A Wireless Multi-Channel Recording System for Freely Behaving Mice and Rats

To understand the neural basis of behavior, it is necessary to record brain activity in freely moving animals. Advances in implantable multi-electrode array technology have enabled researchers to record the activity of neuronal ensembles from multiple brain regions. The full potential of this approach is currently limited by reliance on cable tethers, with bundles of wires connecting the implanted electrodes to the data acquisition system while impeding the natural behavior of the animal. To overcome these limitations, here we introduce a multi-channel wireless headstage system designed for small animals such as rats and mice. A variety of single unit and local field potential signals were recorded from the dorsal striatum and substantia nigra in mice and the ventral striatum and prefrontal cortex simultaneously in rats. This wireless system could be interfaced with commercially available data acquisition systems, and the signals obtained were comparable in quality to those acquired using cable tethers. On account of its small size, light weight, and rechargeable battery, this wireless headstage system is suitable for studying the neural basis of natural behavior, eliminating the need for wires, commutators, and other limitations associated with traditional tethered recording systems.     

Elucidating the responses and role of the cardiovascular system in crocodilians during diving: fifty years on from the work of C.G. Wilber

In 1960, C.G. Wilber in a laboratory-based study described for the first time the changes in heart rate with submergence in the American alligator noting in particular the marked bradycardia that occurred during forced dives. This short review summarizes the major advances in our understanding of diving and the responses and role of the cardiovascular system of crocodilians during submergence in the 50 years since Wilber published his findings. These advances are attributable in part to the technological advances made in physiological monitoring devices and wildlife telemetry that have not only provided greater elucidation of the hemodynamics of the unique crocodilian cardiovascular system but also allowed the natural diving behaviors and heart rates in free-ranging crocodiles to be recorded. Of note, telemetric field-based studies have revealed that wild free-ranging crocodiles typically undertake only short dives, less than 20 min, yet crocodiles are also capable of dives of many hours in duration. In contrast to Wilber's study, dives recorded from free-ranging crocodiles were found to be accompanied by only a modest bradycardia, highlighting the often confounding effects associated with captive animals monitored under laboratory conditions. More recent studies have also documented the complex central flow and pressure patterns of crocodilians, including a pulmonary to systemic shunt that can be initiated by a unique intracardiac valve located in the subpulmonary conus. The role and significance of this cardiac shunt remains controversial and the focus of recent lab-based studies. We contend that elucidation of the role and significance of the cardiac shunt in crocodilians will only be achieved by monitoring telemetrically the central cardiovascular flows and pressures in non-captive animals that are undisturbed and free-ranging. This presents the challenge ahead in the next 50 years.     

Recording of electromyogram in chronic experiments in dogs

A connector for chronic recording of EMG activity located on the dog's neck was developed as a row of male pins fixed on a dense band with attached wires of intramuscular electrodes. The wires were subcutaneously led to the connector. The band of the connector was sutured at the corners to the skin of the dorsal surface of the neck. Then the connector was anchored to the neck by a double adhesive tape wrapping like a collar. Compared to scull connectors, our model provided minimum surgical trauma to the animal, the connector or wires of intramuscular electrodes can be replaced if necessary. Localization of the connector did not impede scull and brain surgical operations. The model has been described and tested in the experiments in seven dogs with 8-20 pairs of implanted EMG-recording electrodes within 2-10 weeks.     

Behavior of rats kept under conditions of a shifted light/dark regimen and receiving melatonin

We studied the effect of shift in the natural light/dark regimen (desynchronosis) and treatment with melatonin on behavioral characteristics of rats with different activity in the open-field test. Experiments were performed on 172 Wistar rats kept under conditions of the natural or shifted light/dark regimen. Some animals were intraperitoneally treated with 1 ml physiological saline or melatonin in doses of 1 and 2 mg/kg, while others did not receive the injections. Desynchronosis altered the normal rhythm of locomotor activity and abolished the differences between daytime and nighttime activity rats not receiving the injections. The influence of melatonin on locomotor activity of rats maintained under normal or shifted light/dark conditions depended on its dose, time of treatment, and initial behavioral characteristics of animals. Our results indicate that the use of melatonin for treatment of disturbances produced by a shift in the light/dark conditions should be performed taking into account individual behavioral characteristics of the organism.     

Contractile activity of the uterus following intrauterine introduction of a polymer

In order to determine the mobile activity of the uterus upon the insertion of a foreign body, 30 mature rabbits were fitted with electrodes in the area of the cervix and then inserted with a polymer, which was made from a small piece of a standard Lippes Loop. 15 of the animals were fitted with electrodes but not administered the polymer in order to serve as a control. The amount of biopotential registration was measured 4-5 days, 7-9 days, and 13-15 days after the insertion. The animals with the polymer were shown to exhibit a much higher and more expressed level of contractile activity than the control animals. This contractile activity is thought to create unfavorable conditions for fertilizations and the implantation of the sperm.     

Validating standardised personality tests under semi‐natural conditions in wild house mice (Mus musculus domesticus)

Personality traits in animals are often measured using standardised behavioural tests for activity, boldness/shyness, sociability, aggression and exploration. These tests are quick and convenient, as well as easy to repeat. As the interest in studying the impact of animal personality on ecological and evolutionary consequences has been growing rapidly, there is increasing focus on cross‐validating measurements taken during these tests with behaviours shown under natural situations. In our experiment, we aimed to study the relationship between standardised measurements for activity, exploration and anxiety‐like behaviour measured in Open Field, Novel Object and Elevated Plus Maze tests with exploration and colonisation in semi‐natural conditions. We carried out a semi‐natural enclosure experiment in parallel with standardised behavioural tests, creating a scenario similar to an invasion or dispersal event. We compared behaviours in standardised tests and in enclosures for animals of two populations of wild house mice (Mus musculus domesticus). Several behavioural variables taken during the standardised tests, such as distance moved and time spent with novel object, were negatively correlated with space‐use in the enclosure while being highly positively correlated among each other. Based on their relationship with space use, we refer to behavioural measurements from standardised tests as activity/exploration. The time spent near the walls in an open field, probably reflecting anxiety, was not correlated to any other variable or the behaviour in the enclosure. In addition, we found differences in activity/exploration behaviour between the two populations in the standardised tests, but not during the colonisation of the novel environment. These results emphasise that researchers have to be careful when trying to extrapolate behaviour shown in standardised laboratory test setups to more natural, ecologically relevant situations. This has to be taken into account in distantly related species but even when studying the wild relative of laboratory rodents, for which these standardised tests have originally been developed.     

Spontaneous K-complexes in behaving rats

The K-complex (KC) is an electrographic rhythmic pattern present in human and cat sleep EEG. In long-term multisite videoEEG recordings in behaving Sprague-Dawley (SD) rats, well-defined spontaneous KCs were observed during sleep. Sprague-Dawley rats were implanted with multiple electrodes bilaterally along the antero-posterior axes at the locations F1, F2, F7, F8, T3, T4, P3, P4, all against a ground reference placed in the midline above the cerebellum. Multiple, closely spaced cortical electrodes allowed two-dimensional surface brain mapping of the power spectra distribution. Two silver wires were also inserted into nuchal muscles to record EMG activity. Each rat was monopolarly recorded from 0900 h to 1500 h in a natural dark-light rodents, we examined the patterns of appearance in various conditions, the progression through a full sleep-waking cycle, the shape, density, spectral components, and spatial distribution in power spectra. The rat KC appears to share similar features with the human and cat KC.     

The Mechanism of Temperature Dependent Sex Determination in Crocodilians: A Hypothesis

Incubation of alligator eggs at 30°C produces 100% females,at 33°C 100% males; temperatures in between produce varyingsex ratios. Wild nests of Alligator mississippiensis show similareffects and the populations are biased towards females. Theincidence and patterns of temperature dependent sex determination(TSD) in other crocodilians are reviewed. Temperature also affectshatchling size and pigmentation patterns, post-hatching growthrates and thermoregulation by juvenile crocodilians. The significanceof temperature sensitive periods defined by temperature shiftexperiments is questioned in relation to a hypothesis to explainthe mechanism of TSD in crocodilians. It is postulated thatthere is an initial sex differentiation mechanism which involvesa quantum period of time and a threshold for a dose of a maledetermining factor. The conditions for induction of males areprecise but exhibit variation between individuals within thepopulation. Females develop by default. The hypothalamus mayhave an important role in a later sex differentiation mechanism.The hypothesis is used to explain the late temperature sensitiveperiods defined by high to low temperature shift experiments,why cooler temperatures are more effective at determining sex,how intermediate temperatures can produce both sexes, the differencesin the pattern between turtles and crocodilians and geographicalsimilarities in the pattern of TSD within crocodilians despitediffering climates. The phylogenetic advantages of TSD in crocodiliansare concerned with the overall reproductive strategy of theanimals. Those crocodilians which are incubated and grow tomaturity under optimal environmental conditions will be bothlarge and male. Larger males are more likely to produce moreoffspring. A review of the effects of the environment on sexdetermination in amphibians and fish suggests that there isa general relationship between size and sex in vertebrates     

Endogenous circannual cycles of ovarian activity and changes in prolactin and melatonin secretion in wild and domestic female sheep maintained under a long-day photoperiod

The present study examines the ovulatory activity of wild and domesticated ewes subjected to either a constant photoperiod of long days (16L:8D) or natural changes in daily photoperiod for 16 mo. The aim was to determine whether an endogenous reproductive rhythm controls seasonal reproductive activity in these sheep, and how the photoperiod might affect this. The effects of long-day photoperiods on long-term changes in prolactin and melatonin secretion were also evaluated. The two species showed changes in reproductive activity under the constant photoperiod conditions, suggesting the existence of an endogenous rhythm of reproduction. This rhythm was differently expressed in the two types of ewe (P < 0.05), with the domestic animals exhibiting much greater sensitivity to the effects of long days. A circannual rhythm of plasma prolactin concentration was also seen in both species and under both photoperiod conditions, although in both species the amplitude was always lower in the long-day animals (P < 0.01). The duration of the nocturnal melatonin plasma concentrations reflected the duration of darkness in both species and treatments. The peak melatonin concentration did not differ between seasons either under natural or long-day photoperiods.     

Multiplexed, high density electrophysiology with nanofabricated neural probes

Extracellular electrode arrays can reveal the neuronal network correlates of behavior with single-cell, single-spike, and sub-millisecond resolution. However, implantable electrodes are inherently invasive, and efforts to scale up the number and density of recording sites must compromise on device size in order to connect the electrodes. Here, we report on silicon-based neural probes employing nanofabricated, high-density electrical leads. Furthermore, we address the challenge of reading out multichannel data with an application-specific integrated circuit (ASIC) performing signal amplification, band-pass filtering, and multiplexing functions. We demonstrate high spatial resolution extracellular measurements with a fully integrated, low noise 64-channel system weighing just 330 mg. The on-chip multiplexers make possible recordings with substantially fewer external wires than the number of input channels. By combining nanofabricated probes with ASICs we have implemented a system for performing large-scale, high-density electrophysiology in small, freely behaving animals that is both minimally invasive and highly scalable.     

Oxidation-reduction potentials in the cecal contents of rats and mice.

The oxidation-reduction potential (ORP) in the cecal contents of conventional rats, germ-free mice, and mice with a Colonization Resistance Factor flora (CRF-mice) was investigated. By using animals that were anaesthetized for a longer period of time, we attempted to eliminate the disturbing influence of oxygen. In addition, measurements were made under anaerobic conditions. For the rats, the ORP values reached a more or less constant level after about 30 min following the insertion of the electrodes. The mean ORP at that time was -458 mV (SD = 45 mV). The mean ORP values for the mice showed a more gradual reduction than was found in the rats. The curves leveled off at about 100 min following the insertion of the electrodes. The mean ORP values 100 min after electrode insertion were: germ-free mice, + 3 mV (SD = 39 mV); CRF-mice, -554 mV (SD = 29 mV). In rats, the ORP value decreased after death; no decrease was observed in mice. No difference was found in the values obtained when measuring under anaerobic or aerobic conditions after death.     

Why the Long Face? The Mechanics of Mandibular Symphysis Proportions in Crocodiles

Background

Crocodilians exhibit a spectrum of rostral shape from long snouted (longirostrine), through to short snouted (brevirostrine) morphologies. The proportional length of the mandibular symphysis correlates consistently with rostral shape, forming as much as 50% of the mandible’s length in longirostrine forms, but 10% in brevirostrine crocodilians. Here we analyse the structural consequences of an elongate mandibular symphysis in relation to feeding behaviours.

Methods/Principal Findings

Simple beam and high resolution Finite Element (FE) models of seven species of crocodile were analysed under loads simulating biting, shaking and twisting. Using beam theory, we statistically compared multiple hypotheses of which morphological variables should control the biomechanical response. Brevi- and mesorostrine morphologies were found to consistently outperform longirostrine types when subject to equivalent biting, shaking and twisting loads. The best predictors of performance for biting and twisting loads in FE models were overall length and symphyseal length respectively; for shaking loads symphyseal length and a multivariate measurement of shape (PC1– which is strongly but not exclusively correlated with symphyseal length) were equally good predictors. Linear measurements were better predictors than multivariate measurements of shape in biting and twisting loads. For both biting and shaking loads but not for twisting, simple beam models agree with best performance predictors in FE models.

Conclusions/Significance

Combining beam and FE modelling allows a priori hypotheses about the importance of morphological traits on biomechanics to be statistically tested. Short mandibular symphyses perform well under loads used for feeding upon large prey, but elongate symphyses incur high strains under equivalent loads, underlining the structural constraints to prey size in the longirostrine morphotype. The biomechanics of the crocodilian mandible are largely consistent with beam theory and can be predicted from simple morphological measurements, suggesting that crocodilians are a useful model for investigating the palaeobiomechanics of other aquatic tetrapods.     

Do crocodilians get the flu? Looking for influenza A in captive crocodilians

It is well established that several wild aquatic bird species serve as reservoirs for the influenza A virus. It has also been shown that the influenza A virus can be transmitted to mammalian species such as tigers and domestic cats and dogs through ingestion of infected birds. Another group of animals that should also be considered as potential hosts for the influenza A virus are the crocodilians. Many crocodilian species share aquatic environments with wild birds that are known to harbor influenza viruses. In addition, many large crocodilians utilize birds as a significant food source. Given these factors in addition to the close taxonomic proximity of aves to the crocodilians, it is feasible to ask whether crocodilian species may also harbor the influenza A virus. Here we analyzed 37 captive crocodilians from two locations in Florida (plus 5 wild bird fecal-samples from their habitat) to detect the presence of influenza A virus. Several sample types were examined. Real-time RT-PCR tests targeting the influenza A matrix gene were positive for four individual crocodilians--Alligator sinensis, Paleosuchus trigonatus, Caiman latirostris and Crocodylus niloticus. Of the seven serum samples tested with the avian influenza virus agar gel immunodiffusion assay, three showed a nonspecific reaction to the avian influenza virus antigen-A. sinensis, P. trigonatus and C. niloticus (C. latirostris was not tested). Viable virus could not be recovered from RT-PCR-positive samples, although this is consistent with previous attempts at viral isolation in embryonated chicken eggs with crocodilian viruses.     

Ectromelia in Morelet's crocodile from Belize

Two Morelet's crocodiles (Crocodylus moreletii) captured on 21 March 1997 and 20 April 1998 in the New River system, Belize exhibited ectromelia of one forelimb. External and radiograph examination appears to indicate limb agenesis of unknown etiology, as there is no apparent scarring or skeletal trauma. These two individuals represent the only cases of missing limbs from 642 individuals captured in this study and to our knowledge, the first reported cases in Morelet's crocodile. Several factors including age and diet of the reproducing female, extremes in nest conditions (egg incubation temperature and humidity), and exposure to environmental contaminants can cause developmental abnormalities in crocodilians and may have contributed to the condition observed in these animals. Survival rates for hatchling crocodilians are generally low, and embryonic malformations such as ectromelia may constitute an added disadvantage to survival. However, both individuals examined in this study were vigorous and appeared in good condition.     

Seasonal variations in circadian rhythms coincide with a phase of sensitivity to short photoperiods in the European hamster

European hamsters (Cricetus cricetus) show pronounced seasonal changes in their physiology and behavior. The present study provides a detailed analysis of the temporal relationship between seasonal cycles of reproduction and body mass and seasonal changes of two circadian parameters, i.e., locomotor activity and 6-sulphatoxymelatonin (aMT6s) excretion, in individual animals kept under natural light conditions. Our results demonstrate a characteristic pattern of locomotor activity and aMT6s excretion observed around the summer solstice, i.e., from mid-May to mid-July. During this time, locomotor activity was characterized by a high level of activity and an early activity onset, while the nightly elevation of melatonin was reduced to baseline levels. These seasonal changes in aMT6s excretion and locomotor activity were only loosely related to changes in the reproductive status of the animals, but correlated well with a period of the annual cycle during which the animals were sensitive to short days. They may therefore reflect a specific state of the circadian pacemaker system within the SCN and can thus be a valuable tool to further characterize molecular and physiological mechanisms of photoperiodic time measurements in European hamsters.     

Acidophilic microbial communities associated with a natural, biodegraded hydrocarbon seepage

Characterization of microbial communities present in a surface petroleum seep in which hydrocarbons have been biodegraded for thousands of years in order to improve the understanding on natural petroleum biodegradation. DNA was extracted from a natural, surface petroleum seep and subjected to culture independent analysis (rRNA gene-based denaturing gradient gel electrophoresis and phylogenetic analysis of clone libraries). Molecular analysis suggested dominance by acidophilic bacteria, especially Alphaproteobacteria (mainly bacteria related to Acidiphilium and Acidocella). Archaea were not detected, but fungi were present. pH of the samples was around 3.5. Acidophilic microbial communities are associated with an acidic petroleum seep. Microbial community structure analysis gives information on the environmental conditions under which petroleum biodegradation occurs. This knowledge could be applied to define conditions for specific cultivation or activity measurements. The activity of acidophilic micro-organisms deserves more attention with respect to their involvement in natural petroleum degradation. This knowledge will contribute to the design of oil bioremediation strategies for polluted acidic settings.     

Genioglossus muscle activity and serotonergic modulation of hypoglossal motor output in obese Zucker rats.

Obese Zucker rats have a narrower and more collapsible upper airway compared with lean controls, similar to obstructive sleep apnea (OSA) patients. Genioglossus (GG) muscle activity is augmented in awake OSA patients to compensate for airway narrowing, but the neural control of GG activity in obese Zucker rats has not been investigated to determine whether such neuromuscular compensation also occurs. This study tests the hypotheses that GG activity is augmented in obese Zucker rats compared with lean controls and that endogenous 5-hydroxytryptamine (5-HT) contributes to GG activation. Seven obese and seven lean Zucker rats were implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and they were implanted with GG and diaphragm wires for respiratory muscle recordings. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid and the 5-HT receptor antagonist mianserin (100 microM). Compared with lean controls, respiratory rates were increased in obese rats across sleep-wake states (P=0.048) because of reduced expiratory durations (P=0.007); diaphragm activation was similar between lean and obese animals (P=0.632). Respiratory-related, tonic, and peak GG activities were also similar between obese and lean rats (P>0.139). There was no reduction in GG activity with mianserin at the hypoglossal motor nucleus, consistent with recent observations of a minimal contribution of endogenous 5-HT to GG activity. These results suggest that despite the upper airway narrowing in obese Zucker rats, these animals have a sufficiently stable airway such that pharyngeal muscle activity is normal across sleep-wake states.     

Environmental regulation of sex determination in reptiles

The various patterns of environmental sex determination in squamates, chelonians and crocodilians are described. High temperatures produce males in lizards and crocodiles but females in chelonians. Original experiments on the effects of incubation at 30 degrees C (100% females) or 33 degrees C (100% males) on development in Alligator mississippiensis are described. These include an investigation of the effect of exposing embryos briefly to a different incubation temperature on the sex ratio at hatching, and a study of the effects of 30 degrees C and 33 degrees C on growth and development of alligator embryos and gonads. A 7-day pulse of one temperature on the background of another was insufficient to alter the sex ratio dramatically. Incubation at 33 degrees C increased the rate of growth and development of alligator embryos. In particular, differentiation of the gonad at 33 degrees C was enhanced compared with 30 degrees C. A hypothesis is developed to explain the mechanism of temperature-dependent sex determination (TSD) in crocodilians. The processes of primary sex differentiation are considered to involve exposure to a dose of some male-determining factor during a specific quantum of developmental time during early incubation. The gene that encodes for the male-determining factor is considered to have an optimum temperature (33 degrees C). Any change in the temperature affects the expression of this gene and affects the dose or quantum embryos are exposed to. In these cases there is production of females by default. The phylogenetic implications of TSD for crocodilians, and reptiles in particular, are related to the life history of the animal from conception to sexual maturity. Those animals that develop under optimal conditions grow fastest and largest and become male. A general association between the size of an animal and its sex is proposed for several types of vertebrate.     

Persistent free-running circannual reproductive cycles during prolonged exposure to a constant 12L:12D photoperiod in laboratory woodchucks (Marmota monax).

Serum levels of gonadal steroid were assayed at approximately 3-month intervals in groups of 5 to 8 male or female woodchucks which were exposed to a natural photoperiod for 1 year as yearlings or 3 years as adults (Study 1), or a constant photoperiod of 12L:12D from birth for 4.5 years (Study 2). After 4.5 years of 12L:12D, food intake was measured in November and compared with that in natural photoperiod animals (Study 3). Other groups of 11 males and 3 females were housed in 12L:12D for 2.5 years after capture at 2 months of age, and gonadal structure and serum steroid levels in November were compared with those of animals at selected times in the normal annual cycle (Study 4). All animals were provided food and water ad libitum and were not induced to hibernate. In Study 1, normal circannual breeding season elevations in testosterone in males and in progesterone in females were detected in most animals maintained in natural photoperiod. In Study 2, similar cycles persisted for 4.5 years in animals exposed to 12L:12D. However, based on quarterly blood samples, obvious asynchrony relative to natural light animals appeared to develop after 2, 3, or 4 years, with apparent free-running intervals of about 10 to 11 months. In Study 3, mean daily food consumption in late autumn for woodchucks in the 12L:12D group was 72% greater than animals in the natural photoperiod. In Study 4, some woodchucks exposed to 12L:12D for only 2.5 years had prematurely increased spermatogenic activity, Leydig tissue development, and elevated serum testosterone levels in November. They were similar in November to those in natural photoperiod animals in March, and significantly greater than those in natural photoperiod animals in November when normal regression and repair of the testis was complete. Likewise, females in the 12L:12D group had luteinized follicles and elevated progesterone in November which were not noted in natural photoperiod animals and which were similar to those observed during the spring in unbred females under normal conditions. The results suggest that circannual cycles of metabolic and reproductive activity in woodchucks persist in the absence of normal changes in photoperiod, are entrained to seasonal changes in the natural photoperiod, and can recede to a periodicity of less than 12 months within 2.5 to 4 years of laboratory maintenance in 12L:12D.     

Intramuscular and esophageal electrode recordings of posterior cricoarytenoid activity in normal subjects

Six normal human subjects were studied to compare intramuscular and esophageal electrode recordings of posterior cricoarytenoid (PCA) muscle activity. A new electromyographic technique was developed to implant hooked wire electrodes into the PCA via a nasopharyngoscope. The esophageal electrode was similar to that used by other investigators to record PCA activity (P. C. Kosch et al. J. Appl. Physiol. 64: 1968-1978, 1988). Simultaneous recordings from the intramuscular and esophageal electrodes were obtained during wakefulness and sleep. Changes in esophageal electrode activity were compared with changes in intramuscular electrode activity under four conditions: 1) voluntary maneuvers, 2) differences in state, 3) nasal airway occlusion during non-rapid-eye-movement sleep, and 4) spontaneous variations in respiratory efforts during non-rapid-eye-movement or rapid-eye-movement sleep. Although similar results were obtained from the esophageal and intramuscular electrodes, differences were present between the two recordings during both wakefulness and sleep. The esophageal electrode recorded activity from surrounding muscles during voluntary maneuvers, vocalization, and quiet breathing in wakefulness. Discrepancies between the two electrode recordings during sleep occurred under conditions of increased and decreased respiratory motor output. The data suggest that the esophageal electrode may not give an accurate assessment of PCA activity during many conditions in wakefulness and sleep.