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Recovery of hippocampal dentate granule cell responsiveness to entorhinal cortical input following norepinephrine depletion.

Hippocampal dentate granule cell responsivity to excitatory input from entorhinal perforant path fibers was examined in the chronic rabbit preparation following norepinephrine (NE) depletion induced with the neurotoxin DSP4. To examine granule cell responsivity as a function of perforant path activation, constant low frequency stimulation (0.1 Hz) was applied to the perforant path using an ascending intensity series. To examine granule cell responsivity to more complex patterns of stimulation, a train of impulses, with a random interstimulus interval (Poisson distribution; mean frequency of 2 Hz), was applied to the perforant path. Both single impulse and random interval impulse stimulation revealed that NE depletion increased the average amplitude of the perforant path-granule cell population spike. The random interval impulse stimulation revealed that NE depletion also increased the magnitude and duration of second order inhibitory interactions. These changes were transient, however, and recovered over the 21 day test period. Hippocampal NE levels were reduced an average of 80% between 23 and 38 days post-DSP4. The activity of the rate-limiting enzyme for NE synthesis, tyrosine hydroxylase (TH), was reduced an average of 60%. That NE levels were reduced to a greater extent than was TH activity is suggestive of increased NE synthesis within the remaining nerve terminals. Such an increase in NE synthesis may reflect a compensatory response underlying the functional recovery of electrophysiological responsiveness following partial NE depletion.

Adrenergic Agents↗

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines.

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4+ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.

Antibodies, Monoclonal↗

A simplified and rapid limiting dilution assay of T lymphocytes.

We report here the development of an alternative limiting dilution assay (LDA) of T lymphocytes (T cells). Blood mononuclear cells were first stimulated for 60 hr with PHA and then cultured in microwells in the presence of recombinant interleukin-2 without feeder cells. After 4 days of culture, wells were scored for proliferation. Clonal expansion of T cells followed the single-hit model of the Poisson distribution. The progenitor frequency (f) in the mononuclear cells and E-rosette-positive cells from normal donors were 0.082 +/- 0.025 (n = 12) and 0.236 +/- 0.029 (n = 5), respectively, but this was markedly decreased in patients who underwent marrow-ablative chemotherapy and autografts with blood hematopoietic stem cells. This LDA system should be of value in routine use for the evaluation of T cell proliferative activities.

Bone Marrow Transplantation↗

A third order iterative procedure for computing exact confidence limits for Poisson expectation values.

The calculation of exact confidence limits for the expectation value of a Poisson distribution when a single observation of K events is given by means of a second order iterative algorithm was recently presented. Here it is shown that the structure of the equations to be solved admits the use of a third order algorithm, thereby significantly reducing computation time, especially for pocket calculators, with practically no additional computational effort.

Computers↗

Progenitors of oligodendrocytes: limiting dilution analysis in fetal rat brain culture.

In this paper, we describe the use of a combination of cell culture techniques and limiting dilution analysis to determine the number of oligodendrocyte progenitor cells and the oligodendrocyte clone size in primary dispersed cultures of 20- to 21-day-old fetal rat brain. Single-cell suspensions (1, 2, 3 x 10(6) cells/ml) were plated in either microwell or 100 mm dishes. After 22 days in culture the number of differentiated oligodendrocytes was ascertained by determining the amount of myelin basic protein by radioimmunoassay. The total amount of myelin basic protein was the same in the two types of dish, indicating that proliferation and differentiation were unaffected when oligodendrocytes were grown in microwells. The fraction (F0) of microwells containing no oligodendrocytes was determined at each cell dilution. F0 decreased exponentially with increasing total cell concentration. The linearity of the plot of ln F0 versus cell number indicates that the number of oligodendrocyte progenitor cells is limiting. From the equation describing the Poisson distribution of progenitor cells in microwells we calculate that, at the time of plating, primary cultures of fetal rat brain contain one oligodendrocyte progenitor cell per 1.3 x 10(5) brain cells, or a total population of 300-500 progenitor cells per brain. The mean oligodendrocyte clone size was determined to be approximately 825 at 22 days and close to 2000 by 35 days in culture. Therefore, each progenitor cell must undergo approximately 11 divisions, on the average, during postnatal development.

Animals↗

Activity of preoptic neurons during synchronization and desynchronization.

Extracellular unit activity from 29 neurons in the preoptic area was recorded together with the cortical EEG in encéphale isolé cats. A majority (55%) of neurons showed alterations in their firing rates during transient changes in the EEG. Among them, a majority (62.5%) showed an increased firing rate during synchronization and the remaining showed an increased firing rate during desynchronization of the EEG. Most neurons showed a Poisson distribution pattern of firing during both the synchronized and the desynchronized phases of the EEG. The changes in the neuronal discharge occurring together with the specific changes in the cortical EEG fits in well with the assigned role of the preoptic area in the sleep-waking cycle.

Animals↗

Computer simulation of the neural discharge carried by the abducens nerve during eye fixation in the cat.

The neural signal carried by the abducens nerve during eye fixations was simulated. The neural discharge was defined by the number of spikes carried by the abducens nerve within each ms. Calculations were based on real neurophysiological data. The computed neural signal showed frequency histograms and variance/mean ratios typical of a Poisson distribution. A peak was obtained in the power spectral density function of the simulated neural signal. This peak appeared in the frequency range corresponding to the firing rate of single motoneurons for each eye position. It remained as a broad spectral peak after filtering by a second-order differential equation simulating the ocular mechanics. The obtained spectra are similar to the described power spectral density function of eye position recordings. Present results add evidence of a possible neural basis for ocular tremor.

Abducens Nerve↗

Quantitative assessment of atherosclerotic lesions in mice.

The well-defined genetic systems of the mouse are proving useful in experimental studies of atherosclerosis. Inbred mouse strains differ in atherosclerosis susceptibility, and several variants of apolipoproteins have been identified and mapped. This report explores the location and timing of lesion formation in the mouse in an effort to provide a basis for quantitatively comparing groups of mice. After 14 weeks on an atherogenic diet containing 1.25% cholesterol, 15% fat, and 0.5% cholic acid, C57BL/6J female mice had aortic lesions at each of the intercostal arteries, at the junction of the aorta to the heart, and in scattered areas covering 1.1% +/- 0.5 (SD) of the aortic surface. After 9 months on the atherogenic diet, those lesions near the heart and intercostal arteries were extensive, 8% +/- 3 (SD) of the remainder of the aorta was involved in lesions, and lesions were found in the coronary arteries. Results indicated that one suitable location for scoring lesions was in a 300 micron area of the aorta just beyond the aortic sinus. The mean number of lesions/mouse in the selected area after 14 weeks on the atherogenic diet was 1.1 +/- 0.3 (SD). The results were reproducible over 10 separate experiments. The number of lesions per mouse fit a Poisson distribution indicating that the presence of one lesion did not predispose the mouse to acquiring a second lesion. Lesion formation and cholesterol levels did not vary with the season of the year as demonstrated by 9 separate experiments over more than 12 months. Methods of evaluating the number and size of lesions were compared including sizing with a microscope eyepiece grid and computer-assisted planimetry. The resulting data provide reproducible methods of quantitatively comparing lesion formation in various strains or groups of mice, thereby increasing the usefulness of the mouse as an experimental system for atherosclerosis research.

Animals↗

Suicide rates among cancer patients in Connecticut.

This study derives age-sex specific standardized mortality ratios (SMR's) among cancer (ca) patients with respect to population suicide rates. Suicide rates were derived for all 144,530 ca patients in the Connecticut Tumor Registry from 1940-1973. We also derived rates by number of years since diagnosis (y-s-dx) and by decade of dx. We hypothesized beforehand that suicide rates will (1) be higher in ca patients than the population; (2) be higher soon after dx than in later y-s-dx; (3) vary directly with spread of disease at dx, highest for metastatic, lowest for local. We address hypotheses (1) and (2) here. After collapsing age groups and y-s-dx in the observed 192 ca patient suicides, SMR's derived for age, sex and y-s-dx were tested with an exact Poisson distribution. Overall, SMR for males was significantly elevated (230, P less than 0.005), but not for females. In males, a test for trend in SMR's with y-s-dx showed a significant decline, supporting hypothesis (2) for males (chi2 = 4.0, df = 1, P less than 0.05). No trend was found for decade of dx. On combining decades, male SMR's were significantly elevated in 10 of the 25 cells of age by y-s-dx, while none of the female cells was significant. We conclude that both hypotheses were supported for males, and neither for females.

Adolescent↗

Limit-dilution assay and clonal expansion of all T cells capable of proliferation.

A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of the expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using [3H]TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.

Animals↗

A new assay suitable for enumeration of murine progenitors of granulo-monocytes and for rapid automated assessment of granulo-monocyte growth factors.

A simple and reproducible assay is described for enumerating the progenitors of granulocytes (G) and monocytes (M) present in either the bone marrow or spleen of mice. This assay is based upon the plating of different dilutions of test cells in the wells of Terasaki plates. Negative and positive wells for presence of G and M are scored 7 days later. Minimal estimates of GM progenitors frequency are obtained by analysis of the Poisson distribution relationship between the percentage of non-responding microcultures and the numbers of cells plated. This liquid microculture assay offers many advantages: (1) the ability to assay directly in situ at clonal level different enzymatic activities like non-specific esterase; (2) the ability to screen within 3 days the presence of GM growth factors by measuring the [3H]thymidine uptake of proliferating responsive cells.

Animals↗

Ishihara test performance and dementia.

Ishihara trail-tracing (TT) and number-naming (NN) tests were administered to a clinical sample of 378 demented patients. Error counts on TT and NN tests were best fit by a negative binomial (overdispersed Poisson) distribution. TT, NN, and combined (TT + NN) error counts were regressed on patient characteristics (sex, age, and education), severity of cognitive impairment (Mini-Mental State Exam: MMSE), dementia stage (Clinical Dementia Rating: CDR), etiology, onset age, and symptom duration in a negative binomial generalized linear model. Patient characteristics, onset age, and symptom duration had no significant effects on any error count. The effects of MMSE, CDR, and etiology, on the other hand, were highly significant and appear to help discriminate vascular dementia from Alzheimer's disease. MMSE (which taps cognitive skills) correlated with both TT and NN errors. CDR (which taps both cognitive and functional skills) correlated only with TT errors and dementia etiology correlated only with NN errors. These distinct correlational patterns reflect differences between the TT and NN tasks (i.e., trail-tracing vs. number-naming) related to specific brain loci and associated functions. This aspect of the phenomenon suggests that Ishihara tests have useful clinical applications in dementia.

Aged↗

Adaptive response of human lymphocytes for the repair of radon-induced chromosomal damage.

In human lymphocytes low doses of X-rays can decrease the number of chromatid deletions induced by subsequent high doses of sparsely ionizing X-rays. Because of the concern with the carcinogenic effects of low doses of alpha-particles from radon in homes, experiments were carried out to see if low doses of X-rays could also decrease the yield of chromosomal aberrations induced by subsequent exposure to radon. Human peripheral blood lymphocytes were irradiated with low doses of X-rays (2 cGy) at 48 h of culture, exposed to radon at 72 h of culture, and analyzed for the presence of chromatid aberrations at subsequent intervals. The frequency of chromatid aberrations induced by radon alone increased with time after exposure, indicating exaggerated differences in the stage sensitivity of cell cycle stages to high-LET radiation. Furthermore, the numbers of aberrations per cell did not follow a Poisson distribution but were over dispersed, as might be expected since high-LET radiations have a high relative biological effectiveness compared with low-LET radiations. Nevertheless, lymphocytes exposed to 2 cGy of X-rays before radon exposure contained approximately one-half the number of chromatid deletions compared with lymphocytes treated with radon alone and analyzed at the same time. Thus, the putative chromosomal repair mechanism induced by low doses of sparsely ionizing radiation is also effective in reducing chromosomal aberrations induced by radon, which hitherto had been thought to be relatively independent of repair processes.

Adaptation, Physiological↗

Induction of chromosomal aberrations by camptothecin in root-tip cells of Vicia faba.

When root-tip cells of Vicia faba were exposed during early and middle interphase to camptothecin (Cpt), the aberrations obtained were exclusively of the chromatid type and tended to be localized in late replicating heterochromatic regions of the chromosomes. In these respects the clastogenic effect of Cpt resembles that of agents that act by an S-phase-dependent mechanism. In contrast to typical S-phase-dependent agents, Cpt produced lesions capable of giving rise to aberrations only in S-phase cells that were synthesizing DNA at the time of treatment. The dependence on ongoing DNA synthesis was suggested in autoradiographic experiments and by the fact that the clastogenic effect of Cpt was strongly suppressed by hydroxyurea, an inhibitor of DNA synthesis. After Cpt treatments, there were many more cells with 3-12 aberrations and far fewer cells with 0, 1 or 2 aberrations than expected on the basis of a Poisson distribution. Cpt further differed from typical S-phase-dependent agents by being capable of inducing lesions in the G2 phase that give rise to chromosomal aberrations in the first mitosis after treatment. This effect was obtained at Cpt concentrations around 10 microM, whereas only 0.03 microM Cpt was required to produce chromatid aberrations in the S phase. Results of G2-phase experiments with Cpt and 5-fluorodeoxyuridine, an inhibitor of DNA synthesis, suggest that DNA synthesis is required for the clastogenic effect of Cpt not only during the S phase, but also during the G2 phase, although the DNA syntheses involved are both quantitatively and qualitatively different.

Autoradiography↗

Quality control in tuberculosis bacteriology. 2. The origin of isolated positive cultures from the sputum of patients in four studies of short course chemotherapy in Africa.

During intensive bacteriological follow-up of 2123 patients who had received short course chemotherapy regimens in 4 controlled clinical trials in Africa and had not had a bacteriological relapse, 405 isolated positive cultures were obtained from 37 429 sputum specimens in 3 East African laboratories. These cultures might have arisen as a result of clerical error, of transfer of bacilli from positive to negative specimens in the laboratory or from the lesions of the patients. Clerical error in the labelling of specimens or the recording of results did not seem a frequent cause, since isolated positive cultures contained many fewer colonies than cultures from other positive specimens being processed at the same time. Several of evidence suggested that some isolated positive cultures arose from the lesions of patients: a decrease in their incidence occurred in successive time periods after chemotherapy; the number of isolated positives per patient departed significantly from the Poisson distribution; they were more often drug resistant than other cultures processed at the time; positive cultures were obtained less frequently from known autoclaved specimens inserted among the the study specimens than from the study specimens themselves; no association was found between the incidence of isolated positive and of specimens containing numerous viable M. tuberculosis being processed at the same time. Nevertheless some of these cultures probably arose by transfer in the laboratory, since the rates at which transfer were known to occur differed in the 3 laboratories and corresponded to the rates of obtaining isolated positive cultures.

Africa, Eastern↗

Foveal cone thresholds.

The method of constant stimuli was used to estimate the psychometric functions for detection of one or two flashes when two light pulses were presented. The test stimulus consisted of two simultaneous 0.5 msec, 1' pulses separated by 17'. Observers reported seeing 0, 1 or 2 flashes. A computer-controlled direct-view apparatus allowed sampling of slightly different foveal locations on each trial. The data were analyzed assuming a binomial probability for sampling of L and M cones and Poisson distributed quantal fluctuation. Under these assumptions, the measurements imply that detection requires a minimum of 5-7 quanta absorbed per cone, and that the effective number of cones illuminated by the 1', 0.5 msec pulse is two. The estimated L/M cone ratio was 1.6 for one observer and 4.0 for the other; each observer's ratio was in general agreement with the value estimated independently by heterochromatic flicker photometry.

Adult↗

Foveal cone detection statistics in color-normals and dichromats.

We measured for six male observers, the psychometric functions for the detection of two simultaneously presented points of light. The test stimuli were two 1 min point sources separated by 17 min arc, and pulsed for 0.5 msec. The stimuli varied in wavelength from 500 to 620 nm. The psychometric functions were fit with a model that assumes ideal detection and the following properties: (1) Poisson-distributed quantal absorptions; (2) binomial sampling of foveal long-wavelength-sensitive (LWS) and middle-wavelength-sensitive (MWS) cones; (3) independent responses of the LWS and MWS cones; and (4) the Smith-Pokorny fundamentals for cone spectral sensitivities. Based on chi 2 fits to the psychometric functions for detecting neither, one or both of the two-point stimuli presented, estimates were derived for the minimum quantal catch by a single cone for detection (C), the number of effective cones illuminated by a point stimulus at threshold (N) and the proportion of central foveal cones of the LWS type (PL). Three observers were color-normal, two were protanopes and one was a deuteranope. A second deuteranope was included in the design but his data were too unreliable for an unambiguous solution. The estimated quantal requirement C was consistently near 5(4-6), and the effective number of illuminated cones always was 1 or 2. The plausible range of PL (98% confidence interval) for the color-normal observers was 0.48-0.68 (observer YY), 0.76-0.90 (observer MW) and 0.77-0.95 (observer DF). The best fitting PL solution for these observers were 0.61, 0.82 and 0.88, respectively. These were comparable to the values obtained from flicker photometric data. The best PL value for each of the protanopes was 0.00 and for the deuteranope the best PL value was 0.98.

Color Perception↗

An oscillation-based model for the neuronal basis of attention.

We propose a model for the neuronal implementation of selective visual attention based on the temporal structure of neuronal activity. In particular, we set out to explain the electrophysiological data from areas V4 and IT in monkey cortex of Moran and Desimone [(1985) Science, 229, 782-784] using the "temporal tagging" hypothesis of Crick and Koch [(1990a) Cold Spring Harbor Symposiums in Quantitative Biology, LV, 953-962; (1990b) Seminars in the neurosciences (pp. 1-36)]. Neurons in primary visual cortex respond to visual stimuli with a Poisson distributed spike train with an appropriate, stimulus-dependent mean firing rate. The firing rate of neurons whose receptive fields overlap with the "focus of attention" is modulated with a periodic function in the 40 Hz range, such that their mean firing rate is identical to the mean firing rate of neurons in "non-attended" areas. This modulation is detected by inhibitory interneurons in V4 and is used to suppress the response of V4 cells associated with non-attended visual stimuli. Using very simple single-cell models, we obtain quantitative agreement with Moran and Desimone's (1985) experiments.

Action Potentials↗