form. Though the lesions induced and the bacilli present are in every way similar to those found in man, their tendency to disappear gradually after a quiescent stage clearly denotes that the tissues of the monkey, although less refractory than the tissues of the animals previously mentioned, still offer resistance to invasion. While mammals react but poorly to inoculations of the leprosy bacillus, this reaction manifests itself in various ways in different species. For example, while multiplication of the organism with the production of lesions occurs in some species, in others that are more refractory, the injected bacilli assume the involuted or beaded forms and do not multiply or produce lesions; in others, still more resistant to the action of the leprosy bacillus, the organisms quickly undergo granular metamorphosis and disappear. Furthermore, in some species the lesions are, in most instances, limited to the site of inoculation, and though presenting all the characteristics of the lesion in man, the nodules and the bacilli disappear after a variable time. This behavior of the leprosy bacillus can be accounted for only by the degree of resistance offered by the tissues of the individual host. Since the morphology of the organism invariably changes from the short coccoid to the large beaded form when placed in insusceptible animals, and conversely, from the long beaded forms to the short coccoid forms when placed in susceptible animals, the deduction can be drawn that the organism varies in morphology and rapidity of growth according to the susceptibility of the host. Examples of similar behavior of Bacillus leprae in the human subject are known to all investigators of leprosy. Ulcers and nodular areas often heal, and the bacilli disappear with little or no treatment. It is true that while older lesions are healing, new ones are constantly appearing, yet the duration of the disease and its undoubted tendency towards healing shows that conditions in the human subject are variable, and suggests that the organism has its natural habitat in some other host. The experiments presented here serve to show that the bacillus of leprosy meets but little or no resistance in the tissues of cold-blooded animals, multiplies in their tissues, and may be harbored by them without apparent discomfort or external evidence of the disease. That no appreciable resistance is offered to the multiplication of the leprosy bacillus by many species of cold-blooded animals is shown by the fact that aside from the trauma produced by the inoculation and the slight initial reaction of the tissues, the organism continues to grow profusely, and to invade the tissues without further reaction. Quite the opposite condition occurs in mammals: in some of these the leprosy bacillus degenerates into a granular mass shortly after inoculation; in others that are less refractory, typical lesions appear, but they seldom extend from the point of inoculation; and while the bacilli multiply slowly, they do not infiltrate the tissues, but disappear after a short time, the lesions healing. That multiplication of Bacillus leprae occurs in the tissues of cold-blooded animals is shown by the fact that while animals examined a few days after inoculation show but a few scattered organisms, those killed at longer intervals show a proportional increase in the number of bacilli. Furthermore, the few bacilli found at the early-period are extracellular and scattered, while after longer periods they tend to be massed and enclosed in large lepra cells. The supposition that these lepra cells are phagocytes has naturally arisen. Duval holds that they are not phagocytes in the true sense of the term, that the bacilli penetrate the cells rather than that the cells engulf them, after which, finding conditions for growth favorable, they multiply without causing serious injury to the cell. The size of the cell depends upon the size of the colony within. The experimental work bears out this view since the decrease in number of the organisms observed in animals killed shortly after inoculation depends not upon phagocytic action nor upon cells which appear later when active lesions are established. In early lesions, the lepra cells are smaller, barely measuring twenty to thirty microns in diameter, and contain but few bacilli; whereas in older ones, they attain a diameter of 100 microns or even more, and contain enormous numbers of bacilli. Were this increase in size due to phagocytic action, some cells would be found in which the limit of their capacity had been reached; and they would either contain a mass of dead and disintegrated bacteria or would themselves show evidence of disintegration. On the contrary, the bacilli, though they occupy most of the cell, show no signs of disintegration, and the nucleus and the cytoplasm of the cell retain normal staining properties. That the invasion and multiplication of the bacilli cause an irritation is evident by the amitotic divisions of the nucleus which occur in the larger cells. The absence of external evidence of invasion by Bacillus leprae in cold-blooded animals, and the apparent lack of discomfort caused by the presence of the organism within their tissues, are points which should be remembered in considering the sources from which leprosy may be transmitted. In not a single instance in the numerous experiments presented here would it have been possible, from any external sign, to suspect that the animals were harboring multitudes of leprosy bacilli. While the evidence in support of the opinion that leprosy may be transmitted from man to man appears sufficiently strong to warrant this belief, the number of cases in which infection can be actually traced to this source is small. Since leprosy is known to be prevalent where fish and sea-food are plentiful, and since the experiments here recorded prove that fish can be infected by being fed cultures of Bacillus leprae, or nodules from human lepers, or bits of fish previously infected with the leprosy organism, account should be taken of the possibility that leprosy, in certain localities, may arise from this source of infection. The question as to how and from what source leprosy bacilli enter the human body may be still regarded as an open one. Isolated examples of direct infection of healthy human beings from lepers have been reported by Arning and Nonne, by Manson, and others. The notion that the agency of infection is already infected human beings, that is lepers, is at the foundation of the modern practice of the isolation and segregation of lepers, which would seem to have brought about a definite decrease in the prevalence of the disease. It is an acknowledged fact, however, that the lepers confined in institutions practically never cause infection of nurses, etc. Some other factor than the human agency may therefore be considered as affecting this issue. It is well known that Jonathan Hutchinson has brought forward the idea that fish are the source of the infection, basing the view on the high prevalence of the disease along the coast countries of Norway and Sweden, and in the Pacific Islands, and in the countries bordering the Mediterranean and Black Seas, in all of which fish furnish the chief food material. No convincing proof was ever adduced in support of this contention. But now that it has been shown that the leprosy bacillus survives and multiplies in cold-blooded animals, at least at room temperature in a warm climate, and since methods have been devised for cultivating and identifying the leprosy bacillus, the question has been opened up to accurate investigation. Duval has shown that the leprosy bacillus in cultures grows better at room temperature than at 37 degrees C., so that growth in cold-blooded animals kept at room temperature is perhaps in some way connected with this phenomenon. What must now be ascertained, in order to test the Hutchinsonian theory more accurately is whether such growth takes place at a temperature corresponding with the average mean temperature of such a body of water as the North Sea and that of the fiords of Norway. Since cold-blooded animals possess the same temperature as their surroundings, they would be suitable media for the cultivation of leprosy bacilli at those temperatures. For the waters of the Mediterranean Sea and the tropical Pacific Ocean, this consideration would count less. But the theory will stand or fall according as it can account for the whole, and not only for a part, of the phenomena to be explained. As the length and shape of the bacilli and the number of chromatin masses are constant for a given species of cold- or warm-blooded animals, which features are governed by the resistance of the individual species, the following conclusions seem justified: that the morphology and rapid multiplication of the leprosy bacillus in cultures and in some species of cold-blooded animals indicate that Bacillus leprae under natural conditions is short, coccoid, and un-beaded, and that the long, slender, beaded variety which occurs in the mammalian species is atypical and the product of an unfavorable environment.

From the foregoing description of the histological changes in the leptomeninx it is quite evident that we are dealing with a chronic, stationary, healing form of tuberculous inflammation. This statement is substantiated, in the first place, by the clinical history. The only reasonable interpretation of the symptoms would establish the duration of the process as four months. The imaginable contingency that there existed first a meningeal syphilitic lesion that was dispersed by the iodide of potassium only to be followed by a tuberculous infection is so remote and unlikely that it need not be discussed. At all events the tuberculous leptomeningitis, which presented a typical distribution, began insidiously, existed at times in a latent condition, and pursued a very anomalous course, marked by a relative mildness of all the symptoms, and thus it came about that when an apparent or real improvement followed the administration of iodide of potassium able observers were induced to make an erroneous diagnosis. Death occurred as a result of an intercurrent infection. The long duration of the process is also shown, anatomically, by the thick layer of firm, translucent and gelatinous material that matted together the structures at the base, and also by the evident adhesions between the pia and the brain. The histological examination furnishes proof positive of the correctness of the conclusion in regard to the peculiar character of this process because it shows:(1) That the tuberculous proliferation is uniform in development and has reached nearly the same stage of evolution throughout the entire extent of the leptomeninx involved; it is not a process that has advanced by exacerbations and irregular extensions; the lesions are, generally speaking, of nearly the same age everywhere and must have begun at about the same time.(2) That only a very limited degree of caseous degeneration is present, pointing to an early arrest of the activity of the tubercle bacillus or to a very decided diminution or attenuation of its virulence.(3) That the subendothelial intimal proliferations of epithelioid cells, so generally found in acute tuberculous leptomeningitis,* have in this case become more or less completely changed into distinct fibrous tissue in which but very slight, if any, direct evidence of its tuberculous origin can be found. It is only by recognizing that the chronic endarteritis is most marked in correspondence with the most advanced adventitial tuberculous changes, and by finding an imperfect, much altered giant cell in one district of intimal thickening, that we were able to establish the direct kinship of the endovascular changes with those of the pia in general.(4) That acute inflammatory changes, in the form of emigration of polymorphonuclear leucocytes and of fibrinous exudation, are entirely absent in all parts of the district involved. The presence of a turbid serous fluid is of course not at all inconsistent with the view that the anatomical changes are of long duration.(5) That the granulation tissue present is, in general, undergoing fibrillation and contains a rich supply of enabryonal capillary vessels as well as of larger blood-vessels of evidently new formation. The absence of any considerable extent of polymorphonuclear leucocytic infiltration in this tissue has already been referred to. The cells in the granulation tissue correspond to the cells of embryonal or formative connective tissue. Vacuolation is rarely present.(6) That the unusually large number of giant cells present are remarkably free from evidences of necrosis and degeneration of the character ordinarily observed in tuberculous proliferations, that they do not contain in demonstrable form tubercle bacilli, and that the majority of the giant cells seem to be separating into individual cells and smaller masses often with, but sometimes also without, evidences of nuclear disintegration. The possibility that these phenomena may signify fusion instead of the sundering of cells will be discussed below. For these reasons there can be no doubt that the general claim that we are dealing with an instance of chronic, healing tuberculous meningitis must be regarded as established beyond dispute. The growth of tubercle bacilli in the glycerine-agar tubes, inoculated with the fluid from the pial meshes, and the demonstration of tubercle bacilli, though in very small numbers, between the cells of the embryonal tissue, furnish the positive evidence that we are actually dealing with a tuberculous process due to living and not to dead bacilli. The degree of virulence of the cultures of tubercle bacilli was, unfortunately perhaps, not studied. The presence of living tubercle bacilli in a tissue free from active and acute changes characteristic of tuberculosis demonstrates that, whatever the actual degree of virulence of the bacilli may have been, the tissue in which they were found was at this time relatively immune from their action. The manner in which this immunity was produced, and in which the process of healing was initiated, need not be discussed at this time any further than to again direct attention to the fact that the bacilli lost their virulency as regards the cells in this leptomeninx before these cells underwent any marked degree of degeneration. The cells of the tuberculous proliferations survived the further action of the bacilli whose original effect it was to initiate cell accumulation or proliferation; the cells also retained sufficient vitality to develop, in some instances at any rate, into formative cells according as their origin would dictate, e. g. into fibroblasts. That fibroblasts are formed only by embryonal connective tissue cells, and not by wandering cells, such as the large mononuclear leucocytes, we are well aware, is possibly still a disputable assumption, and we do not consider it pertinent to discuss the question any further in connection with this study, but would only emphasize the point that some of the cells of tuberculous proliferations may, under favorable circumstances, become formative cells, and, furthermore, that the amount of formative tissue produced may be far in excess of what is actually needed for purposes of repair only. Surely the appearances here noted indicate that the bacillus of tuberculosis has the power to stimulate fixed cells to multiply, unless one assumes that all, or almost all, the formative cells here seen are derived from wandering cells attracted by the presence of the bacillus and its products. As to the ultimate fate of the formative and other cells in this healing tuberculous tissue no final statements can be made. It must be remembered that it is only one stage in the process of healing that is dealt with. The well marked evidences of fibrillation, the quite extensive formation of new vessels, the absence of evidences of degenerative changes in the uninuclear cells, all point to the production of new fibrous tissue as sure to occur, but it seems quite probable that occasional epithelioid cells may undergo or have undergone dropsical or other forms of degeneration, although it is certainly apparent that so far as the small cells are concerned the involution of the tuberculous tissue is not occurring through disintegration. Perhaps the most interesting feature in this case is the opportunity it affords to study the changes in the giant cells of healing, non-degenerated tuberculous tissue. In the first place, the large number of giant cells is quite remarkable. The general characters of the tissue in which they are found recall the fact that giant cells are regarded as quite constant elements in chronic mild tuberculosis; often the giant cells are the only cells that contain bacilli (Koch). In this instance the giant cells do not contain bacilli that are demonstrable by the usual methods; neither do they contain bodies that can be definitely interpreted as degenerate forms of bacilli such as those found by Metchnikoff, Stchastny, Weicker, and others, in the giant cells of Spermophilus guttatus, in avian and in human tuberculosis. Metchnikoff states, however, that he knows of the occurrence of such degenerate forms only in the Spermophilus guttatus under the circumstances mentioned, and in the rabbit and guinea-pig in mammalian tuberculosis, but not in man; consequently, the manner in which the giant cells rid themselves of the bacilli undoubtedly present in their interior at some time during their existence, must as yet remain without any explanation. In the description of the histological changes the various appearances presented by the giant cells are described somewhat minutely. The essential observations made concern, in my opinion, the further fate of giant cells which are still found to persist in healing nondegenerated tuberculous tissue. It was, I believe, quite conclusively shown that the consecutive changes appear to consist in the breaking up of the nuclei, the removal of the detritus by phagocytes, and the formation of a few apparently viable uninuclear cells in the case of more degenerated, exhausted giant cells, while other, and, as it would seem, better preserved or younger giant cells, separate into a number of individual, uninuclear cells with but little or no nuclear disintegration. Objection might be raised to this interpretation of the appearances in the giant cells. While no one could very well dispute the view that part of the giant cells are undergoing retrogressive and absorptive changes with the production of some viable cells, a question might well be raised concerning the nature of the process taking place in those giant cells that have been spoken of as splitting up or dividing into uninuclear cells and smaller multinucleated masses without much evidence of nuclear disintegration. It might be claimed that the process is one of fusion of many cells to form giant cells, and not one of division of fully formed giant cells into small cells. But a broad view of the processes described speaks against fusion. In the first place we are not dealing with a stage of tuberculous proliferation (Baumgarten), or cell accumulation (Metchnikoff), in which one would look for the production of giant cells, no matter which view concerning the histogenesis of tubercle be assumed as the correct one, because it has been demonstrated that, from whichever point of view the lesions are examined, the same positive conclusion that they are in the process of healing is reached; there is, therefore, no occasion for the formation of new giant cells in such wide-spread degree throughout the district involved. It might he claimed that the cells became arrested and, as it were, fixed in the act of fusion which was taking place in the early stage of the meningitis, but it would be difficult to understand the nature of the stimulus that could hold the cells together in such a peculiar manner for such a long time. It must be remembered that bacilli or bacillary detritus could not be found among the incomplete or in the complete giant cells. In the second place the difference between the cells that are undergoing disintegration and those regarded as dividing is essentially, to a certain extent at any rate, one of degree, because in the first instance there is not much, if any, doubt but that viable smaller cells are also formed, and in the second instance some, though often very slight, evidence of nuclear fragmentation is nearly always present; it would also be correct to infer that in advanced subdivision of a giant cell much, and perhaps all, of the nuclear detritus produced might have been removed up to the last trace; finally, the two extremes of these changes in the giant cells are connected by transition stages passing by gradation from the one to the other. Hence it is justifiable to conclude, for the time being, that in healing non-degenerated tuberculous tissue, the multinucleated giant cells may in part disintegrate and undergo absorption, in part form viable small cells; that both these changes may, and usually do, affect the same cell, but that in one class of cells-presumably the older or the more exhausted-the retrogressive process is predominant, while in a second class of cells-presumably the young and vigorous-the progressive changes are the more marked. In this connection it may be pointed out that while there cannot very well be any question but that we are dealing only with dividing and not coalescing cells, yet if this conclusion should be disputed and found incorrect, then the only remaining alternative would be to infer that this tissue furnished a unique and striking example of the formation of plasmodial masses by fusion in human tuberculosis, a conclusion to which many pathologists would refuse to subscribe, if for no other reason than because it is not in accordance with the almost universally accepted teachings of Baumgarten and Weigert in regard to the mode of formation of the giant cells in tuberculosis. Believing as I do that the giant cells under consideration are in the act of division and not at all of fusion, there remain to be discussed some of the histological and other features presented by the dividing cells. Many of the giant cells, perhaps the majority, contain larger and smaller vacuoles in the protoplasm. The exact significance of this vacuolation is not always clear. When the vacuolation accompanies an evident solution of the nucleus (karyolysis), there cannot be any doubt but that we are in the presence of a distinctly retrogressive process. Vacuoles are also most numerous in the giant cells that present other evidences of degeneration, such as coarseness of the granules in the protoplasm and extensive nuclear disintegration, but they occur as well around nuclei that stain deeply, around cells that seem to be separating from the giant cell, and even about nuclei that present mitoses. The formation of vacuoles seems to be responsible, to a certain extent at any rate, for the diminution in the volume of disintegrating and dividing giant cells, as shown by the clear spaces that form about them; these spaces are too large and occur too uniformly to be attributed solely to artificial shrinking produced by the hardening in alcohol. Further undoubted evidence of retrogression in certain giant cells is the occurrence of nuclear disintegration, or karyorhexis, which sets free larger and smaller chromatin masses that are recognized in the giant cell as well as in the interior of the phagocytes usually found around such cells. Almost all the polymorphonuclear leucocytes found in this tissue are met with around giant cells with broken-up nuclei. In many nuclei of disintegrating giant cells can be noted appearances that correspond well to certain stages in the complicated karyorhexis observed in anaemic necrosis by Schmaus and Albrecht; some of the nuclei with budding processes correspond particularly well with those in certain of their drawings; the interior of giant cells of tuberculous tissue may, it would seem, present conditions favorable to the development of this series of postnecrotic nuclear change. Vacuolation, karyolysis and karyorhexis are the essential steps that lead to destruction of the whole or parts of some of the giant cells; associated with these processes there is usually observed a splitting up of the body of the giant cell into irregular fragments with as well as without nuclei; and, as described, more or less phagocytosis of the resulting remnants of various kinds is seen. But evident degenerative and necrotic processes in a giant cell may be associated with progressive changes. While some nuclei undergo vacuolation or break up, others seem to become richer in chromatin and to stain more deeply at the same time that they seem to acquire cell bodies quite distinct from the protoplasm of the giant cells: this hyperchromatosis does not, therefore, seem to be a stage in karyorhexis. A very few but undoubted karyokinetic figures were found, together with evidences of division of the cell body formed in the giant cell protoplasm. Precisely similar changes are described by Klebs in healing pulmonary tuberculosis of the guinea-pig; the nuclei of the giant cells became rich in chromatin and karyokinetic figures occurred. Krückmann among others has found occasional mitoses in giant cells around foreign bodies, as well as elsewhere, but it would seem that such mitoses have always been interpreted as indicating the probable mode of formation of the giant cells rather than of their involution. The question of mitosis in existing multinucleated cells has recently been studied by Krompecher, who concludes that the individual nuclei of such cells may undoubtedly divide by mitosis, either simultaneously or at separate times. Division by amitosis can also occur, but mitosis is the only progressive form of division, amitosis being a retrogressive, disintegrating process that must be looked upon as an evidence of degeneration of the nucleus. Ziegler states that in division of giant cells whose nuclei have multiplied by mitosis it may happen that the separating cell remains enclosed in the protoplasm of the mother cell. A singular phase in the involution of the giant cells in this pia is to be found in the existence of progressive changes side by side with nuclear necrosis and with degeneration; this finding indicates that giant cells may contain many independent elements which, though apparently fused into one large cell, may preserve their individuality so that while some nuclei die, others proliferate and perhaps feed on the remnants of their dead brethren and form new, viable small cells. The nuclei in giant cells may be looked upon as representing independent centres, capable at times of existing even though the cell protoplasm is disintegrated. Many of the giant cells separate into individual cells, unaccompanied or unassociated with much evidence of necrosis. These cells may be regarded as the more vigorous forms. Here also are observed occasional mitoses-but on the whole extremely few-and very constantly an evident increase in the amount of chromatin in the nuclei of the new cells as compared with the amount ordinarily found in the nuclei of giant cells. These deductions concerning the persistence of the vitality of some of the nuclei, even in the presence of molecular and morphological changes in the cytoplasm and in other nuclei of the giant cell that lead to disintegration, are not entirely without the support of previous observations on cells, which, although made under different conditions, are nevertheless, it would seem, applicable to cells in general. Thus the brilliant investigations of Loeb upon the effects of various unfavorable surroundings, such as absence of oxygen or reduction of the amount of water, upon the cleavage of eggs of many kinds, show that the conditions which arrest development are qualitatively alike for nucleus and protoplasm, but quantitatively less for the protoplasm; when the irritability of the protoplasm is suspended the nucleus may segment without segmentation of the protoplasm, but upon re-establishment of favorable conditions the protoplasm may divide into about as many spheres as there are nuclei preformed-the nucleus persists, preserves the irritability of the cell and stimulates the protoplasm to segmentation. From the appearances of the giant cells here described it would seem, then, that some nuclei are able to maintain their vitality longer than others in the same cell, and under certain conditions to stimulate parts of the protoplasm to segment; in other cells all the nuclei have, as a rule, preserved their irritability. The groups of cells formed by the dividing of the giant cells can be traced by studying the process at the different stages in the different parts of the tissue. They assume an oval or spindle-shaped form, becoming more and more like the formative and endothelioid cells of young connective tissue, but their ultimate fate cannot be determined because it concerns essentially only one limited period in the involution of the tissue. It may be said with reasonable certainty, however, that the new cells do not form blood-vessels, but as regards their forming lymph-vessels nothing definite can be concluded. It would not be safe to draw any definite conclusions, from the appearances described, with regard to the origin and the mode of formation of the giant cells. The resulting small cells in general resemble very much endothelial and formative cells, but some of them are, at certain stages at any rate, not unlike large mononuclear leucocytes; their final fully developed or mature condition being unknown, no positive inference can be drawn as to their pre-giant-cell origin. The evidence points to the fact that the most probable origin of the giant cells, as indicated by their form and the apparent future career of their descendants, would be the fixed mesoblastic cells of the pia. In regard to the mode of formation of the giant cells it is quite clear that it must involve some process which is not incompatible with the viability of the small cells which may spring from the giant cells. Whether this would speak more in favor of formation by fusion than by karyokinesis of a single cell without division of the cell body cannot be well determined, and as long as authors are not agreed upon the question of the production of living, procreative cells by amitosis (direct segmentation, direct and indirect fragmentation) it would not be profitable to discuss the compatibility or incompatibility of the views of those investigators who trace the origin of giant cells to amitotic division, with the progressive changes that giant cells have been shown to be capable of. The fact that giant cells in tuberculous tissue, under certain conditions, undergo progressive changes and separate into small, living cells proves that they are not, as claimed by Baumgarten, Weigert and others, necrobiotic elements that are doomed to destruction from their very inception. On the other hand it lends more strength, if that were necessary, to the teleological view urged by Metchnikoff that they are living, defensive cells (whatever their origin may be), formed for the distinct purpose, like plasmodial masses in general, of isolating and removing foreign, harmful bodies, in this case the tubercle bacillus, and, having accomplished their object without being destroyed or exhausted, or the cause of their formation being removed or neutralized in some way, they, or their nuclei, may retain enough irritability to form a larger or smaller number of living, small, uninuclear cells.

Age changes of NADPH-diaphorase activity were studied histochemically in the ventral horn motor neurons at different segmental levels of the spinal cord of rats aged 3-90 days both under normal conditions and in the model of deafferentation (by intraperitoneal capsaicin injection). Wave-like age changes of motor neuron enzyme activity were detected at the level of T(II), L(IV) and S(II) spinal segments with its increase by day 60 followed by a significant decrease to day 90. Age dynamics of NADPH-diaphorase activity development in the spinal cord motor neurons of intact rats characterizes the constructive processes in neurons, while the changes found after the deafferentation are indicative of the motor neuron damage and are manifested by an abrupt increase of the enzyme activity at the age of 90 days.

On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of kymograph columns. The applicability of the method was measured on simulated kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR.

During normal vertebrate development, Hoxd10 and Hoxd11 are expressed by differentiating motoneurons in restricted patterns along the rostrocaudal axis of the lumbosacral (LS) spinal cord. To assess the roles of these genes in the attainment of motoneuron subtypes characteristic of LS subdomains, we examined subtype complement after overexpression of Hoxd10 or Hoxd11 in the embryonic chick LS cord and in a Hoxd10 loss-of-function mouse embryo. Data presented here provide evidence that Hoxd10 defines the position of the lateral motor column (LMC) as a whole and, in rostral LS segments, specifically promotes the development of motoneurons of the lateral subdivision of the lateral motor column (LMCl). In contrast, Hoxd11 appears to impart a caudal and medial LMC (LMCm) identity to some motoneurons and molecular profiles suggestive of a suppression of LMC development in others. We also provide evidence that Hoxd11 suppresses the expression of Hoxd10 and the retinoic acid synthetic enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In a normal chick embryo, Hoxd10 and RALDH2 are expressed throughout the LS region at early stages of motoneuron differentiation but their levels decline in Hoxd11-expressing caudal LS segments that ultimately contain few LMCl motoneurons. We hypothesize that one of the roles played by Hoxd11 is to modulate Hoxd10 and local retinoic acid levels and thus, perhaps define the caudal boundaries of the LMC and its subtype complement.

We describe an algorithm for the efficient annotation of events of interest in video microscopy. The specific application involves the detection and tracking of multiple p ossibly overlapping vesicles in total internal reflection fluorescent microscopy images. A st atistical model for the dynamic image data of vesicle configurations allows us to properly weight various hypotheses online. The goal is to find the most likely trajectories given a sequence of images. The computational challenge is addressed by defining a sequence of coarse-to-fine tests, derived from the statistical model, to quickly eliminate most candidate positions at each time frame. The computational load of the tests is initially very low and gradually in creases as the false positives become more difficult to eliminate. Only at the last step, state variables are estimated from a complete time- dependent model. Processing time thus mainly depends on the number of vesicles in the image and not on image size.

The proximodistal axonal transport of choline acetyltransferase (ChAc) has been studied in the chick sciatic nerve in the absence of any experimental manipulation. The phenomenon utilized by us is a physiological transient fall in the activity of ChAc. This fall occurs in most areas of the chick central nervous system after hatching, and moves toward the periphery along the nerves. Having ruled out the presence of transient inhibitors, the movement of the fall towards skeletal muscles has been considered to correspond to the proximodistal transport of ChAc. The calculated average velocity is 7.86 mm/day, corresponding to the slow rate of transport. Nevertheless, the kinematics study suggests that the velocity varies along the nerve from a state of rest to an intermediate rate of transport (16 mm/day). The possible influence of variations in the resistance of the conductor on these changes in velocity is discussed.