Remote monitoring of electroencephalogram, electrocardiogram, and behavior during controlled atmosphere stunning in broilers: implications for welfare.

This study examined the welfare implications of euthanizing broilers with 3 gas mixtures relevant to the commercial application of controlled atmosphere stunning (CAS). Birds were implanted/equipped with electrodes to measure brain activity (electroencephalogram, EEG) and heart rate. These signals were recorded using a purpose-built telemetry-logging system, small enough to be worn by each bird in a spandex backpack. The birds were euthanized in a scaled-down CAS apparatus consisting of a conveyor belt passing through 2 compartments. Three gas environments were applied (8 birds per treatment): 1) anoxia (N(2) with <2% residual O(2), in both compartments), 2) hypercapnic anoxia (N(2) with 30% CO(2) and <2% residual O(2), in both compartments), and 3) a 2-phase approach with a hypercapnic hyperoxygenated anesthetic phase (40% CO(2), 30% O(2), and 30% N(2), in the first compartment, 80 s) followed by a second euthanasia phase (80% CO(2) in air, in the second compartment). All 3 CAS approaches effectively achieved nonrecovery states, and time to loss of consciousness for each bird was determined by visual determination of isoelectric EEG and by calculation of the correlation dimension of the EEG. Hypercapnic anoxia resulted in rapid unconsciousness and death; both anoxic treatments were associated with early onset prolonged wing flapping and sustained tonic convulsions as displayed in the electrophysiological recordings. These responses were seen in the period when consciousness remained a possibility. Hypercapnic hyperoxygenation (the 2-phase approach) was associated with respiratory disruption, but this treatment eliminated initial clonic convulsions in the stunning process, and tonic convulsions were not seen. These results suggest that the presence of O(2) in the first stage of CAS is associated with an absence of potentially distressing behavioral responses. The respiratory discomfort associated with hypercapnic hyperoxygenation is an issue. We propose that this may be compensated by a more gradual induction to unconsciousness, which eliminates the impact of other potentially negative experiences.

N150 in amygdalar ERPs in the rat: is there modulation by anticipatory fear?

The hypothesis was tested whether the amygdalar N150 of rats, a slow, negative component in the event-related potential from the lateral amygdala, is sensitive to a state of anxious anticipation. A conditioning procedure was applied in which a series of six auditory stimuli was followed by a shock when presented alone, but not when the auditory stimuli were preceded by a visual stimulus. Heart rate recordings confirmed that the auditory stimulus train induced a state of increasing anticipatory fear and that this condition was modulated by the visual stimulus. During behavioral training, a N150 appeared in the amygdalar event-related potential evoked by the auditory stimuli, replicating previous findings. However, the amplitude of the N150 was not affected by whether or not the visual stimulus had been presented before. These results failed to support the idea that the N150 is related to the expectancy of an aversive event. An alternative interpretation, emphasizing the increase in arousal and attention that is inherent to aversive learning, is discussed.

Are there different mechanisms of synchronization in the course of spike-wave discharges (SWDs) burst development in WAG/Rij rats?

In WAG/Rij rats the pair linear correlation r was calculated for bipolar recordings in fronto-temporal, fronto-occipital and occipito-temporal zones of both hemispheres as well as in paleocerebellar cortex (culmen). It was shown that development of SWD bursts resulted in interhemispheric decreases of correlation between the right occipito-temporal cortical region on one side, and left fronto-temporal on the contralateral side. Towards the end of SWD, we found an increased interhemispheric correlation between left fronto-temporal and right fronto-occipital cortical zones, as well as, between both fronto-temporal zones. Paleocerebellum correlates at a weak to moderate level during different periods of SWD burst generation.

Behavioral transitions modulate hippocampal electroencephalogram correlates of open field behavior in the rat: support for a sensorimotor function of hippocampal rhythmical synchronous activity.

A clear relationship exists between moment-to-moment behavioral elements and hippocampal rhythmical synchronous activity (RSA) (theta rhythm). However, behavioral elements are not isolated events but are part of behavioral sequences in a context of behavioral activity. By concurrently monitoring open field behavior and hippocampal EEG, EEG correlates of open field behavior in relation to preceding and following behavior were studied in Sprague Dawley rats to determine whether the behavioral context influences EEG correlates of behavior. Results show that preceding and subsequent behavioral patterns influenced the spectral power correlates of behavior. RSA power was increased when a "type 1 behavior" (voluntary movement) preceded the behavior compared with when a "type 2 behavior" (automatic movement, awake immobility) preceded it. The modulating effect of behavioral transitions was shown for several types of behaviors, and systematic modulation of hippocampal EEG correlates of behavior was demonstrated. The present report shows that the strong and systematic relationship between hippocampal RSA and behavior is modulated by the behavioral-sequential context. Thus, in addition to the well established relationship between RSA and motor activity, a second nonmotor process seems to contribute to hippocampal RSA. A likely candidate is a sensory process, which is in accordance with theories on the sensorimotor function of hippocampal RSA.

The relationship between hippocampal EEG theta activity and locomotor behaviour in freely moving rats: effects of vigabatrin.

The relationship between hippocampal electroencephalogram (EEG) theta activity and locomotor speed in both spontaneous and forced walking conditions was studied in rats after vigabatrin injection (500 mg/kg i.p.). Vigabatrin increased the percentage of time that rats spent being immobile. During spontaneous walking in the open field, the speed of locomotion was increased by vigabatrin, while theta peak frequency was decreased. Vigabatrin also reduced the theta peak frequency during forced (speed controlled) walking. There was only a weak positive correlation (r=0.22) between theta peak frequency and locomotor speed for the saline condition. Furthermore, vigabatrin abolishes the weak relationship between speed of locomotion and theta peak frequency. Vigabatrin and saline did not differ in the slope of the regression line, but showed different offset points at the theta peak frequency axis. Thus, other factors than speed of locomotion seem to be involved in determination of the theta peak frequency.

Audiogenic seizures associated with a cortical spreading depression wave suppress spike-wave discharges in rats.

To study the role of the cortex and sub-cortical structures in the generation of epileptic spike-wave discharges in more detail, cortical and striatal activity was eliminated by the induction of spreading depression in a non-invasive way. EEG and DC potentials were recorded from the cortex and striatum of WAG/Rij rats. Several of these rats show two forms of generalised epilepsy: spontaneously occurring non-convulsive absence seizures, together with convulsive audiogenic seizures. The latter can be evoked by a brief sound stimulation, provoking a fit of wild running, which is regarded as the first phase of an audiogenic seizure. In a majority of fits the cortical DC potential does not show main changes, while the spontaneously occurring spike-wave discharges are briefly suppressed for some minutes. In a minority of fits, however, audiogenic seizures are associated with a spreading depression wave, clearly expressed in the cortical DC potential. This wave is bilaterally initiated in the cortex and propagates to the caudate nucleus of the striatum. In these cases spontaneously occurring spike-wave discharges are fully suppressed for about 1 h. It is suggested that cortical spreading depression, triggered by a short audiogenic seizure, induces a long-lasting suppression of spike-wave discharges. These results are in line with the concept that spike-wave discharges are originally initiated in the cortex, as proposed by the 'cortical focus' theory. The precise role of the striatum remains less clear, although this structure seems not to play a pivotal role in spike-wave generation.

Ictal stimulus processing during spike-wave discharges in genetic epileptic rats.

In the present experiment it was investigated whether and to what extent auditory information processing is possible during the presence of spike-wave discharges in rats. To that end, WAG/Rij rats which are an animal model for absence epilepsy, were provided with cortical electrodes for the registration of the electroencephalogram (EEG). The animals were first trained in an appetitively motivated conditioning paradigm to learn to discriminate between two auditory stimuli with equal duration and frequency but with different intensities. Next, the stimuli were presented in the test phase in pseudorandom order during spike-wave discharges. The reactivity of the ongoing EEG was analysed. It was found that the presentation of the reinforced stimulus induced a larger number of aborted spike-wave discharges than the non-reinforced stimulus, regardless of the intensity of the stimuli. This implies that during generalised spike-wave discharges the brain is still capable of evaluating the meaning of an ictally presented stimulus. It also shows that sensory, attentional and mnemonic processes are at least partially intact during the occurrence of a spike-wave discharge. The results of the present study are largely in agreement with results on human spike-wave activity-related cognitive disturbances. Moreover, they may lead to a refinement of the concept of epileptic consciousness and may emphasise the heuristic value of rodent models for studying both ictal and interictal information processing.

Enhanced re-habituation of the orienting response of the human event-related potential.

Previous studies found the amplitude of the orienting response (OR) of the human event-related potential to decrease with repeated stimulus presentations. This decrease has been suggested to reflect short-term habituation and/or long-term habituation, both of which are learning processes. However, this earlier research failed to provide direct evidence supporting this claim. The present study attempted to show that the OR pattern shares one important feature of habituation: an enhanced response decrement across stimulus-presentation blocks (enhanced re-habituation). Participants received four blocks of 25 auditory stimulus presentations and showed an OR decrement both within (short-term habituation) and across (long-term habituation) blocks. Importantly, the OR decreased more rapidly during later than initial trial blocks, suggesting enhanced re-habituation. The latter result supports the notion that the amplitude decrement reflects an elementary learning process.

Strain differences in hippocampal EEG are related to strain differences in behaviour in rats.

To date, EEG studies towards strain differences have focussed on pharmacologically altered or pathological EEG activity, but only few studies have investigated strain differences and normal EEG activity. A strong relation between behaviour and EEG activity has been demonstrated, especially for hippocampal EEG activity. This relation is known to be similar across species and strains, but no direct comparisons between rat strains within one study have been made. This study compared two rat strains (Sprague-Dawley and Long-Evans) with regard to open-field behaviour and concurrent hippocampal EEG recordings. A detailed behavioural analysis was made and spectral power was calculated for corresponding EEG activity in eight frequency bands. The two strains differed in exploratory activity and in spectral power in the 9-10-Hz frequency band (high frequency rhythmical slow activity [RSA] 6-10 Hz). Long-Evans rats showed higher exploratory activity and higher 9-10 Hz spectral power for voluntary movement and sniffing behaviours. Our results demonstrated these behaviour-specific strain differences in RSA power, although the relation between EEG and behaviour within each strain was similar. The strain differences in EEG were interpreted in relation to strain differences in exploratory behaviour, attributing the differences to a main motor component but also to a smaller sensory component integrated in exploratory behaviour. This is in accordance with theories on the sensory-motor function of the hippocampus and hippocampal theta activity.

Animal models for information processing during sleep.

Information provided by external stimuli does reach the brain during sleep, although the amount of information is reduced during sleep compared to wakefulness. The process controlling this reduction is called 'sensory' gating and evidence exists that the underlying neurophysiological processes take place in the thalamus. Furthermore, it is clear that stimuli given during sleep can alter the functional state of the brain. Two factors have been shown to play a crucial role in causing changes in the sleeping brain: the intensity and the relevance of the stimulus. Intensive stimuli arouse the brain, as well as stimuli having a high informational impact on the sleeping person. The arousal threshold for important stimuli is quite low compared to neutral stimuli. A central question in sleep research is whether associative learning, or in other words the formation of new associations between stimuli, can take place in a sleeping brain. It has been shown that simple forms of learning are still possible during sleep. In sleeping rats, it is proven that habituation, an active, simple form of learning not to respond to irrelevant stimuli, can occur. Moreover, there is evidence for the view that more complex associations can be modulated and newly formed during sleep. This is shown by two experimental approaches: an extinction paradigm and a latent inhibition (pre-exposure) paradigm. The presentation of non-reinforced stimuli during sleep causes slower extinction compared to the same presentation of these stimuli during wakefulness. Consistently, the suppressive capacity of a stimulus in the latent inhibition paradigm is less when previously pre-exposed during sleep, as compared to pre-exposure during wakefulness. Thus, while associative learning is not completely blocked during sleep, aspects of association formation are clearly altered. However, animal studies also clearly indicate that complex forms of learning are not possible during sleep. It is hypothesised that this restriction in information processing during sleep is due to the considerable reduction of incoming information by the sleeping brain. This reduction may serve to protect the sleep process.

Causes and consequences of pathogenic processes in evolution: implications from experimental epilepsy in animals.

Examples from experimental epilepsy in animals are used to illustrate the view that a crucial role of the transfer of mechanisms from compensatory into pathogenic (e.g. lethal ones in the course of a disease), is played by the power of pathologic stimuli. In the genesis of epilepsy it is suggested that a critical increase of endogenous factors may underlie the conversion of the absence form of epilepsy into a generalized self-supporting form. The ability to precipitate endogenous self-augmenting mechanisms of diseases may have increased in the course of evolution. The lethal result of a serious pathogenic process leads to the suggestion that organisms cope with the disease by dying. This prevents spreading of the putative infectious disease within the population. This mechanism of disease aggravation could play a role in the survival of the species and in further evolutionary progress. This may explain why certain species may have survived in evolution and supports the theory of synthetic evolution.

Dualism and uniformism in sleep.

The phenomenological evidence for distinguishing between REM and NREM sleep is overwhelming. However, this difference has only been found thanks to electrophysiological analytical methods, and is practically non existent in phenotypic terms, i.e., observable with the naked eye. It is well accepted that the selective pressure determining evolutionary changes can only work upon phenotypic differences. Hence, it follows that the differences between REM and NREM could not have been selected through evolution and this implies that, in functional terms, both states could be equivalent.

Rat models of genetic absence epilepsy: what do EEG spike-wave discharges tell us about drug effects?

Electroencephalographic studies in the WAG/Rij rats of Nijmegen and genetic absence epileptic rats of Strasbourg (GAERS), two genetic models for human generalized absence epilepsy, illustrate the usefulness of drug-electroencephalogram (EEG) interaction studies. In the EEG of both types of rats, spontaneously occurring spike-wave discharges are present. For drug discovery, a model with predictive validity is imperative, and both the WAG/Rij and the GAERS models seem adequate. The present paper discusses effects on spike-wave discharges of various compounds that are clinically used. Not only new antiepileptic drugs, such as remacemide, loreclezole, lamotrigine, tiagabine, gabapentin, progabide and levetiracetam are evaluated, but also drugs used for other purposes, such as etomidate and fentanyl-fluanisone for anesthesia, opioidergic drugs and drugs used for strokes. It is shown that some new antiepileptic drugs, such as tiagabine, have spike-wave discharge-increasing properties, while other drugs are worth studying in clinical trials for antiabsence treatment. Furthermore, it is shown that many commonly used drugs such as analgesics, anesthetics and drugs to treat stroke generally enhance spike-wave discharges. It can be concluded that EEG monitoring is imperative for the discovery and development of potentially antiepileptic compounds and that genetic rat models such as the WAG/Rij or GAERS, to a large extent, can reliably predict clinical efficacy of various types of compounds as well as alert us of potentially adverse effects.

[The cortico-thalamic theory for generalised spike-wave discharges]. / Kortikotalamicheskaia teoriia proiskhozhdeniia generalizovannykh pik-volnovykh razriadov.

The origin of generalized absence epilepsy is still not known. In the last century, four theories have dominated the debate about the origin of the bilateral synchronous generalized spike-wave discharges associated with absence seizures: the "centrencephalic" theory [Penfield and Jasper], the "cortical" [Bancaud, Niedermeyer, Luders], the "cortico-reticular" theory [Gloor, Kostop[oulos, Avoli] and the "thalamic clock" theory [Buzsaki]. There is now some evidence that absence epilepsy, as studied in the WAG/Rij model, is a corticothalamic type of epilepsy. A new hypothesis is proposed which suggests that a cortical focus in the somatosensory cortex is driving the widespread corticothalamic networks during spontaneous absence seizures. This modern theory was given the name "hot spot' theory" [Meeren et al., 2002]. According to the present view three brain structures are critically involved and their integrity seems a minimal and sufficient condition for the occurrence of spike-wave discharges. Firstly, the reticular thalamic nucleus is involved and most likely its rostral pole. Secondly, the thalamocortical relay cells in the ventrobasal complex play a role and, thirdly and most importantly, the cerebral cortex with its epileptic zone. The zone in which the epileptic focus seems to be localised is located on the somato-sensory cortex, and more precisely in the area on which the peri-oral region including the upper lip, projects.

Electroencephalogram functional connectivity between rat hippocampus and cortex after pilocarpine treatment.

The muscarinic agonist pilocarpine has been shown to increase the duration and total number of episodes presenting theta rhythm-simultaneously in hippocampus and cortex-in rats during the waking states. Theta waves are suggested to be involved in the flow of information between hippocampus and cortex during memory processes. This work investigates this functional interdependence using the spectral and phase synchronization analysis of the electroencephalogram (EEG) theta band recorded in these brain structures of rats after pilocarpine treatment. Pilocarpine was used at doses devoid of epilepticus-like seizures effects in conscious freely moving rats. The results showed that pilocarpine administration significantly increased the relative theta power during the waking states in the cortex, but not in the hippocampus of rats. Additionally, the EEG coherence between the hippocampal EEG theta band and that arising at the frontal cortex increased after pilocarpine treatment but only during the waking states. This result reveals an increase of the linear correlation between the theta waves of these two brain structures after pilocarpine treatment during the waking states. Moreover, phase synchronization results showed an effective phase locking with non-zero phase difference between hippocampus and frontal cortex theta waves that remained after pilocarpine treatment. Therefore, pilocarpine seems to reinforce the neural transmission waves from the hippocampus toward the cortex during waking. In conclusion, the present EEG study could suggest an effect of the muscarinic cholinergic agonist pilocarpine on the hippocampal-cortical functional connectivity.

Effects of pilocarpine on the cortical and hippocampal theta rhythm in different vigilance states in rats.

It has been suggested that theta rhythm gates the flow of information between the hippocampus and cortex during memory processes. The cholinergic system plays an important role in regulating vigilance states and in generating theta rhythm. This study aims to analyse the effects of the muscarinic agonist pilocarpine (120 and 360 microg, i.c.v.) on hippocampal and frontal cortical theta rhythm during several vigilance states in rats. Pilocarpine injection increased the duration and number of episodes with theta activity, particularly when theta rhythm appeared during waking states in the cortex and hippocampus simultaneously. It seems that the effects of pilocarpine are related to the appearance of cortical theta activity in waking states, and suggest that pilocarpine could modify the transference rate of information from the hippocampus to cortex in rats during wakefulness states, in relation to the postulated effect of cholinergic system modulating memory consolidation.

An effective correlation dimension and burst suppression ratio of the EEG in rat. Correlation with sevoflurane induced anaesthetic depth.

BACKGROUND AND OBJECTIVE: Anaesthesiologists need parameters that measure the depth of anaesthesia. In the context of this need, the present study investigated in rats how two variables from the electroencephalogram, the burst suppression ratio and effective correlation dimension correlated with a measure of anaesthetic depth as measured in the strength of a noxious withdrawal reflex. METHODS: Eight rats were exposed to different inspiratory concentrations of sevoflurane, each rat in two separate experiments. In the first experiment, spontaneously breathing animals could move freely and no painful stimuli were applied. In the second experiment, in mechanically ventilated restrained anaesthetized rats, the withdrawal reflex was measured every 80 s. In both experiments the electroencephalogram was continuously recorded. The concentration in the effector compartment was estimated using a first order two compartment model. Correlation dimension was computed following the Grassberger/Procaccia/Takens approach with optimized parameter settings to achieve maximum sensitivity to anaesthetic drug effects and enable real-time computation. The Hill, equation was fitted to the data, describing the effect as a function of sevoflurane concentration. RESULTS: Good correlations of Depth of Anaesthesia with correlation dimension as well as burst suppression ratio were established in both types of experiments. Arousal by noxious stimuli decreased burst suppression ratio and increased correlation dimension. The effective sevoflurane concentration associated with 50% of the maximum effect (C50) was higher in experiment II (stimulation) than in experiment I (no stimulation): i.e. for correlation dimension 2.18% vs. 0.60% and for burst suppression ratio 3.07% vs. 1.73%. The slope factors were: gammaCD = 4.15 vs. gammaCD = 1.73 and gammaBSR = 5.2 vs. gammaBSR = 5.4. Correlation dimension and burst suppression ratio both correlated with the strength of the withdrawal reflex with correlation coefficients of 0.46 and 0.66 respectively (P < 0.001). CONCLUSIONS: Both correlation dimension and burst suppression ratio are related to anaesthetic depth and are affected by noxious stimuli. The relationship between anaesthetic depth and burst suppression ratio is confirmed and the potential of correlation dimension is demonstrated.

Genetic animal models for absence epilepsy: a review of the WAG/Rij strain of rats.

Based on the reviewed literature and the data presented in this paper, conclusions can be drawn with respect to the validity of the WAG/Rij strain of rats as a model for absence epilepsy in humans. The view that the WAG/Rij model has "face validity" is supported by the simultaneous presence of clinical and electroencephalographic signs characterizing absences in rat and humans, by the decrease in responsiveness during the presence of spike-wave discharges in both species, by the agreement between model and patient with respect to the preferential occurrences of spike-wave discharges at transitions in states of vigilance, by the corresponding modulation of spike-wave discharges by physical and mental activities in both and, finally, by the fact that in both humans and rats absence epilepsy is inherited. Against this view, however, argue two points. In rats, absences appear after puberty and are maintained during life, while in humans the seizures occur before puberty and then disappear or convert to more serious forms of epilepsy. The second point is the frequency difference of the spikes and waves in the discharge train: 8-10 Hz in the rat and 3 Hz in the human (though there are no a priori reasons why the frequency of spike waves in the burst must be the same in all species). The absence model also has predictive validity, based on pharmacological data that demonstrate the specificity of certain drugs as being effective in convulsive epilepsies and not in absence epilepsy. So far, all drugs affect spike-wave activity the same way in rats and humans, with lamotrigine being, perhaps, the only exception. Furthermore, sleep deprivation is a powerful provocation for the initiation of spike-wave discharges in both rats and humans. Potential explanations for the presence of absence seizures in rats have been found at the levels of activities in networks and nuclei; of neurons, membrane properties, and ion channels; of proteins and enzymes; and, finally, of genes and chromosomes. Further descriptions of the cellular processes can be found extensively in the literature (e.g., McCormick and Contreras, 2001) and those of the thalamo-cortico-thalamic network in this review as well as in others (Avanzini et al., 1999). Considering the extensive involvement of the phenomena under study with theoretical issues such as the relationship between sleep spindles and spike-wave discharges, and the origin of seizure activity, it can be concluded that the model also has construct validity as far as the present neurobiological theories holding for absence epilepsy in humans are concerned. The WAG/Rij model can therefore be recommended for continued use in evaluating antiepileptic drugs for monotherapy and polytherapy, as well as for the toxicological side effects of putative new antiabsence drugs.

[Pregnancy in WAG/Rij rats--changes in the levels of progesterone, estradiol and generalized absence epilepsy]. / Beremennost' krys linii WAG/Rij--izmeneniia urovnei progesterona i éstradiola i generalizovannoi absansnoi épilepsii.

Progesterone and oestradiol serum level was investigated in WAG/Rij rats with genetically determined absences. Blood samples were drawn before and after the pregnancy following the parturition. The serum concentration of progesterone increased after the 3rd day of pregnancy. There is no increasing of oestradiol during pregnancy as large as this. The progesterone is kept high to the 18th day of pregnancy and drastically decreased before the parturition. Common duration of absences--spontaneous spikewave discharges (SWD), frequency and the duration of every SWD decreased from 3rd to 19th days of pregnancy before the parturition. On the basis of these data and modern investigations, regulation of GABAA receptor expression during pregnancy by progesterone (Brusaartd A. B. et al., 1999) it can be assumed that the changes in the parameters of SWD are possibly correlated with the progesterone changes in serum during pregnancy in WAG/Rij rats.