Observational methods to measure behaviors of captive cotton‐top tamarins (Saguinus oedipus)

The purpose of this study was to identify efficient sampling methods for establishing accurate activity budgets for zoo animals. Seven cotton‐top tamarins (Saguinus oedipus) from two zoos were videotaped for multiple 90 min sessions, 3 to 4 days per week for 12 weeks. An activity budget was constructed for each animal using a continuous sampling method to analyze 30 hr of video recording of each animal. These master datasets, reflecting actual behavior, were re‐sampled using interval sampling lengths of 0.25, 0.5, 1, 2, 4, 5, 7, 10, 15 and 20 min, and cluster sampling protocols (periodic sessions of continuous sampling) of 10 min×3, 15 min×2, 20 min×1, 15 min×1 and 10 min×1 (min×repetitions/90 min sample period) to construct additional activity budgets for each animal. The Canberra similarity index was used to determine the statistical relationship between these activity budgets and those based on the master datasets. As interval length increased, there was a consequent decrease in the accuracy of the associated activity budgets as compared with the master dataset. No cluster sampling protocols yielded activity budgets as accurate as the four shortest interval lengths, but all cluster sampling protocols were more accurate than the three longest interval lengths. All the tested protocols varied in ability to accurately portray animal behavior. Overall, interval sampling provided superior behavioral representations at lower observer input. Results from this study will potentially facilitate the standardization of behavior monitoring protocols at zoos. Zoo Biol 29:416–431, 2010. © 2009 Wiley‐Liss, Inc.     

Trade-off between number and length of remote videos for rapid assessments of reef fish assemblages

Remote underwater videos are widely employed to assess the structure and composition of reef fish assemblages but the sampling effort employed on each survey differs considerably, indicating that both the number of assessments and video length could be optimized. We searched for this optimal sampling effort in remote video samples to conduct rapid assessments of community composition and discussed the relation between number of replicates and video length, and how it impacts the method's efficiency to characterize species assemblages. Remote video recordings from tropical reefs in northeastern Brazil were used to investigate how fish species richness and composition builds across time and number of assays. Videos as short as 5 min successfully recorded species richness, requiring about five repetitions to record most species that compose 80% of the total biomass. Recording species composition required even less time in these reefs, setting a minimum of 3 min with the same five videos. By comparing the detected richness per analysed time unit, we found several shorter videos recorded for more species than a few longer videos, indicating that increasing the sampling coverage in the reef area might be better than just extending the video length for rapid assessments.     

The problem of the acts duration measurement in relation to the “focal-animal” technique of behavioral recording

The focal-animal technique assumes the continuous recording of the behavior of an individual during a certain time interval. The length of this interval (sampling unit) can be problematic when estimating the duration of behavioral acts. Two acts from the behavioral repertoire of the ant Leptothorax fuenteiwere focused on in this work at different ranges of temporal scales. Analyzing these acts we show the possibility of existence of a sampling artifact, in such a way that the estimates of the durations of the acts would be forced to depend upon the length of the sampling unit that is being used.     

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact,Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.Download video file.^{(145M, mp4)}     

Observation time and sampling intervals for measuring behavior and interactions of chimpanzees in the wild

This study examined two problems in the measurement of chimpanzee behavior: (1) comparability among data sets varying in length of total observation time; and (2) the longest interval for scoring reliable numbers of sample points with instantaneous sampling (this required procedures for evaluating the chi-square statistics of the sampled data). During a 4.5-month field study conducted at the Mahale Mountains National Park, Tanzania, one adult male was observed as a focal animal for about 300 hr with continuous recording. His behavior was classified into five categories. Data sets varying in total time were prepared by extraction from the raw data. Comparability among the data sets was evaluated using Pearson's correlation coefficients and Kendall's coefficients of concordance calculated from two kinds of measures obtained from the raw and simulated data sets: (a) the percentages of time spent by the focal animal in each behavior category; and (b) those of the time spent by adult males in his proximity. The results revealed that observation time of 25 hr was the critical length for scoring the above measures reliably. Sample points for the focal animal's behavior categories and for adult males in his proximity were simulated with intervals of various lengths for data sets differing in total time. The longest interval was measured by comparing the simulated scores with confidence limits calculated for the number of sample points to be scored with the respective intervals. It was found that the interval for sampling should be set at 3 min or shorter, and that chi-square statistics calculated from the data sampled with such an interval should be evaluated after their modification into the values to be obtained from the data sampled with a 5-min interval. These results may not be directly applicable to studies dealing with other behavior categories, other age/sex classes of focal animals, etc. However, the above problems should be examined widely in studies attempting to measure animal behavior, and the methods employed in this study are applicable to such studies.     

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.Download video file.^{(129M, mp4)}     

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. Download video file.^{(92M, mp4)}     

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents. The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.Download video file.^{(217M, mp4)}     

Size Effects on Correlation Measures

The detection and quantification of long-range correlations in time series is a fundamental tool to characterize the properties of different dynamical systems, and is applied in many different fields, including physics, biology or engineering. Due to the diversity of applications, many techniques for measuring correlations have been designed. Here, we study systematically the influence of the length of a time series on the results obtained from several techniques commonly used to detect and quantify long-range correlations: the autocorrelation analysis, Hursts analysis, and detrended fluctuation analysis (DFA). Using the Fourier filtering method, we generate artificial time series with known and controlled long-range correlations and with a broad range of lengths, and apply on them the different correlation measures we have studied. Our results indicate that while the DFA method is practically unaffected by the length of the time series, and almost always provides accurate results, the results from Hursts analysis and the autocorrelation analysis strongly depend on the length of the time series.     

Effects of Temporal Sampling Patterns of All-occurrence Recording in Behavioural Studies: Many Short Sampling Periods Are Better than a Few Long Ones

All-occurrence recording is generally thought to yield unbiased results. However, its accuracy and power to detect real effects may depend on the temporal patterning of the observation periods. Although several studies evaluated the suitability of time-sampling methods (one-zero and instantaneous sampling), there has not been an evaluation of various temporal patterns of the ‘all-occurrence’ method to date. Here we present an empirical comparison of time-sampling and all-occurrence recording methods, based on a large data set, with special reference to various temporal patterns of all-occurrence recording. The three different sampling methods were imposed on raw data protocols. The scores, obtained by the use of each method and temporal pattern, were compared with true values from the corresponding continuous video recordings. The accuracy and power of each method was estimated by comparison of medians and magnitudes of effect (r_m), respectively. We were able to confirm results of earlier studies on the suitability of time-sampling methods (one-zero and instantaneous sampling), but we found that different temporal patterns of all-occurrence sampling yielded magnitudes of effect that differed from true value magnitudes. We conclude that behavioural studies may reveal different results, even when all conditions are the same, only because of power differences of different temporal pattern of the observation periods, for example 1 × 15 min versus 15 × 1 min. Recommendations are made about appropriate application of methods.     

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.Download video file.^{(95M, mp4)}     

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane ¹. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette ^2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.Download video file.^{(166M, mp4)}     

Body electrical loss analysis (BELA) in the assessment of visceral fat: a demonstration

Background

Parkinson's disease (PD) is a neurodegenerative disorder resulting in motor disturbances that can impact normal gait. Although PD initially responds well to pharmacological treatment, as the disease progresses efficacy often fluctuates over the course of the day, and clinical management would benefit from long-term objective measures of gait. We have previously described a small device worn on the shank that uses acceleration and angular velocity sensors to calculate stride length and identify freezing of gait in PD patients. In this study we extend validation of the gait monitor to 24-h using simultaneous video observation of PD patients.

Methods

A sleep laboratory was adapted to perform 24-hr video monitoring of patients while wearing the device. Continuous video monitoring of a sleep lab, hallway, kitchen and conference room was performed using a 4-camera security system and recorded to hard disk. Subjects (3) wore the gait monitor on the left shank (just above the ankle) for a 24-h period beginning around 5 pm in the evening. Accuracy of stride length measures were assessed at the beginning and end of the 24-h epoch. Two independent observers rated the video logs to identify when subjects were walking or lying down.

Results

The mean error in stride length at the start of recording was 0.05 m (SD 0) and at the conclusion of the 24 h epoch was 0.06 m (SD 0.026). There was full agreement between observer coding of the video logs and the output from the gait monitor software; that is, for every video observation of the subject walking there was a corresponding pulse in the monitor data that indicated gait.

Conclusions

The accuracy of ambulatory stride length measurement was maintained over the 24-h period, and there was 100% agreement between the autonomous detection of locomotion by the gait monitor and video observation.     

Cytochalasin effects on structure and movement in the pollen tube of Iris

Summary Cytochalasins B and D in the concentration range 0.5–10 g ml^–1 produced similar effects on growth, movement and cytoplasmic structure in the pollen tubes of Iris spp. cultured in vitro. Continuous video recording showed that at 5 g ml_–1, CB was capable of stopping organelle circulation in as short a period as 20 s. The usually elongated vegetative nuclei were also arrested, and subsequently contracted irregularly. Generative cells were not radically changed in shape, but occasionally moved erratically before being halted. Detailed examination of CD- and CB-treated tubes regarded as being capable of recovering growth upon transfer to normal medium revealed several characteristic effects on cytoplasmic structure. Fibrils presumed to consist of, or contain, microfilament bundles are readily visible in the older parts of the living tube where they form the pathways of organelle movement; these were either condensed into amorphous columns or fragmented by treatment. In the distal parts of the tube, the cytoplasm had contracted into amorphous masses which continued to show very slow shape changes. With the arrest of extension growth, pectin accumulated over the tube tip and in patches along the flanks. In a medium containing 1 mM ATP, recovery from treatment was achieved in some instances within l min. Organelle circulation in the younger tubes was resumed, and fresh adventive tube tips were formed. The fibrillar system of the older tubes was not restored, however; instead, the cytoplasm in these zones formed aggregates which underwent continuous amoeboid movement, the organelles within moving rapidly in irregular trajectories with no indication of the resumption of the original long-range cyclotic flux. Some possible implications of the results are briefly discussed.     

Neonatal non-contact respiratory monitoring based on real-time infrared thermography

Background

The duration of bronchoscopy examinations varies considerably depending on the diagnostic and therapeutic procedures used. It can last more than 20 minutes if a complex diagnostic work-up is included. With wide access to videobronchoscopy, the whole procedure can be recorded as a video sequence. Common practice relies on an active attitude of the bronchoscopist who initiates the recording process and usually chooses to archive only selected views and sequences. However, it may be important to record the full bronchoscopy procedure as documentation when liability issues are at stake. Furthermore, an automatic recording of the whole procedure enables the bronchoscopist to focus solely on the performed procedures. Video recordings registered during bronchoscopies include a considerable number of frames of poor quality due to blurry or unfocused images. It seems that such frames are unavoidable due to the relatively tight endobronchial space, rapid movements of the respiratory tract due to breathing or coughing, and secretions which occur commonly in the bronchi, especially in patients suffering from pulmonary disorders.

Methods

The use of recorded bronchoscopy video sequences for diagnostic, reference and educational purposes could be considerably extended with efficient, flexible summarization algorithms. Thus, the authors developed a prototype system to create shortcuts (called summaries or abstracts) of bronchoscopy video recordings. Such a system, based on models described in previously published papers, employs image analysis methods to exclude frames or sequences of limited diagnostic or education value.

Results

The algorithm for the selection or exclusion of specific frames or shots from video sequences recorded during bronchoscopy procedures is based on several criteria, including automatic detection of "non-informative", frames showing the branching of the airways and frames including pathological lesions.

Conclusions

The paper focuses on the challenge of generating summaries of bronchoscopy video recordings.     

Pathway phase waveform characteristics correlated with length and rate of stylet advancement and partial stylet withdrawal in AC electrical penetration graphs of adult whiteflies

A technique was developed for measuring the length of stylet insertion during adult whitefly probing. The distance that the labium shortens during a probe was shown to be equal to the length of stylets that were inserted into the plant tissue. The length of labial shortening then was measured in high-magnification video recordings of adult female silverleaf whitefly, Bemisia argentifolii, in conjunction with recording electrical penetration graphs (EPGs – AC method). Using a split-screen device, video images of the whitefly's labium during a probe and the EPG waveforms produced during the probe were recorded simultaneously on the same video tape. On playback, changes in labial length could be measured during specific EPG waveforms to determine the length of stylet insertion that occurred during the waveforms. The focus of the study was on two characteristics of the pathway phase sawtooth waveform: the frequency of voltage peaks and the increase in voltage level that occurs over time during sawtooth waveforms. The rate of stylet penetration was significantly and positively correlated with frequency of sawtooth waveform voltage peaks (r ²=0.33) and the length of stylet penetration was significantly and positively correlated (second-order polynomial) with the relative difference in voltage level between the beginning and end of the sawtooth waveform (r ²=0.43). Stylet advancement did not appear to occur during the few low-flat waveforms (unknown behavioral correlation) and high-flat waveforms (phloem phase) that were observed. Voltage drops occur sporadically during sawtooth waveforms, and these were associated with partial stylet withdrawal (indicated when the labium increased in length, but the probe was not terminated) with an accuracy of 99%.     

Pollinator abundance,morphology, and flower visitation rate: analysis of the “quantity” component in a plant-pollinator system

Summary Abundance and flower visitation rate of the pollinators of Lavandula latifolia (Labiatae), an insect-pollinated shrub, were studied over a 6-year period. The objective was to elucidate interspecific patterns in the quantity component of the plant-pollinator interaction. A total of 54 insect taxa are considered in the analyses, including hynenopterans, dipterans and lepidopterans. Most pollinators were comparatively scarce, with a few taxa acounting collectively for the majority of individuals. Pollinators differed broadly in flower visitation rate (0.2–30 flowers/min). Most of this variation was explained by differences in flower handling time (HT). Regardless of proboscis length, hymenopterans had intrinsically shorter handling times than lepidopterans. Within each group, HT decreased exponentially with increasing proboscis length. Abundance and visitation rate were uncorrelated across pollinator taxa. The total number of visits that each pollinator contributed to the plant (NFV) was estimated as the product of abundance x visitation rate. NFV values spanned four orders of magnirade. A small, taxonomically diverse group of species (1 moth, 1 butterfly, 4 bees) accounted for most visits and thus could effectively exert some selection on floral features. Nevertheless, the morphological diversity represented in this group of dominant pollinators probably constrains plant specialization, as they will most likely select for different floral features or in opposing directions on the same traits.     

Sleep fragmentation in mentally retarded people decreases with increasing daylength in spring

We studied the sleep–wake behavior of mentally retarded people from late winter to early summer at 60°N. During this time the daylength increased 8 h 51 min. The data were collected by observing the sleep–wake status of 293 subjects at 20-min intervals for five randomized 24h periods (=recording days). The intervals during which the individual recording days of the same order (1st, 2nd, etc.) were carried out, were called recording periods. Consequently, there were five recording periods, each containing 293 individual recording days. Even though there was overlap among the recording periods, the median daylength from one period to another increased approximately by 100 min. In the initial statistical analysis, the number of wake–sleep transitions was found to differ significantly among the five recording periods (Friedman test, p<0.001). The mean ranks in the Friedman test suggested that the number of wake–sleep transitions was highest during the 1st and lowest during the 5th recording period. In further statistical analyses using a program for mixed effects regression analysis (mixor 2.0) it was found that the increase in daylength during the study period was associated with a simultaneous decrease of approximately 0.5 wake–sleep transitions in the whole study population (p<0.001). The decrease in the number of wake–sleep transitions was significant only in the subgroups of subjects with a daylength change of more than 350 min between the 1st and 5th recording days (Wilcoxon tests, p<0.005). This suggests that after a marked prolongation of the natural photoperiod, the reduction in sleep episodes was more probable than after smaller changes in daylength. It is concluded that the sleep of mentally retarded people living in a rehabilitation center at a northern latitude is more fragmented in winter than in early summer and that the change is related probably to the simultaneous increase in the length of the natural photoperiod. The sleep quality of persons living in institutional settings might be improved by increasing the intensity and/or duration of daily artificial light exposure during the darker seasons.     

MaleHeliothis virescens maintain upwind flight in response to experimentally pulsed filaments of their sex pheromone (Lepidoptera: Noctuidae)

Aspects of the intermittent fine structure of an odor plume were mimicked and experimentally modified in the wind tunnel using an air-pulsing device. Filaments of a behaviorally active blend of six sex-pheromone components created by the device in a temporally regular fashion elicited sustained upwind flight and source location in male Heliothis virescens.Males did not exhibit sustained upwind flight in significant numbers until a frequency of 4 filaments/s was delivered, at a loading of 1 g of the major component, Z11-16: Ald, with the other components loaded at their appropriate ratios. A loading of 10 g Z11-16: Ald was found to be optimal at this filament delivery rate. Electroantennogram recordings to different filament delivery rates of the complete blend indicated that a stationary male antenna can respond to the pulse rates used in this study. Importantly, when a main component necessary for upwind flight, Z9-14:Ald, was isolated into its own filaments and pulsed alternately against filaments of the five other components (including the other component essential for upwind flight, Z11-16: Ald), upwind flight to the source was significantly reduced (9%) compared to upwind flight and source location in response to filaments composed of the entire blend (30%), indicating that the complete pheromone blend must arrive on the antenna simultaneously for optimal evocation of sustained upwind progress. Neurophysiological evidence from other studies suggests that higher-order interneurons whose phasic response is enhanced when the entire blend is presented simultaneously may be of importance in explaining this behavioral difference stemming from synchronous vsasynchronous arrival of the components.     

Entropies of short binary sequences in heart period dynamics

Dynamic aspects of R-R intervals have often been analyzed by means of linear and nonlinear measures. The goal of this study was to analyze binary sequences, in which only the dynamic information is retained, by means of two different aspects of regularity. R-R interval sequences derived from 24-h electrocardiogram (ECG) recordings of 118 healthy subjects were converted to symbolic binary sequences that coded the beat-to-beat increase or decrease in the R-R interval. Shannon entropy was used to quantify the occurrence of short binary patterns (length N = 5) in binary sequences derived from 10-min intervals. The regularity of the short binary patterns was analyzed on the basis of approximate entropy (ApEn). ApEn had a linear dependence on mean R-R interval length, with increasing irregularity occurring at longer R-R interval length. Shannon entropy of the same sequences showed that the increase in irregularity is accompanied by a decrease in occurrence of some patterns. Taken together, these data indicate that irregular binary patterns are more probable when the mean R-R interval increases. The use of surrogate data confirmed a nonlinear component in the binary sequence. Analysis of two consecutive 24-h ECG recordings for each subject demonstrated good intraindividual reproducibility of the results. In conclusion, quantification of binary sequences derived from ECG recordings reveals properties that cannot be found using the full information of R-R interval sequences.