Webinar Q&A Report - InsideScientific

Circadian Rhythms of Food Intake: Are You Seeing The Whole Picture?

Webinar Q&A Report

Q: Can a system like this record 10 or 20 mice simultaneously? If yes, how does one configure such a system? (J. Lighton): Yes. Most manufacturers produce systems that can technically sample “simultaneously.” Yet, when discussing “simultaneous” measurement, it is important to distinguish “rapid succession sampling” from “continuous” sampling. There are two ways to measure the metabolic rates of multiple animals. A multiplexed system switches a gas analyzer chain rapidly from cage to cage in rapid succession. If the sampling rate is frequent enough for the purpose of the study, some researchers consider it the equivalent of “simultaneous sampling,” although the actual time between samples from any one animal can be separated in legacy systems by a pause of 10 to 20 minutes. The Sable Systems Promethion multiplexed system can sample metabolic data from all 8, 16, 24 etc. cages in the system in as little as 2.5 minutes, from cage 1 through all other cages and back to cage 1, irrespective of the number of cages. And, unique to the Promethion systems, all cage sensors (food intake, position, activity, etc.) are monitored every second and stored for all cages simultaneously. A continuous system monitors the metabolic signals of each cage with its own gas analyzer chain and flow path. How truly “continuous” a system is will depend on the actual sampling rate. Manufacturers systems vary in their sampling capacity. Most are constrained to every 40 seconds or longer. Sable’s Promethion-C system samples from all cages every second, simultaneously and in sync with the 1 second sampling rate from its food, water, body mass and activity sensors. Obviously the multiplexed systems are more economical. But if very high temporal resolution is a key requirement, a continuous system should be considered. The Promethion Continuous system offers 10to 60-fold better temporal resolution than any other “continuous” system. Configuration of either the multiplexed or continuous Promethion system is designed to simplify unnecessary complexities involved in legacy system setup. It is straightforward, involving simple tubing runs and a single daisy-chained data cable. Depending on the system size and installation environment, time from system installation to production of data is typically less than a day.

Q: How modular is the system? Can I start with a subset of its sensors and add others as I need them? (J. Lighton): Modularity of components is ideal as it provides an opportunity to use available funds and gather data “now”, then expand the system later. Most manufacturers offer systems with some level of modularity or “add on” capacity. However, unless a system has all of its parts designed by the same manufacturer, the trade-off for expandability is a high space requirement and the additional upselling cost of extraneous aggregators and computer cards. We purposefully designed Promethion expandability to be easy and economical. You can start with a small subset of sensors, and add other capabilities as your research requirements evolve. All Promethion instrumentation is connected by a single daisychained cable using a patented, high-speed error correcting digital network designed for mission-critical applications such as aerospace. Moreover, expanding the system incurs no per-sensor software charges, and compatibility with new sensors (such as our new environmental/intrusion monitor, which senses temperature, relative humidity, barometric pressure, light level, sound level, and human presence via PIR) is guaranteed. Q: Does the system have operant conditioning capability? If yes, please explain… (J. Lighton): The Promethion system has effective operant conditioning capability in the ingestive behavior arena. For example, the ability to couple food availability to distance run on the in cage running wheel. An assortment of operant conditioning features incorporating suites of sensors and effectors are in development for release in the near future.

Q: How would someone run a “fast and re-feeding” protocol? For instance, a 24 hour duration of fasting followed by 48 hours of refeeding. (J. Lighton): This is as simple as running a suitable food access control script. Q: Can this system measure urine and feces output by the test subjects (rats or mice, e.g.)? If yes, how is this achieved? (J. Lighton): No “home/low-stress” cage is suitable for truly quantitative urine and feces output measurement. This is because truly quantitative measurements require using a “metabolic cage”, in which the animal is kept in a restricted space with a grid floor above a urine/feces separator. Such cages are known to trigger significant stress responses. As a lower-stress alternative we can supply our standard cage with a wire grid platform in place of the usual bedding, allowing feces to fall through for counting and measurement, and allowing urine to be absorbed into bibulous paper. If use of a metabolic cage is required, we have a cooperative agreement with the Hatteras Instruments company which is generally recognized as the best metabolic cage manufacturer in the market. Our metabolic measurement systems can be used with their cages simply by swapping the lid, as can our activity quantification systems (XY arrays). Thus, if the high stress environment of a metabolic cage is warranted by a requirement for truly quantitative urine and feces measurement, simultaneous metabolic and activity measurements can still be made with the Promethion system.

Q: Regarding water consumption and measuring this over time, what concerns should one have regarding evaporation? Is there a way to manage this, and account for evaporation in the water consumption measurements? (J. Lighton): Because water intake volumes can be quite small, it is very important, if a gravimetric system is used, that the system be able to resolve small mass changes, or low-volume water intake events will be missed. In the case of Promethion, intake events down to 2 µL can be resolved. In our case, intake measurements are intrinsically differential, and involve comparing the mass of the dispenser immediately prior to, and immediately after, an intake event or a series of intake events. Each intake event is marked by substantial mass disturbances which are lacking from more gradual signals such as evaporation, and which by default trigger an intake measurement during data analysis. Thus, the system will ignore evaporation unless specifically directed not to do so. Because Promethion stores all raw data, you can choose to analyze intake via a naïve mass difference across a given time span (equivalent to manual weighing at intervals), or use the more sophisticated and versatile differential technique which is immune to gradual mass changes caused by evaporation or by changes in food hydration. Q: If someone is to use powdered feed to minimize caching, what hopper or instrumentation do they need? (J. Lighton): For food, a versatile hopper with an interchangeable grille is very helpful. To minimize caching, or for use with powdered food, a grille with fine to very fine spacing can be used, while with coarse pelleted food, especially if caching is not a concern, a more open grille spacing would be preferred. A tray immediately below the grille collects any food crumbs that might otherwise spill into the bedding. We are also happy to work with Promethion users to develop customized dispensers on an as-needed basis. The unique design of our mass measurement module allows for almost unlimited flexibility without adding significant headspace about the cage or dead volume within it. Coupled with the extraordinary resolution of our mass measurements (0.002 g, 10-fold finer than with legacy systems), intake measurements can be made with extraordinary precision. Q: Do the grids in the feed hopper prevent the mouse from removing a whole feed pellet and subsequently chewing on it in the cage environment? (J. Lighton): See the answer to the above question. Using a fine grille spacing reduces the ability to remove a substantial portion of a food pellet, and using powdered food may likewise reduce caching. That said, it would be dishonest to claim that caching can be eliminated - at best, it can be reduced to tolerable or even negligible levels. Caching is a deeply conserved aspect of rodent ingestive behavior with significant evolutionary advantages, and is difficult to defeat. When caching behavior occurs, intelligent use of the entire cage sensor suite can, however, make it easy to detect. For example, food intake from a hopper during a caching event is easily distinguished from food intake during normal feeding, and a sudden increase in body mass in the absence of a measurable food or liquid intake event may correspond to consumption of a cached food source.

Q: What software do you use to analyze the data? Can someone use other data acquisition software like matlab for instance? (J. Lighton): The Promethion data format is open, and raw or processed data can be imported into a variety of statistical environments, for example R or MATLAB, for further processing. We provide all necessary technical support to allow users of the Promethion system to reach any analytical goals they may have. Raw Promethion data are typically analyzed using Sable System’s Expedata, a sophisticated, menu-and script-driven analytical environment optimized for metabolic measurement and metabolic phenotyping applications, and capable of automated operation. Expedata then exports data as required to other packages, either directly (in the case of Excel), via .CSV files, or via the clipboard. Because Promethion records the raw data from the entire system every second, analytical flexibility is practically limitless and all final results can be traced back to the original instrument and sensor data. Q: I recently received a dataset and am very new to this. For an animal on a high fat diet, is it normal for VO2 to be higher and RQ lower, indicative of more fat as metabolic fuel? (J. Lighton): Animals on a high-fat diet will usually tend towards a lower RQ, indicating preferential catabolism of lipids. Because RQ is the quotient of VCO2/VO2, the lower RQ will necessarily reflect a proportionate increase in VO2 or decrease in VCO2. The metric of most interest with regard to overall metabolism is generally not VCO2 or VO2, but rather energy expenditure or EE, which is derived from both VCO2 and VO2. A change in dominant catabolic substrate, for example from carbohydrates to lipids, will usually result in a negligible change in EE at equivalent levels of activity, but a significant change in RQ. Bear in mind that RQ is very sensitive to activity levels, and rapidly increases as the animal becomes more active and its catabolic substrate shifts more towards carbohydrates. In addition, if an animal gains weight via de novo lipogenesis its RQ may temporarily increase, even to superficially “nonphysiological” levels above 1.0. We provide Promethion users with expert, PhD-level technical and scientific support “by scientists, for scientists” to address questions of expectation and interpretation. Q: I am interested to know if zbreak/rearing is related to food intake? In your experience do rats need to rear for consumption or are these separate? (J. Lighton): Mechanically, mice usually need to rear during food intake from Promethion food hoppers. As for rats, in my experience rearing is an almost invariable accompaniment to food intake. The reverse is not necessarily true; if the rat is rearing, it is not necessarily feeding. Of course, the operational definition for “rearing” in any given system depends on the height of the Z axis, which the operator can set to detect anything from marginal elevation to full stretching.

Q: Can this cage system be integrated in to a group housed environment? (J. Lighton): The system is compatible with group housing, with a couple of significant caveats. For metabolic measurements, the summed metabolic rates of all animals in the cage will be recorded. For intake measurements, likewise, the food and water intake totals will reflect the summed intakes of all cage inhabitants. For activity measurements, beam break counts will again reflect the overall activity of all animals in the cage. Q: I am wondering if your software can somehow calculate what would be resting EE, let's say in fasted mice. B y correcting for activity, drinking & maybe lean mass? Some experts suggest ANCOVA.

(J. Lighton): The software can calculate EE according to a wide variety of parameters, including absolute level windows (high or low), degree of variation, amount of activity and time since the last intake event. Thus, extracting resting EE is very straightforward. Correcting for body mass is also possible, but it must be borne in mind that EE scales allometrically with body mass, rather than isometrically as is commonly and misleadingly assumed in legacy metabolic phenotyping systems. This problem can be solved by using analysis of covariance (ANCOVA), which I have been using in my published metabolic research since 1985 but which has only recently come to the attention of the metabolic phenotyping community. I advocated for its use in my metabolic measurement textbook published by Oxford University Press in 2008. Promethion users obtain access to expert advice on the analysis and presentation of metabolic parameters because of our unique background in metabolic research. Q: When it comes to measuring VO2, RQ, and the importance of resolution. What about the low resolution of a O2 sensor, like a fuel cell? (J. Lighton): It is a myth that fuel cell O2 sensors have low resolution. This myth derives from products made by other manufacturers but is not correct in our hands. Our fuel cell analyzers are designed specifically for flow-through respirometry and use cutting-edge, proprietary digital and analog signal processing techniques that are unique to Sable Systems. The highest resolution O2 analyzer in the world is currently the Sable Systems FC-2, widely used in climatological atmospheric analysis, with a peerreviewed, published resolution limit of 0.3 ppm (0.00003%) against a background of 209,400 ppm O2. It is a fuel cell analyzer, like the analyzer used in Promethion, and uses similar proprietary Sable Systems technology. The noise figure of the Promethion O2 analyzer is typically 1 ppm (0.0001%), which is 10 to 100 times superior to O2 analyzer technologies used in legacy metabolic phenotyping systems. Sable Systems also produces paramagnetic O2 analyzers, but we chose our fuel cell analyzers for Promethion because of their extraordinarily high resolution, stable readings and absence of cross-sensitivity to other gases. Sable Systems is unique among metabolic phenotyping manufacturers for producing stand-alone analyzers that are regarded as cutting-edge and sought after by demanding customers such as NIST and NASA. The high resolution of our gas analyzers allows us to utilize much higher flowrates than other systems, leading to better accuracy, excellent atmospheric integrity within the cage, compliance with IACUC air exchange requirements, and lower CO2 buildup (which can cause physiological side effects and cognitive impairment at the high levels commonly encountered in legacy systems).

Do you recommend using laser O2 sensor technology? They have more resolution than others sensor technology like fuel cell, zirconia, etc. (J. Lighton): Interesting question. Laser O2 sensor technology has very low resolution even compared with other low-resolution technologies such as most zirconia-cell analyzers. Laser O2 analyzers depend on an extremely weak and very narrow O2 optical absorbance band, thus requiring a high-volume, folded-path optical bench design that adds noise. Worse, they suffer from nonlinear Doppler band broadening during pressure fluctuations (not STP-correctable), and Lorentz band broadening due to vibrational interaction of O2 with larger molecules such as CO2. These undesirable and difficult-tocorrect problems combined with their high noise figures make laser O2 sensors unsuitable, in my opinion as an acknowledged expert in metabolic measurement, for small-animal use if resolution and accuracy are important considerations. Q: Pull mode respirometry has some inconveniences: for example you cannot control the room air that enters into the cage. What alternatives do you suggests to this issue? (J. Lighton): Animal facilities are required by care and use regulations to have excellent HVAC systems with substantial air exchange, yielding a very stable incurrent O2 and CO2 concentration. Slow fluctuations in the concentration of either gas species are corrected with practically perfect accuracy by frequent measurement and correction of incurrent concentrations. In some studies, it is necessary to expose animals to other defined gas mixtures, and this is easily achieved in a pull mode system by maintaining the cages in a cabinet flushed with the desired incurrent gas mixture. Such manipulations of incurrent gas concentrations are only required for a tiny minority of studies, and are easily achieved. Push mode systems, unlike pull mode systems, are easy and cheap to produce. However, they require drying of incurrent air for optimum accuracy, low flow rates (because of low resolution gas analyzers), and completely sealed cages. Because of these and a host of other disadvantages that detract from measurement accuracy and animal welfare, I designed the Promethion system to operate in native pull mode with no requirement for a tight seal, thus exposing the animals to ambient pressure and humidity, reducing stress and improving the reproducibility of experimental data. Is your system compatible with video analysis and/or telemetry? (J. Lighton): Yes. We have cooperative agreements with two leading manufacturers of state-of-the-art video analysis systems, Cleversys and Noldus. Likewise, we have similar agreements with Data Sciences International and with Starr Life Sciences. Telemetry data from either company can be incorporated into the Promethion data stream. Our corporate philosophy is to excel in the areas of high resolution metabolic, intake and activity measurement, and to forge win-win agreements with experts in other areas for the benefit of scientific researchers.

If you have additional questions for Sable Systems International or Dr. John Lighton regarding content from his presentation, or wish to receive additional information about solutions for preclinical metabolic and behavioral monitoring please contact them by phone or email:

Dr. John Lighton, PhD President and Chief Scientist – Sable Systems International Email: [email protected]

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