Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats: comparative effects on selected behavioural and blood variables

We compared the behaviours of rats, and measured various blood parameters, after three blood sampling techniques: orbital puncture while they were under diethyl-ether anaesthesia, blood collection by tail vein puncture under O2-N2O-halothane anaesthesia and puncture of the saphenous vein without anaesthesia. Twelve rats were subjected to the three treatments according to a Latin square design. After each treatment, the behaviour of the rats was automatically monitored using the so-called LABORAS method, which discriminates between grooming, locomotion and inactivity in rats. Based on excitation scores and urine production, it was found that induction of diethyl-ether anaesthesia combined with orbital puncture caused more distress than did the other two blood sampling techniques. The three techniques had no differential effects on the behaviours of grooming, locomotion and inactivity. Collecting 0.5 ml of blood by orbital puncture was +/-7 times faster than doing so by saphenous vein puncture and +/- 15 times faster than collecting blood by tail vein puncture while the rats were under O2-N2O-halothane anaesthesia. The levels of some haematological and plasma variables differed significantly between the three blood collection techniques. These observations may help to select the most appropriate technique of blood sampling with respect to anticipated discomfort in the animals.     

Endocrine stress response in rats subjected to singular orbital puncture while under diethyl-ether anaesthesia.

In an attempt to assess possible discomfort in rats subjected to orbital puncture while under diethyl-ether anaesthesia, their endocrine stress response was determined. Concentrations of corticosterone, adrenaline and noradrenaline were measured in plasma obtained via a jugular catheter from rats subjected to diethyl-ether anaesthesia with or without orbital puncture. No statistically significant differences were found between the punctured and non-punctured rats as to peak levels of plasma corticosterone and adrenaline as well as for the times required by the increased concentrations to return to baseline values. The rate by which the plasma noradrenaline level returned to baseline values was somewhat decreased by orbital puncture. Diethyl-ether anaesthesia alone produced a marked endocrine response when compared with handling and novelty stress associated with the induction of anaesthesia. It is concluded that diethyl-ether anaesthesia causes pronounced increases in the plasma levels of the selected stress hormones and that orbital puncture does not amplify this response. It is suggested that diethyl-ether anaesthesia masks any effects of orbital puncture.     

Histological changes in the orbital region of rats after orbital puncture.

To contribute to the assessment of the degree of discomfort in rats after orbital puncture, we have examined the histological changes in the intraorbital tissues caused by this technique of blood sampling. Orbits were studied from rats euthanized either within 1 min, 4 days, 28 days or 56 days after puncture while under diethyl-ether anaesthesia. The techniques of 2 animal technicians were compared, one using a broken haematocrit capillary and the other using an intact Pasteur's pipette. Non-punctured orbits served as controls. Microscopic slides containing the eye in situ at 2 horizontal levels in the orbital region were examined for 37 parameters; the slides were scored blind and in random order. Orbital puncture caused haemorrhages in the puncture track and, depending on the technique used, also in the periosteum. Four days after puncture, inflammatory reactions were present in the puncture track. Depending on the technique of puncture, these reactions were also seen in the eye muscles and periosteum or in the Harderian gland. Within 4 weeks after puncture, the lesions had healed without detectable scars. The different histological effects of the 2 techniques of orbital puncture are discussed in the light of the characteristics of these techniques.     

The effects of buprenorphine on behaviour in the ACI and BN rat inbred strains

Buprenorphine is a partial mu, kappa agonist that has been shown to influence spontaneous behaviour in animals. Previously, we have demonstrated significant differences in the analgesic response to buprenorphine between the August Copenhagen Irish (ACI)/SegHsd and the Brown Norway (BN)/RijHsd inbred rat strains. The purpose of this study was to determine whether these strains also differed in their behavioural response to buprenorphine in order to provide an additional parameter for the genetic analysis and localization of genes involved in this response. Male and female rats of both strains were used (n = 6/strain/sex) for this study. Each rat was subjected, respectively, to three treatment regimens at 15:00 h: (A) unchallenged; (B) intravenous saline; (C) intravenous buprenorphine (0.05 mg/kg) according to a crossover design. The relative duration (s/h) of locomotion, grooming, drinking and eating behaviour was subsequently determined from 15:30 to 07:00 h using the automatic registration system, Laboratory Animal Behaviour Registration and Analysis System(trade mark). Significant strain differences were observed in unchallenged behaviour between the ACI and the BN rats. ACI rats, but not BN rats, responded to buprenorphine treatment with decreased levels of locomotion, drinking and eating behaviour. The same treatment resulted in an increased grooming behaviour in both strains. Slight but significant sex differences were observed for locomotion and eating in the analysis of variance procedure, but did not reach the level of statistical significance in the multiple comparison procedure. The results of this study emphasize the possibility that strain-specific effects must be taken into account when using behavioural parameters for the assessment of the analgesic effects of buprenorphine in rats.     

Validation of a new system for the automatic registration of behaviour in mice and rats

A newly developed behaviour registration system, Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS) for the automatic registration of different behavioural elements of mice and rats was validated. The LABORAS sensor platform records vibrations evoked by animal movements and the LABORAS software translates these into the corresponding behaviours. Data obtained by using LABORAS were compared with data from conventional observation methods (observations of videotapes by human observers). The results indicate that LABORAS is a reliable system for the automated registration of eating, drinking, grooming, climbing, resting and locomotion of mice during a prolonged period of time. In rats, grooming, locomotion and resting also met the pre-defined validation criteria. The system can reduce observation labour and time considerably.     

Endocrine stress response in jugular-vein cannulated rats upon multiple exposure to either diethyl-ether, halothane/O2/N2O or sham anaesthesia

The main objective of this study was to assess the endocrine stress response to multiple anaesthesia followed by sham anaesthesia in order to detect any memory effects. For this purpose, jugular-vein cannulated rats were subjected to either sham, diethyl-ether or halothane/O2/N2O anaesthesia, and their plasma ACTH, corticosterone, glucose, adrenaline and noradrenaline levels measured. The study had three separate experiments, each consisting of a control and treatment group. In two experiments, the rats were exposed to high or low concentrations (40-15%) of diethyl ether, using either a jar containing cotton soaked in diethyl ether or a vaporizer. In the third experiment, rats were exposed to halothane/O2/N2O. Control animals underwent sham anaesthesia. Blood samples were taken 6 min before and at 5, 15 and 55 min after starting the exposure (t = 0 min). For each variable, the dt5 (level at t = 5 min minus that at t = -6 min) and the cumulative levels over the one-hour period as determined by the area under the curve (AUC) were calculated. Further, the peak levels (Cmax) were determined. The mean time needed to induce anaesthesia was 68, 121 and 55 s for exposure to high and low concentrations of diethyl ether and to halothane/O2/N2O, respectively. Increased noradrenaline and adrenaline dt5 levels were observed only after the first exposure to the high concentration of diethyl ether. Multiple anaesthesia sessions using either diethyl ether or halothane/O2/N2O did not clearly influence adrenaline and noradrenaline levels. Diethyl ether induced a sharp rise in plasma ACTH and glucose levels, irrespective of the concentration used. The response of the ACTH and glucose was similar for single and multiple exposure. An increased response of ACTH, corticosterone and glucose to sham anaesthesia following multiple induction of anaesthesia was observed for the high concentration of diethyl ether only. Halothane/O2/N2O raised plasma glucose without differences between single and multiple anaesthesia sessions. Upon sham anaesthesia following multiple exposures to halothane/O2/N2O, glucose levels were significantly increased. This study indicates that repeated anaesthesia in rats can elicit an increased stress response during subsequent handling and change of environment.     

Behavioural functions of grooming in male Aedes albopictus (Diptera: Culicidae), the Asian tiger mosquito

Flight, walking and resting behaviours of male Aedes albopictus (Skuse) were observed in a flight cage. These behaviours were also associated with repetitive acts of grooming. The effects of sugar meal and period of the day on the total duration, the frequency and the duration of each individual grooming behaviour were studied. Fed or unfed males were individually released in a transparent plastic flight cage and direct visual observations using Observer software were carried out for 2?h during the morning and 2?h in the afternoon. There was no significant difference in the grooming behaviour between morning and afternoon observations. Fed male mosquitoes groomed longer than unfed males. The sugar meal seemed an important influencing factor of the grooming behaviour by giving more energy and making different parts of the mosquito sticky. The functions of grooming behaviour are discussed in regard to other behaviours such as mating, copulation, sensory detection and flight.     

Effect of anaesthesia on the pattern of breathing.

The pattern of breathing of male rats was studied after stimulating respiration with carbon dioxide at different levels of general anaesthesia. Anaesthesia was induced by the inhalation of halothane or by the i.p. injection of urethane. Ventilation values were measured in intubated rats in body plethysmograph. It was found that a linear relationship between minute ventilation and tidal volume was maintained during the decrease of minute ventilation due to deepening of anaesthesia. The slope of the relationship after stimulating respiration with carbon dioxide also diminished during deeper anaesthesia. The duration of inspiration did not alter significantly, despite marked changes in tidal volume. Tidal volume correlated with the duration of expiration at different anaesthesia levels. In vagotomized rats, the duration of expiration shortened as ventilation was depressed by deepening anaesthesia.     

Differences in aerial and terrestrial visual scanning in captive black tufted-ear marmosets (Callithrix penicillata) exposed to a novel environment

Aerial and terrestrial visual scanning were investigated in captive black tufted-ear marmosets, Callithrix penicillata, exposed to a novel environment. Naive adult subjects (n=24) were individually exposed to a figure-eight maze during seven 30-min trials, 48 h apart. Habituation to the maze was observed, as indicated by the significant decrease in locomotion. The frequency of aerial scanning, however, remained elevated throughout the 7 trials, while its duration rapidly increased to high levels. Frequency and duration of terrestrial scanning persisted at constant low rates, differing significantly from aerial scanning. Males and females did not differ significantly. The different impact of aerial versus terrestrial predators could have a significant influence on vigilant behaviour in this species. Thus, visual scanning is an important and highly organized antipredation strategy in marmosets.     

Dose-response effects of orexin-A on food intake and the behavioural satiety sequence in rats

Although intracerebroventricular (i.c.v.) administration of orexin-A has been reported to stimulate food intake and/or feeding behaviour in rats, mice and goldfish, little attention has thus far been paid to its effects on normal patterns of feeding. In the present study, a continuous monitoring technique was used to characterise the effects of this novel neuropeptide on the microstructure of rat behaviour during a 1-h test with palatable wet mash. Particular attention was devoted to the behavioural satiety sequence, in which feeding is followed by grooming and resting. Although results confirmed the hyperphagic effects of orexin-A (3.33-30.0 microg i.c. v.), gross behavioural analysis failed to reveal any reliable effects of peptide treatment on eating, drinking, sniffing, grooming, resting, locomotion or rearing. However, microstructural analysis revealed behavioural effects of orexin-A that are both dose- and time-dependent. At lower doses (3.33-10.0 microg), orexin-A primarily delayed behavioural satiety, i.e. the normal transition from eating to resting. In contrast, the 30 microg dose initially induced a sedative-like effect, significantly suppressing eating and other active behaviours for the first 15-20 min of the test period. This sedative-like effect resulted in a phase-shifting of the entire behavioural sequence with higher than control levels of eating, grooming, locomotion, rearing and sniffing observed over the second half of the test session. Present findings illustrate the advantages of microstructural behavioural analysis and suggest that the hyperphagic response to low doses of orexin-A results largely from a delay in behavioural satiety while that seen in response to high doses may occur in rebound to initial behavioural suppression. Further studies will be required to confirm the identity of the specific orexin receptors (i.e. OX(1) or OX(2)) involved in mediating the dose-dependent behavioural effects reported.     

QTL and systems genetics analysis of mouse grooming and behavioral responses to novelty in an open field

The open field is a classic test used to assess exploratory behavior, anxiety and locomotor activity in rodents. Here, we mapped quantitative trait loci (QTLs) underlying behaviors displayed in an open field, using a panel of 53 BXD recombinant inbred mouse strains with deep replication (10 per strain and sex). The use of these strains permits the integration and comparison of data obtained in different laboratories, and also offers the possibility to study trait covariance by exploiting powerful bioinformatics tools and resources. We quantified behavioral traits during 20‐min test sessions including (1) percent time spent and distance traveled near the wall (thigmotaxis), (2) leaning against the wall, (3) rearing, (4) jumping, (5) grooming duration, (6) grooming frequency, (7) locomotion and (8) defecation. All traits exhibit moderate heritability making them amenable to genetic analysis. We identified a significant QTL on chromosome M.m. 4 at approximately 104 Mb that modulates grooming duration in both males and females (likelihood ratio statistic values of approximately 18, explaining 25% and 14% of the variance, respectively) and a suggestive QTL modulating locomotion that maps to the same locus. Bioinformatic analysis indicates Disabled 1 (Dab1, a key protein in the reelin signaling pathway) as a particularly strong candidate gene modulating these behaviors. We also found 2 highly suggestive QTLs for a sex by strain interaction for grooming duration on chromosomes 13 and 17. In addition, we identified a pairwise epistatic interaction between loci on chromosomes 12 at 36–37 Mb and 14 at 34–36 Mb that influences rearing frequency in males.     

Chronobiological perspectives on myocardial electrophysiological parameters under three types of general anaesthesia in a rat model

The specific aim of the present study, with respect to dependence on the light–dark (LD) cycle under in vivo conditions in spontaneously breathing rats was to review initial state in electrophysiological parameters that may predict the development of heart rhythm disorders in pentobarbital (40 mg/kg), ketamine–xylazine (100 + 15 mg/kg) and zoletil (30 mg/kg) anaesthetized animals. The study was performed using female Wistar rats that were adaptated to an LD cycle (12 h:12 h). Heart rate, PQ and QT intervals were evaluated for their dependence on the LD cycle. The longest PQ interval duration is under zoletil anaesthesia in the light period and the longest QT interval duration is under ketamine–xylazine anaesthesia in both light periods. We concluded that the most significant predisposition toward the development of ventricular arrhythmias originating from disorders of impulse production and conduction occurred under zoletil anaesthesia in the light period; those resulting from disorders in the dispersion of refractory periods occurred under ketamine–xylazine anaesthesia in both the light periods.     

Diel behaviour and time budget of the adult pine weevil Hylobius abietis

The pine weevil [Hylobius abietis (L.); Coleoptera: Curculionidae] has a high economic impact on forest regeneration in Europe. The general biology of the pine weevil has received considerable attention, although there is insufficient knowledge about its diel behaviour and time budget. Therefore, in the present study, the feeding and locomotion behaviour of individual adult weevils on Norway spruce seedlings [Picea abies (L.) Karst.] is observed for 24‐h periods in the laboratory. Both girdled and nongirdled seedlings are used to assess how the behaviour of weevils is influenced by the physiological response of plants to the girdling. The locomotion pattern shows a distinct maximum during the beginning of the dark phase, whereas most feeding occurs during the second half of the dark phase and the first hours of the subsequent light phase. The girdling treatment increases the time that weevils spend on the seedlings during the first part of an observation session, although it has no effect on their feeding pattern. The time budgets of weevils on girdled and nongirdled seedlings are similar. On average, weevils spend 34% of their time in locomotion and 6% on feeding. Females spend more time feeding than males (7.1% versus 4.2%), possibly because they have higher food requirements (e.g. for egg production). Females also spend more time in total on the seedlings than males (26.3% versus 7.0%). The present study reveals, in high temporal resolution, the diel feeding and locomotion behaviour and time budget of male and female pine weevils.     

Effects of carbon-dioxide anaesthesia on the number of instars,larval duration and adult body size of the German cockroach, Blattella germanica

Effects of repeated carbon-dioxide anaesthesia during larval development on the number of moults, larval duration, and the body size of resulting adults were investigated. Concentration of carbon dioxide administered was 70%, and each period of anaesthesia lasted for 3 min. Anaesthesia administered twice a week until the 3rd instar increased the number of instars required to reach maturity. However, repeated anaesthesia after the 4th instar scarcely influenced the number of instars. On the other hand, repeated anesthesia prolonged larval duration whenever administered during the early or late instars. It is thus suggested that anaesthesia by carbon dioxide during larval development has two distinct effects: increasing the number of moults and prolonging larval duration. The former is stage specific and effective only during early instars, whereas the latter is effective during any particular instars until the last instar. The body size of resulting adults remained almost the same as for controls even after the repeated carbon-dioxide anaesthesia as many as 14 times. Some further observations concerning correlations between the number of moults, larval duration and adult body size are shown, and differences between the sexes regarding the number of instars and larval duration are also presented.     

Chronobiological study of the pharmacological response of rats to combination ketamine-midazolam

Ketamine is commonly administered in combination with benzodiazepines to achieve surgical anaesthesia in rats. The aim of the present study was to analyze the pharmacological response of the combination ketamine-midazolam injected intraperitoneally at different times of day to rats. The study was conducted in July 2003, during the winter in the Southern hemisphere. Female prepuberal Sprague-Dawley rats synchronized to a 12h light:12h dark cycle (light, 07:00-19:00h) were used as experimental animals. A combination treatment of ketamine (40 mg/kg) and midazolam (2 mg/kg) was administered to five different clock-time groups of rats (n=7/group). Duration of the latency period, ataxia, loss-of-righting reflex (LRR), post-LRR ataxia, and total pharmacological response were assessed by visual assessment. Significant treatment-time differences were detected in the duration of LRR, post-LRR ataxia, and total pharmacological response duration. The longest pharmacological response occurred in rats injected during the light (rest) phase, and the shortest pharmacological response occurred in rats injected during the dark (activity) phase. Cosinor analysis documented circadian rhythmicity in the duration of post-LRR ataxia. The findings of the study indicate the duration of CNS-depression of the ketamine-midazolam combination exhibits treatment-time-dependent variation in the rat.     

Age-related changes in activity of cerebellum Purkinje cells,shape of the complex spike,and locomotion of Wistar rats under effect of ethanol

The work deals with study of peculiarities of effect of ethanol upon the Purkinje cell activity, shape of the complex spike, and locomotion of rats at different stages of ontogenesis, such as the stage of the morphofunctional maturation of the cerebellar cortex, the mature stage, and in the process of aging. The experiments were carried out on three age groups of Wistar rats: rat pups (2 weeks), adult rats (3–6 months), and senile animals (22–26 months). The administration of ethanol has been established to produce an increase in frequency of simple spikes, a decrease in frequency of complex spikes, a shortening of duration of depression of simple spikes, a decrease in the total duration of the complex spike, the number and frequency of its impulses as well as reduction of the motor activity of animals of all age groups. The change of the majority of the studied parameters occurred by the common temporal scheme. The earliest responding were the rat pups, later-the adult rats, and the last-the animals of the senior group. The stronger effect of ethanol was observed in adult rats. Their differences of all studied parameters, as compared with rat pups and senile animals, were characterized on the whole by the longer period of time and by the higher percent of changes relative to the initial values. Analysis of the obtained results has shown that the most pronounced changes in parameters of the cerebellum Purkinje cell activity and of the complex spike shape corresponded to the more significant decrease in the locomotion level, i.e., were recorded in adult rats. Thus, the work has demonstrated different sensitivity to administration of ethanol in the Wistar rats at different stages of ontogenetic development.     

Effects of basolateral or corticomedial amygdaloid lesions on grooming,consummatory, and locomotor behaviours in rats

Grooming behaviour in rats was induced by limited water access and by water spray before and after corticomedial or basolateral amygdaloid lesions or control operations. Corticomedial lesions produced some attenuation of grooming induced by limited water access but increased grooming induced by water spray. Basolateral lesions did not consistently affect grooming. There was an increase in ambulation time following basolateral lesions and a relative decrease in feeding time following corticomedial lesions. There were no effects of either lesion on drinking or rearing. Detailed examination of both grooming and non-grooming behaviours provided little evidence for lesion induced disruption of response sequencing.     

Comparative studies with cyclic and linear somatostatin on active avoidance behaviour and open-field activity in rats

The action of cyclic and linear somatostatin on active avoidance and open-field behaviour was investigated in rats. Both peptides inhibited the extinction of the avoidance behaviour and slightly but not significantly increased the intertrial activity (ITR). Both peptides increased ambulation and rearing activity in a dose related fashion, while the grooming and defecation rates were not changed. Since both peptides are capable of increasing the locomotor performance this action might contribute to the effect exerted on the extinction of active avoidance behaviour.     

Chronobiological Study of the Pharmacological Response of Rats to Combination Ketamine–Midazolam

Ketamine is commonly administered in combination with benzodiazepines to achieve surgical anaesthesia in rats. The aim of the present study was to analyze the pharmacological response of the combination ketamine–midazolam injected intraperitoneally at different times of day to rats. The study was conducted in July 2003, during the winter in the Southern hemisphere. Female prepuberal Sprague-Dawley rats synchronized to a 12 h light:12 h dark cycle (light, 07:00–19:00 h) were used as experimental animals. A combination treatment of ketamine (40 mg/kg) and midazolam (2 mg/kg) was administered to five different clock-time groups of rats (n = 7/group). Duration of the latency period, ataxia, loss-of-righting reflex (LRR), post-LRR ataxia, and total pharmacological response were assessed by visual assessment. Significant treatment-time differences were detected in the duration of LRR, post-LRR ataxia, and total pharmacological response duration. The longest pharmacological response occurred in rats injected during the light (rest) phase, and the shortest pharmacological response occurred in rats injected during the dark (activity) phase. Cosinor analysis documented circadian rhythmicity in the duration of post-LRR ataxia. The findings of the study indicate the duration of CNS-depression of the ketamine–midazolam combination exhibits treatment-time-dependent variation in the rat.