Decapitation in rats: latency to unconsciousness and the 'wave of death'

The question whether decapitation is a humane method of euthanasia in awake animals is being debated. To gather arguments in this debate, obsolete rats were decapitated while recording the EEG, both of awake rats and of anesthetized rats. Following decapitation a fast and global loss of power of the EEG was observed; the power in the 13-100 Hz frequency band, expressing cognitive activity, decreased according to an exponential decay function to half the initial value within 4 seconds. Whereas the pre-decapitation EEG of the anesthetized animals showed a burst suppression pattern quite different from the awake animals, the power in the postdecapitation EEG did not differ between the two groups. This might indicate that either the power of the EEG does not correlate well with consciousness or that consciousness is briefly regained in the anesthetized group after decapitation. Remarkably, after 50 seconds (awake group) or 80 seconds (anesthetized group) following decapitation, a high amplitude slow wave was observed. The EEG before this wave had more power than the signal after the wave. This wave might be due to a simultaneous massive loss of membrane potentials of the neurons. Still functioning ion channels, which keep the membrane potential intact before the wave, might explain the observed power difference. Two conclusions were drawn from this experiment. It is likely that consciousness vanishes within seconds after decapitation, implying that decapitation is a quick and not an inhumane method of euthanasia. It seems that the massive wave which can be recorded approximately one minute after decapitation reflects the ultimate border between life and death. This observation might have implications in the discussions on the appropriate time for organ donation.     

Evaluating methods of mouse euthanasia on the oocyte quality: cervical dislocation versus isoflurane inhalation

Cervical dislocation is a commonly used method of mouse euthanasia. Euthanasia by isoflurane inhalation is an alternative method which allows the sacrifice of several mice at the same time with an anaesthesia, in the aim to decrease pain and animal distress. The objective of our study was to assess the impact of these two methods of euthanasia on the quality of mouse oocytes. By administering gonadotropins, we induced a superovulation in CD1 female mice. Mice were randomly assigned to euthanasia with cervical dislocation and isoflurane inhalation. Oviducts were collected and excised to retrieve metaphase II oocytes. After microscopic examination, oocytes were classified into three groups: intact, fragmented/cleaved and atretic. Intact metaphase II oocytes were employed for biomedical research. A total of 1442 oocytes in the cervical dislocation group were compared with 1230 oocytes in the isoflurane group. In the cervical dislocation group, 93.1% of the oocytes were intact, versus 65.8% in the isoflurane group (P ≤ 0.001). In light of these results, we conclude that cervical dislocation is the best method of mouse euthanasia for obtaining intact oocytes for biomedical research.     

Effects of chlorpromazine on pattern and flash ERGs and VEPs compared to oxazepam and to placebo in normal subjects

Antidopaminergic drugs delay the pattern-reversal VEP (P-VEP) and the flash VEP (F-VEP) and, in separate studies, reductions in the amplitude and increases in the latencies of scotopic ERGs have been reported. This study investigated the effects of chlorpromazine (CPZ) on the pattern ERG (P-ERG), P-VEP, flash ERGs and VEPs and oscillatory potentials (OPs). Normal volunteers (N = 15) were administered a placebo, or a single dose of CPZ 100 mg or oxazepam (OZP) 15 mg at weekly intervals, in a double-blind crossover design. A gold foil-ipsilateral ear derivation and an Oz′-Fz derivation were used for the ERG and VEP recordings, respectively. The latencies of ‘mixed’ and cone ERGs were significantly prolonged after CPZ compared to both placebo and to OZP. Amplitudes of rod- and cone-dominated ERGs were reduced following CPZ administration. All components of the OPs were significantly delayed after CPZ administration. No significant intertreatment differences were found in the F-VEP results. The P-ERG P50 peak and the P-VEP N70 and P100 peaks were significantly delayed after CPZ in the case of 28′ checks but not 55′ checks. Retinocortical times and P-ERG and P-VEP amplitudes were not significantly affected. In contrast to CPZ, the administration of OZP had virtually no significant effects compared to placebo. These findings suggest that the antidopaminergic CPZ has a primary effect on retinal electrophysiology. Similar findings have been reported in Parkinson's disease and in animal models.     

Scalp-recorded oscillatory potentials evoked by transient pattern-reversal visual stimulation in man

Replicable oscillatory potentials, time-locked to pattern stimuli (9.0° central; counterphase reversal at 2.13 Hz) were dissociated from conventional, broad-band VEPs recorded in healthy volunteers at occipital scalp locations by high-pass digital filtering at 17.0–20.0 Hz. Nine consecutive wavelets were identified with a 56.4 ± 8.4 msec mean latency of the first replicable wavelet and mean peak-to-peak amplitude varying between 0.9 and 2.0 μV. The first 2 wavelets had significantly shorter latencies than wave N70 of unfiltered VEP, whereas the last 2 wavelets had longer latencies than N145. Latency and amplitude values varied as a function of contrast and spatial frequency of the stimulus, with shorter latencies and larger amplitudes at 60–90% contrast level and tuning of amplitude at 5.0 c/deg. All wavelets were correlated with wave P100 of unfiltered VEP, while a correlation with N70 of VEP was observed only for those wavelets with latencies in the range of wave P100. Two patients with documented brain lesions involving the visual system are described as examples of oscillatory responses occurring irrespective of filter bandpass and instead of the expected conventional VEP when the generation of these is interfered with by brain pathology. A substantial cortical contribution to the origin of the oscillatory response is conceivable. It is suggested that the oscillatory response to pattern-reversal stimulation reflects events in the visual system that are parallel to, and partly independent of, the conventional VEP, with potential application in research or for clinical purposes.     

Multichannel visual evoked potentials in migraine

Multichannel recordings of visual evoked potentials (VEPs) have proved to be useful in the evaluation of visual field defects. We studied the topographic distribution of transient VEPs in 15 migraine patients (8 with visual aura and 7 without) and 15 age-matched controls during the migraine-free interval. All the subjects included in the study had normal visual fields. VEPs were recorded from 9 electrodes placed on the posterior scalp. Stimuli were full-field and hemifield reversing square wave grating patterns of medium spatial frequency (4 c/deg). The groups did not show significant differences in latencies and amplitudes of the major components (N70, P100) recorded from the midline. However, migraine patients with visual hemianopic aura showed definite asymmetries in the VEP amplitude distribution. Significantly reduced, absent or polarity-invered VEP responses were recorded ipsilateral to the side of the prodromic visual symptoms. Direct comparison of affected and unaffected hemispheres by partial field stimulation confirmed these findings. According to the VEP cortical generator theory, these abnormalities suggest a functional anomaly consistent with the clinical syndrome and detectable also in the migraine-free interval. None of the migraine patients without aura or the controls showed VEP amplitude asymmetries. We conclude that multichannel VEP recordings may discriminate between different subtypes of migraine and contribute important physiopathological information to the study of this disease.     

Effect of H2-receptor antagonists, cimetidine and ranitidine on reproductive functions in male mice.

Na+,K(+)-ATPase and Ca(2+)-ATPase in testis were inhibited with an oral administration of cimetidine and ranitidine. Cimetidine at dose level of 100 and 30 mg while ranitidine at 70 and 10 mg per kg body wt inhibited the enzyme activities, 24 hr after single administration or daily administration for 15-days. Mg(2+)-ATPase activity was increased with cimetidine while ranitidine inhibited the enzyme. Michaelis-Menten kinetic characteristics revealed mixed type of inhibition for Na+,K(+)-ATPase with cimetidine, whereas it was noncompetitive for Ca(2+)-ATPase with cimetidine as well as ranitidine administration. Inhibition of Na+,K(+)-ATPase with ranitidine was also of noncompetitive type. Mg(2+)-ATPase behaved differently with administration of ranitidine at both the time points used i.e. noncompetitive type of inhibition after 24 hr and mixed type after 15-days. Histologically, signs of degeneration of testicular elements appeared after administration of cimetidine with a significant decrease in tubular diameter and germinal epithelial cell height. Ranitidine administration did not produce any change in the seminiferous tubules of testis. Scanning electron microscopy of spermatozoa from cimetidine-treated mice exhibited distinct departure from the normal morphology such as, (i) breaks at various places along distal portion of the tail, (ii) roughening, wrinkling and disorganization of plasma membrane of the head region, (iii) decapitation of the head and (iv) changes in shape of cytoplasmic droplet. Ranitidine administration showed normal morphology of the spermatozoa.     

Mode of death and post-mortem time effects on 3,3',5-triiodothyronine levels--relevance to elevated post-mortem T3 levels in SIDS

Post-mortem T3 levels have been reported to be increased in victims of SIDS. Recent animal studies suggest, however, that elevated T3 in SIDS may be a non-specific post-mortem phenomenon. Therefore, we studied the possible effects of post-mortem time on T3 levels in 10- and 20-day-old rats killed by various methods including: Sodium pentobarbital overdose, injection of KCl, cervical dislocation or asphyxia with 100% N, 95% N-5% CO2 or 100% CO2. In both age groups T3 remained unchanged or increased slightly when the animals were killed with Na Pentobarbital or KCl. Greater increases were observed when rats were killed by cervical dislocation or asphyxia (100% N, 95% N-5% CO2 or 100% CO2). T3 levels did not become elevated in asphyxiated adult rats in which the inferior vena cava was ligated immediately following death. By extension to the human infant, the results of this study support the possibility that elevated T3 levels in SIDS victims may result from post-mortem processes. However, these results also suggest that the post-mortem elevation in T3 levels may be directly related to the mode of death.     

Use of injectable potassium chloride for euthanasia of American lobsters (Homarus americanus)

Potassium chloride (KCl: 330 mg/ml) was assessed as an euthanasia agent in American lobsters (Homarus americanus). Two groups of 10 lobsters (408.2 to 849.9 g) were maintained at 11.9 to 12.1 degrees C ('warm') and 1.5 to 2.5 degrees C ('cold') to evaluate the possible effect of ambient temperature on response to KCl. Death was defined as time of cardiac arrest, as viewed and measured by use of ultrasound. The KCl solution was injected (100 mg of KCl/100 g of body weight) at the base of the second walking leg to flood the hemolymph sinus containing the ventral nerve cord with potassium. Disruption of this 'central nervous system' was immediate, followed by cardiac arrest within 60 to 90 seconds. Group median ( +/- SD) baseline heart rate was 42 +/- 14 'warm' and 36 +/- 5 'cold' beats per minute. Time until cardiac arrest ranged from 35 to 90 (57 +/- 18) seconds in the 'warm' group and from 40 to 132 (53 +/- 34) seconds in the 'cold' group. There was no significant difference between group medians for either parameter. Histologic lesions were limited to mild to moderate acute degeneration, characterized by cell swelling, loss of contraction bands, and occasional mild cytoplasmic vacuolation of skeletal muscle at the injection site. Injectable KCl solution was an effective, reliable method for euthanasia of H. americanus.     

Mouse embryo yield and viability after euthanasia by CO2 inhalation or cervical dislocation

Efficient production of transgenic mice requires high yields of viable, healthy embryos. Cervical dislocation (without prior anesthesia) rather than CO2 inhalation as a means of euthanasia has been justified on the basis of the increased yield of viable ova, but controlled studies have not directly supported this contention. The American Veterinary Medical Association (AVMA) and Canadian Council on Animal Care (CCAC) Guides, and respective Institutional Animal Care and Use Committees (IACUC) have supported the use of CO2 as a preferred, humane method. The study reported here was undertaken to determine the relative yields of viable embryos from mice euthanized either by inhalation of 100% CO2 or by cervical dislocation. Inbred and hybrid mouse strains, representative of common strains used in genetic engineering experimentation included C57BL/6, FVB/N, and B6SJLF1. There was no difference in the embryo yields in comparisons using the two methods of euthanasia (P = 0.534). Decisions regarding the method of euthanasia can be made on the basis of criteria other than those associated with embryo yield and viability.     

Dependence of the Parameters of Visual Evoked Potentials on the Parameters of a Complex Visual Stimulus (Tests on Humans)

We recorded visual evoked potentials, VEP, elicited by presentation of a reversal chess pattern; 35 healthy persons were examined. A recording electrode was in point Oz, according to the 10-20 system; dimensions of the chess pattern square fields and their contrast varied from 1.8 to 120 and from 2 to 100%, respectively. Peak latencies, PL, of the N75 component depended on the contrast of stimulus elements in a U-like manner with the minimum within a 20-25% range. The PL of the P100 component monotonically increased with a decrease in the contrast, while the dependence of the PL of N145 was complex: a trend toward a decrease with a decrease in contrast to 30%, a local maximum in about 25%, a decrease and stabilization within a 15-4% range, and a sharp increase at the minimum contrast. The amplitude of N75 decreased with decrease in contrast down to 20-25% and then stabilized; the amplitude of P100 decreased in an S-like mode, with a plateau within 25-4%. The dependence of the N145 amplitude was complex, with a plateau-like maximum at 30-15% contrast. The amplitudes of the negative VEP components, N75 and N145, increased with an increase in the square dimensions to 7.5 and then decreased. Changes in the P100 amplitude included two phases, with a local minimum within an angular dimension range of 30-15^O. The PL of all analyzed components in general increased with a decrease in the angular dimension of the pattern components. The above data are discussed considering concepts on the genesis of different VEP components and on the existence of specialized channels in the visual system, which are responsible for detection of different parameters of a visual object. The complex dynamics of the PL of the analyzed VEP components and of the P100 amplitude related to variation of the contrast and angular dimensions of the elements of a complex stimulus are considered to be determined by a module principle of the organization of the elements constituting different levels of the visual system and by transformations of the receptive fields of these elements on the level of cortical modules, which are related to changes in the stimulus parameters.     

Peculiarities of Ca<Superscript>2+</Superscript>-regulation of functional activity of myocardium of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

To elucidate role of intra- and extracellular Ca²⁺ in regulation of rhythm and strength of frog heart contractions, there were studied ECC and isometric contraction of myocardium preparations in response to verapamil, adrenaline, and blockers of α- and β-adrenoreceptors. It has been shown that after an intramuscular injection of verapamil (6 mg/kg), bradycardia develops, the heart rate (HR) decreasing by 50–70%. Further, the cardiac arrest occurred; however, administration to the animals of adrenaline (100 mg/kg) restored the cardiac rhythm for a short while. After an intramuscular injection of adrenaline at doses of 0.1–10 mg/kg, no essential changes were observed in the potential action amplitude and HR; an increase of the administered adrenalin concentration to 100 mg/kg was not accompanied by the cardiac rhythm stimulation, as this takes place in homoiothermal animals and human; on the contrary, an essential HR deceleration was revealed. Phentolamine (5 mg/kg) gradually decelerated HR rhythm by 32–45%. The potential amplitude changed insignificantly. A subsequent intracardiac injection of adrenaline (100 mg/kg) on the background of block of α-adrenoreceptors produced acceleration of the rhythm (by 15–21%) and fall of the electrogram amplitude. These results can indicate that in the frog heart phentolamine interacts predominantly with α 1-adrenoreceptors. An intracardial administration of propranolol (1 mg/kg) to frogs promoted inhibition of β-adrenergic receptors and produced a gradual cardiac rhythm deceleration. In experiments on assessment of verapamil effect on the character of contractions this preparation at a concentration of 150 μM was established to produce a significant dose-dependent decrease of the contraction strength. A rise of verapamil concentration in the sample to 200 μM led to a decrease of the amplitude, on average, by 68–70% and in individual preparations—by 80–85%; however, administration into the sample of adrenaline (10 μM) restored the cardiac contraction strength. Adrenaline (1 nM–100 μM) increased markedly the contraction amplitude. Phentolamine (10 μM) did not inhibit transmission of contractile signal to cardiomyocytes; this was manifested in that the contraction amplitude after addition of adrenaline (10 μM) into the sample was approximately the same as in the sample containing no phentolamine. Propranolol (10 μM) eliminated the stimulatory action of adrenaline (10 μM). The results of these experiments indicate that in the frog ventricular cardiomyocytes the main adrenaline acceptors are β-adrenoreceptors.     

Euthanasia via CO2 inhalation causes premature cortical granule exocytosis in mouse oocytes and influences in vitro fertilization and embryo development

Generation of high quality mouse metaphase II oocytes is an integral part for efficient in vitro fertilization (IVF), and subsequent embryo production for reproductive studies and genome banking. The main objectives of this study were to investigate the impact of various euthanasia methods on IVF, embryo development, and subcellular structures of MII mouse oocytes. Following superovulation regimen, female mice were euthanized by high flow CO₂ (H CO₂), low flow CO₂ (L CO₂), or cervical dislocation (CD). The MII oocytes obtained from these mice were evaluated for subcellular integrity by assessing their cortical granules and F‐actin. Furthermore, fertilization and subsequent embryonic development competence up to blastocyst stage were also evaluated in vitro. The oocytes collected from females euthanized by CD resulted in significantly higher two‐cell development rates (p = 0.028) and subsequently lead to in higher embryo development rates (p = 0.027) compared with oocytes from females euthanized by L CO₂. The cortical granule integrity analysis revealed significantly higher rate of premature cortical granules exocytosis (PCGE) for L CO₂ group compared with CD and H CO₂ groups (p < 0.001). These data collectively suggest that CO₂ associated PCGE during euthanasia procedure is the main cause of decreased IVF rates and CD is the optimal euthanasia method for the purpose of obtaining good quality MII oocytes for mouse IVF and other reproductive studies.     

Pharmacological Mechanisms of Cortical Enhancement Induced by the Repetitive Pairing of Visual/Cholinergic Stimulation

Repetitive visual training paired with electrical activation of cholinergic projections to the primary visual cortex (V1) induces long-term enhancement of cortical processing in response to the visual training stimulus. To better determine the receptor subtypes mediating this effect the selective pharmacological blockade of V1 nicotinic (nAChR), M1 and M2 muscarinic (mAChR) or GABAergic A (GABA_AR) receptors was performed during the training session and visual evoked potentials (VEPs) were recorded before and after training. The training session consisted of the exposure of awake, adult rats to an orientation-specific 0.12 CPD grating paired with an electrical stimulation of the basal forebrain for a duration of 1 week for 10 minutes per day. Pharmacological agents were infused intracortically during this period. The post-training VEP amplitude was significantly increased compared to the pre-training values for the trained spatial frequency and to adjacent spatial frequencies up to 0.3 CPD, suggesting a long-term increase of V1 sensitivity. This increase was totally blocked by the nAChR antagonist as well as by an M2 mAChR subtype and GABA_AR antagonist. Moreover, administration of the M2 mAChR antagonist also significantly decreased the amplitude of the control VEPs, suggesting a suppressive effect on cortical responsiveness. However, the M1 mAChR antagonist blocked the increase of the VEP amplitude only for the high spatial frequency (0.3 CPD), suggesting that M1 role was limited to the spread of the enhancement effect to a higher spatial frequency. More generally, all the drugs used did block the VEP increase at 0.3 CPD. Further, use of each of the aforementioned receptor antagonists blocked training-induced changes in gamma and beta band oscillations. These findings demonstrate that visual training coupled with cholinergic stimulation improved perceptual sensitivity by enhancing cortical responsiveness in V1. This enhancement is mainly mediated by nAChRs, M2 mAChRs and GABA_ARs. The M1 mAChR subtype appears to be involved in spreading the enhancement of V1 cortical responsiveness to adjacent neurons.     

The effect of mouse euthanasia technique on subsequent lymphocyte proliferation and cell mediated lympholysis assays

The purpose of this study was to determine the effects that specific euthanasia methods have on mitogen induced lymphocyte proliferation (LP) and the induction of alloantigen specific cytolytic T-lymphocytes (CTL). Mice were euthanatized by cervical dislocation (CD), or anesthesia with methoxyflurane or pentobarbital followed by CD (M-CD or P-CD respectively), CO2 overexposure (CO2-OD) or halothane overexposure (H-OD). Mitogenic lymphoproliferation was increased in cells derived from mice euthanatized by M-CD and P-CD. In contrast, the cytolytic profile of CTL derived from mice euthanatized by P-CD, CO2-OD and H-OD was decreased. The results of this study show that euthanasia techniques involving the use of methoxyflurane, pentobarbital, CO2 and halothane affect in vitro lymphoproliferation and CTL function. We conclude that the method of euthanasia influences certain immunologic parameters and selection of a particular technique should be given careful consideration.     

Left ventricular mechanoenergetics after hyperpolarized cardioplegic arrest by nicorandil and after depolarized cardioplegic arrest by KCl

We hypothesized that there are no differences in left ventricular (LV) mechanoenergetics between after hyperpolarized cardioplegic arrest by nicorandil (nicorandil arrest) and after depolarized one by high potassium chloride (KCl arrest). The aim of the present study was to test this hypothesis using LV curved end-systolic pressure-volume relation (ESPVR) and linear pressure-volume area (PVA)-myocardial oxygen consumption per beat (VO2) relation. All hearts underwent 30 min global ischemia (30 degrees C) after infusion of 5 ml of cardioplegia. Cardioplegia consisted of either 30 mmol/l KCl (7 hearts) or nicorandil (100 micromol/l) in Tyrode solution (6 hearts). After a 30-min blood reperfusion, ESPVR and VO2-PVA relation were assessed again. Mean end-systolic pressure (ESP(mLVV)) and mean PVA at midrange LV volume (PVA(mLVV)) significantly (P < 0.05) decreased to 79.1 +/- 13.4% and 85.4 +/- 17.1% of control after KCl arrest and to 85.3 +/- 14.8% and 86.4 +/- 16.9% of control after nicorandil arrest. There were no significant differences in both decreases of mean ESP(mLVV) and PVA(mLVV) between each arrest. The slopes of VO2-PVA relations were also unchanged after each arrest. There was a significant (P < 0.005) difference in the decreases of mean VO2 intercepts of VO2-PVA relations between post-KCl arrest (73.9 +/- 8.2% of control) and post-nicorandil arrest (99.2 +/- 10.1% of control), however. Proteolysis of alpha-fodrin due to Ca2+ overload was significantly marked after KCl arrest. The present results indicate that the total calcium handling in excitation-contraction coupling is transiently impaired after KCl arrest, whereas it is unchanged after nicorandil arrest. This suggests the possibility that nicorandil is a better cardioplegia than KCl.     

Peculiarities of Ca2+ regulation of functional activity of myocardium of frog Rana temporaria

To elucidate role of intra- and extracellular Ca2+ in regulation of rhythm and strength of frog heart contractions, there were studied ECC and isometric contraction of myocardium preparations in response to verapamil, adrenaline, and blockers of alpha- and beta-adrenoreceptors. It has been shown that after an intramuscular injection of verapamil (6 mg/kg), bradycardia develops, the heart rate (HR) decreasing by 50-70 %. Further, the cardiac arrest occurred; however, administration to the animals of adrenaline (100 mg/kg) restored the cardiac rhythm for a short while. After an intramuscular injection of adrenaline at doses of 0.1-10 mg/kg, no essential changes were observed in the potential action amplitude and HR; an increase of the administered adrenalin concentration to 100 mg/kg was not accompanied by the cardiac rhythm stimulation, as this takes place in homoiothermal animals and human; on the contrary, an essential HR deceleration was revealed. Phentolamine (5 mg/kg) gradually decelerated HR rhythm by 32-45 %. The potential amplitude changed insignificantly. A subsequent intracardiac injection of adrenaline (100 mg/kg) on the background of block of alpha-adrenoreceptors produced acceleration of the rhythm (by 13-21%) and fall of the electrogram amplitude. These results can indicate that in the frog heart, phentolamine interacts predominanty with alpha-adrenoreceptors. An intracardiac administration of propranolol (1 mg/kg) to frogs promoted inhibition of beta-adrenergic receptors and produced a gradual cardiac rhythm deceleration. In experiments on assessment of verapamil effect on the character of contractions this preparation at a concentration of 150 microM was established to produce a significant dosedependent decrease of the contraction strength. A rise of verapamil concentration in the sample to 200 microM led to a decrease of the amplitude, on average, by 68-70 % and in individual preparations -- by 80-85 %; however, administration into the sample of adrenaline (10 microM) restored the cardiac contraction strength. Adrenaline (1 nM--100 microM) increased markedly the contraction amplitude. Phentolamine (10 microM) did not inhibit transmission of contractile signal to cardiomyocytes; this was manifested in that the contraction amplitude after addition of adrenaline (10 microM) into the sample was approximately the same as in the sample containing no phentolamine. Propranolol (10 microM) eliminated the stimulatory action of adrenaline (10 microM). The results of these experiments indicate that in the frog ventricular cardiomyocytes the main adrenaline acceptors are beta-adrenoreceptors.     

Cerebral oxidized and reduced nicotinamide-adenine dinucleotide phosphate and glucose 6-phosphate dehydrogenase in mice during exposure to high oxygen pressure.

NADP+, NADPH and glucose 6-phosphate dehydrogenase were determined in the cerebral cortex of mice exposed to high O2 pressure for 0, 8 and 16 min. These time intervals corresponded to 0, 50 and 100% of the CT50 (the time taken for 50% of the mice to convulse). Cerebral NADP+, NADPH and glucose 6-phosphate dehydrogenase also were determined in O2-exposed mice exhibiting hyperactivity, convulsions, and in mice killed 10s after convulsions. Similar increases in cortical NADP+ and decreases in NADPH were found in mice exposed to 610kPa (6 atm.) of 100% O2 for 0, 50 and 100% of the CT50, during hyperactivity, onset of seizure and 10s after convulsions. The NADP+/NADPH ratio increased approx. 25% at 0% of the CT50, and remained at this increased value at all O2-exposure periods including the hyperactive state, onset of seizure and 10s after convulsions. Identical changes in cerebral NADP+ , NADPH and the NADP+/NADPH ratio were found in mice exposed for 16min to 100% O2 at 100, 350 or 610kPa. No change in cerebral glucose 6-phosphate dehydrogenase was found in mice exposed to 610kPa of 100% O2 during the various stages of O2 toxicity. Only in the 10s post-convulsive group was a statistically significant decrease in glucose 6-phosphate dehydrogenase observed. Disulfiram [bis(diethylthiocarbamoyl) disulphide], an effective O2-protective agent, did not prevent the O2-induced increase in cerebral NADP+ and the NADP+/NADPH ratio, or decrease in NADPH.     

GLYCOLYTIC SUBSTRATE UTILIZATION AND ENERGY CONSUMPTION IN THE CEREBRAL HEMISPHERES OF THE CHICK EMBRYO DURING THE PERIOD OF EEG DEVELOPMENT

Abstract— The glycolytic substrate utilization and energy consumption of chick embryo hemispheres aged 15 and 19 days, respectively, were studied by converting the heads into a closed system. After transforming the brain into a closed system by decapitation, spontaneous bio-electrical potential changes, as indicated by EEG measurements, stayed normal in the hemispheres until at least 30 s after the procedure. Although the two developmental stages studied represented the start and the final phase of functional development, no changes in carbohydrate utilization and energy consumption were observed when comparing hemispheres of the two stages mentioned. The utilization of substrates other than glucose during embryological development is discussed. The disappearance of the EEG is not related to the overall depletion of high energy phosphates in the hemispheres since ATP is still present at nearly its original level after 2 min of ischaemia, despite the low level of P-creatine occurring at 30 s after decapitation. A compartmentation of the major pools of P-creatine and ATP is suggested.     

Are spontaneous activity and supraspinal control co-responsible for functional development of the generator of embryonic motility?

The effect of chronic administration of phenobarbital (from the 4th to the 16th day of incubation) combined with chronic spinal decentralization (decapitation at stage 11-13) on the development of spontaneous motor activity (recorded on the 17th day of incubation) was studied in 4 to 17-day-old chick embryos. 1. Combination of the two experimental treatments led to summation of their negative effect on development; spontaneous motor activity fell to 15.2% of the control value (to 26.9% after the isolated administration of phenobarbital and to 30.5% after isolated decapitation). 2. Metrazol activation of motor activity (100 mg/kg egg weight) after combination of the two factors was relatively no different from the results after their isolated use. 3. The acute administration of GABA (100 mg/kg e.w.) likewise induced relatively the same depression of spontaneous motility as after isolated chronic decapitation and the isolated chronic administration of phenobarbital. The results confirm the hypothesis of the significance of spontaneous activity of the spinal motoneurones for their survival and their actual functional development during the embryonic period.     

Effect of dihydroxyphenylalanine, imipramine and coffeine on the light induced decrease in nocturnal serotonin N-acetyltransferase in the rat pineal gland.

The s.c. administration of 150mg L-dihydroxphyenylalaine/kg b.w. 15 min before the decapitation prevents the light induced decrease in nocturnal serotonin N-acetyltransferase activity in the rat pineal gland. The s.c. administration of 50mg imipramine/kg b.w., resp. 100mg/kg b.w., 15 min before the decapitation, slows down, or prevents the light induced fall in the activity. The maintenance of a sufficient level of active norepinephrine on beta-receptors, either by displacement of norepinephrine in the nerve endings by dopamine, or by the inhibition of norepinephrine reuptake by imipramine, thus slows down or prevents the decrease in serotonin N-acetyltransferase activity after exposure to light during the night. The i.p. administration of a phosphodiesterase inhibitor coffeine citrate in a dose 200mg/kg 90 min after switching off the light for the night stimulated serotonin N-acetyltransferase activity 270 min after the light and been switched off, but did not influence the abrupt decrease induced in nocturnal activity by exposure to light.