Effects of a novel melatonin analog on circadian rhythms of body temperature and activity in young, middle-aged, and old rats.

Circadian rhythms of body temperature and activity were recorded in young, middle-aged, and old rats. A new melatonin analog, S20242, was administered daily around the onset of darkness for a 2-week period. Compared to the young animals, there was a significant age-related reduction in the amplitude and stability of body temperature and activity in both the middle-aged and old rats. In these two groups there was an improvement of the circadian rhythm of body temperature as a result of daily application of the melatonin analog.

Circadian rhythms in spontaneous neuronal discharges of the cultured suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is believed to play a major role in the generation and control of circadian rhythms in mammals. In order to obtain further evidence concerning this, single and multiple neuronal discharges were continuously recorded over a period of several days in neonatal rat SCN explants. These organotypic explants, which had been cultured for several weeks in a chemically defined medium, showed alternating high and low levels of spontaneous neuronal discharges with a periodicity around 24 h. Such explants can serve as a useful model to study the neuronal mechanisms underlying the generation of mammalian circadian rhythms.

The role of cAMP in regulation of electrical activity of the neuroendocrine caudodorsal cells of Lymnaea stagnalis.

The neuroendocrine caudodorsal cells (CDCs) of the pond snail Lymnaea stagnalis release a number of peptides, including the ovulation hormone, caudodorsal cell hormone (CDCH), during a period of high electrical activity (the CDC-discharge). Earlier studies have shown that during the CDC-discharge adenylate cyclase activity is increased, and that the cyclic adenosine monophosphate (cAMP) analogue 8-chlorophenylthio (8-CPT)-cAMP induces exocytosis and release of peptides from the CDCs. Here, we have investigated the role of cAMP, adenylate cyclase and phosphodiesterase in determining the state of excitability of the CDCs. The cAMP analogue 8-CPT-cAMP induced long-lasting discharges in CDCs. Simultaneous inhibition of the phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX) and activation of the adenylate cyclase by forskolin gave similar results. These agents also induced discharges of CDCs in dissociated cell culture, indicating that the responses to an increase of cAMP were an endogenous property of the cells. The CDC-afterdischarge can be induced by a period of repetitive electrical stimulation. Inhibition of the phosphodiesterase-activity by IBMX did not change the resting membrane potential, but increased the proportion of preparations that responded to this stimulation with an afterdischarge by more than 200%. It is suggested that cAMP-regulating enzymes are involved in stimulus-response coupling of the afterdischarge in CDCs. The induction of an after discharge probably requires both a low phosphodiesterase-activity and the activation of the adenylate cyclase. The low excitability of the CDCs following an afterdischarge might originate from a refractoriness in the activation of the adenylate cyclase.

Chemically mediated positive feedback generates long-lasting afterdischarge in a molluscan neuroendocrine system.

The peptidergic neuroendocrine caudodorsal cells (CDCs) of Lymnaea stagnalis control egg laying. The CDC network consists of 100 electrotonically coupled neurons that form two clusters in the cerebral ganglia. Upon prolonged, repeated, intracellular stimulation of one CDC, excitation spreads over the network and leads to a 30-min period of spiking activity: the afterdischarge. During the afterdischarge a number of peptides, including the ovulation hormone, are released. When two ganglia rings from different animals were pinned down next to each other, an afterdischarge initiated in the CDCs of one CNS activated the CDCs of the other CNS, indicating that excitation spreads in the absence of physical contact between the CDCs. A single isolated intercerebral commissure (COM), the neurohaemal area of the CDCs, displayed the same discharge-inducing capability when brought in the vicinity of a second, intact, CNS. Other parts of the CNS did not possess this property. CDC afterdischarges could also induce repetitive spiking in adjacent isolated CDC somata showing that the effect can be directly on the CDCs themselves. The discharge-inducing factor was well separated from the ovulation hormone on a Bio-Gel P-6 column. The factor was pronase-degradable and inhibitors of proteolytic enzymes increased the factor's longevity. It is concluded that, contingent upon the CDC-discharge, a small (less than or equal to 1500 Da) excitatory peptide is released that acts directly on the CDCs. Its function is argued to be: (1) the spread of excitation from a subset of CDCs, receiving external input, over the entire CDC network; and (2) to provide a positive feedback to generate a maximum (all-or-none) response.

Decrease of rapid-eye-movement sleep in the light by intraventricular application of a VIP-antagonist in the rat.

Vasoactive intestinal polypeptide (VIP) has been shown to increase the amount of time spent in rapid-eye-movement (REM) sleep both in cats and in rats. In the present study we examined the effect of a newly available competitive VIP-antagonist ([4Cl-D-Phe6-Leu17]-VIP) on sleep-wake patterns in male rats during both the light and the dark phase of 24 h. Continuous intracerebroventricular application of this VIP-antagonist reduced by 44% the amount of time spent in REM sleep during the light period. It is concluded that VIP may play a role in the generation and maintenance of REM sleep.

Neonatal clonidine treatment results in long-lasting changes in noradrenaline sensitivity and kindling epileptogenesis.

In the present experiment we tested the hypothesis that early interference with noradrenaline transmission can have permanent consequences for brain function in adulthood. Neonatal depletion of noradrenaline by daily subcutaneous injections of clonidine results in supersensitivity to noradrenaline in adult hippocampal CA1 cells as shown in our previous microiontophoretic study. These findings were confirmed and extended here with dose-response curves. Furthermore, we tested whether this form of neonatal interference with noradrenaline also permanently affects long-lasting plasticity as revealed in kindling epileptogenesis in adulthood. The initiation of the epileptic activity after the kindling stimulation was significantly delayed in the clonidine-treated group, and all measured parameters of seizure expression tended to be retarded in comparison with saline-treated control rats. This indicates that noradrenaline supersensitivity induced by neonatal clonidine treatment retards kindling development in adulthood.

Neurons of the rat suprachiasmatic nucleus show a circadian rhythm in membrane properties that is lost during prolonged whole-cell recording.

The suprachiasmatic nucleus is commonly considered to contain the main pacemaker of behavioral and hormonal circadian rhythms. Using whole-cell patch-clamp recordings, the membrane properties of suprachiasmatic nucleus neurons were investigated in order to get more insight in membrane physiological mechanisms underlying the circadian rhythm in firing activity. Circadian rhythmicity could not be detected either in spontaneous firing rate or in other membrane properties when whole-cell measurements were made following an initial phase shortly after membrane rupture. However, this apparent lack of rhythmicity was not due to an unhealthy slice preparation or to seal formation, as a clear day/night difference in firing rate was found in cell-attached recordings. Furthermore, in a subsequent series of whole-cell recordings, membrane properties were assessed directly after membrane rupture, and in this series we did find a significant day/night difference in spontaneous firing rate, input resistance and frequency adaptation. As concerns the participation of different subpopulations of suprachiasmatic nucleus neurons expressing circadian rhythmicity, cluster I neurons exhibited strong rhythmicity, whereas no day/night differences were found in cluster II neurons. Vasopressin-containing cells form a subpopulation of cluster I neurons and showed a more pronounced circadian rhythmicity than the total population of cluster I neurons. In addition to their strong rhythm in spontaneous firing rate they also displayed a day/night difference in membrane potential.

Cortical neuron sensitivity to neurotransmitters following neonatal noradrenaline depletion.

In order to test the functional significance of rapid eye movement (REM)-sleep and noradrenergic activity for cerebral cortex maturation, rat pups were daily injected with clonidine from 8 to 21 days of life. Previous studies have shown that this treatment reduces the amount of time spent in REM-sleep and the level of noradrenaline turnover in the brain. For long-term consequences of such treatment in adulthood, cortical neuron responses to micro-iontophoretically applied neurotransmitters were studied. No significant differences were found in the single cell responses to glutamate, GABA or noradrenaline in the cerebral cortex of clonidine treated rats as compared with age matched controls. However, the magnitude of GABAergic depression of glutamate induced neuronal responses was greater in the clonidine than in the control group.

Spectral analysis of the electroencephalogram in neonatal rats chronically treated with the NMDA antagonist MK-801.

In order to study the involvement of NMDA-receptor activation in brain development, rat pups were chronically treated with the non-competitive NMDA antagonist MK-801 during the neonatal period. We recorded the cortical EEG at various vigilance states throughout the treatment period. Spectral analysis of the EEG showed reduced power in the delta (delta) frequency range (1.5-4 Hz) during quiet sleep and less power in the theta (theta) range (4-7 Hz) during REM-sleep in MK-801 animals than in controls. No significant differences were found for the total time spent in each of the different vigilance states. We conclude that chronic MK-801 treatment probably causes a developmental retardation in state-related brain activities.

Chronic neonatal MK-801 administration leads to a long-lasting increase in seizure sensitivity during the early stages of hippocampal kindling.

Persistent effects of chronic neonatal administration of the N-methyl-D-aspartate (NMDA) antagonist MK-801 were investigated by measuring susceptibility to CA1 kindling epileptogenesis in adulthood. Rat pups were chronically treated with MK-801 from postnatal day 8 through day 19. Hippocampal kindling showed an increase in electrical seizure duration in the MK-801-treated group as compared with controls along with a more severe expression of behavioral seizures during the first few kindling stimulations. These results show that neonatal interference with NMDA receptor function leads to a long-lasting increase in hippocampal excitability.

Effects of excitatory and inhibitory amino acids on neuronal discharges in the cultured suprachiasmatic nucleus.

The influence of amino acids on neuronal activity was studied microiontophoretically in the cultured suprachiasmatic nucleus (SCN). Three types of SCN neurons could be characterized: silent (glutamate responsive), irregular, and regular neurons. Glutamate excited about 70% of the regular and 60% of the irregular cells. GABA inhibited both the spontaneous and the glutamate-evoked activity of more than 90% of all three types of SCN neurons. MK-801 partially blocked glutamate responses. N-acetyl-aspartyl-glutamate (NAAG), a new neurotransmitter found in the retinohypothalamic fibers, directly increased firing rate and potentiated glutamate responses in the SCN neurons that were studied. These results indicate the potential significance of the amino acids in neuronal transmission within the biological clock.

Effect of light intensity on diurnal sleep-wake distribution in young and old rats.

During the aging process, the amplitude of the circadian rhythms of many physiological variables is reduced. It has been hypothesized that increasing light intensity during the light phase of the light-dark cycle might result in a reduction of age-related changes in the circadian rhythms. Indeed, in the present sleep-wake study in young and old rats it was found that (a) various parameters, such as the light-dark differences and total amounts of each behavioral state responded positively to changes in environmental light intensity (i.e., age-related trends were reversed), (b) in both age groups, the logarithm of light intensity appeared to have a linear dose-response relationship with light-dark differences of the sleep-wake states, (c) the light-dark difference of active wakefulness and quiet sleep of old rats under high light intensity were comparable to those of young rats under low light intensity. The results of the present study suggest that, under appropriate conditions, light could be of clinical use in reducing age-related circadian sleep disturbances in humans. This may, in turn, reduce the use of sedatives in elderly people.

Circadian rhythm generation in the cultured suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the hypothalamus is involved in the generation and entrainment of circadian rhythms. The results of a series of experiments in long-term cultured organotypic SCN slices suggest that (1) some but not all SCN neurons display circadian rhythmicity in their extracellular discharges. To the extent they could be studied, these neurons did not usually show synchronized high/low levels of activity; (2) simultaneous daily depolarization of these neurons (K+ pulses) to some extent influenced the distribution of the firing rate of SCN neurons around the time of expected daily pulses; (3) extracellular Ca++ and synaptic input is required for the pacemaker activity of the SCN. We conclude that the mammalian biological clock is a heterogeneous neuronal system in which the circadian pacemaker rhythm is generated and entrained via complex interactions among SCN neurons.

Hippocampal neuronal responsiveness to different neurotransmitters in the adult rat after neonatal interference with noradrenaline transmission.

In order to test the functional significance of noradrenergic neurotransmission for brain maturation, rat pups were injected daily with clonidine from 8-21 days of postnatal life. In adulthood, single cell responses to microiontophoretically applied neurotransmitters were studied in different layers of the hippocampus. No significant differences were found in neuronal responsiveness to glutamate, GABA, or acetylcholine in the hippocampus of clonidine-treated rats as compared with age-matched controls. However, there was a significantly stronger depression of glutamate-evoked activity of CA1 pyramidal neurons by noradrenaline in the clonidine-treated rats.

The effect of old age on the free-running period of circadian rhythms in rat.

The free-running period is regarded to be an exclusive feature of the endogenous circadian clock. Changes during aging in the free-running period may therefore reflect age-related changes in the internal organization of this clock. However, the literature on alterations in the free-running period in aging is not unequivocal. In the present study, with various confounding factors kept to a minimum, it was found that the free-running periods for active wakefulness, body temperature, and drinking behavior were significantly shorter (by 12-17 min) in old than in young rats. In addition, it was found that the day-to-day stability of the different sleep states was reduced in old rats, whereas that of the drinking rhythm was enhanced. Transient cycles were not observed, nor were there any age-related differences in daily totals of the various sleep-wake states. The amplitudes of the circadian rhythms of active wakefulness, quiet sleep, and temperature were reduced, whereas those of paradoxical sleep and quiet wakefulness remained unchanged.

Na+- and Ca2+-dependent components in action potentials of the ovulation hormone producing caudo-dorsal cells in Lymnaea stagnalis (Gastropoda).

Action potentials in the afterdischarge of the ovulation hormone producing caudo-dorsal cells (CDC) of Lymnaea stagnalis are strikingly different from electrically evoked spikes in the silent resting and inhibited states of these cells. Spikes evoked in the silent states consist of one fast peak (80-100 mV; 10-15 ms). The overshoot in Na+- and Ca2+-dependent. Spikes are blocked in Na+-free saline and by TTX. Repolarization is retarded by TEA. Co2+ increases the overshoot. Active state action potentials (60-80 mV) last up to 125 ms, due to activation of a slow component following the TTX-sensitive spike. The slow component is Na+- and Ca2+-dependent. In normal saline it is blocked by Co2+ and La3+. In Ca2+-free saline the remaining part of the slow component is blocked by La3+ only. The slow component is voltage-dependent in a graded fashion. Activation is bound to the active state in which the CDC are depolarized by 20 mV. TEA and Ca2+-free saline greatly increase spike duration in the active state. This suggests that, in addition to the classical TEA-sensitive channel, a Ca2+-dependent K+ channel is involved in repolarization of active state action potentials. The underlying membrane properties and the functional significance are discussed in relation to the pacemaking mechanism of the CDC.

Pacemaking mechanism of the afterdischarge of the ovulation hormone-producing caudo-dorsal cells in the gastropod mollusc Lymnaea stagnalis.

The ovulation hormone-producing caudo-dorsal cells (CDC) of Lymnaea stagnalis have three states of excitability (active, inhibited, and resting), which are related to the egg-laying cycle. Active state CDC produce a firing pattern of prolonged spiking activity (1 spike/2 s), which in the animal occurs shortly before egg laying. In preparations it is evoked as an afterdischarge upon repetitive stimulation of CDC. The afterdischarge is not synaptically driven, but rests on a pacemaking mechanism. CDC are silent in the inhibited and resting states, which follow egg laying. In these states the membrane potential is mainly dependent on [K+]0. In the active state the ratio of the K+, Na+, and Ca2+ permeabilities has changed considerably, probably resulting from an increased permeability to Na+ and Ca2+. The firing rate in the afterdischarge is dependent on the membrane potential, which is confirmed experimentally by varying [K+]0. [Na+]0 and [Ca2+]0 directly influence the firing rate. Firing stops in Na+-free saline, but is enhanced by Ca2+-free or high-Mg2+ saline. TTX does not affect firing. Relatively high concentrations of Co2+ and La3+ (2 x 10(-3) M) strongly inhibit CDC. Regular firing can be changed into bursting by various means, such as high K+ or addition of 1 mM Ba2+. Bursting normally occurs at the beginning of the afterdischarge. Postburst hyperpolarizations are reduced in Ca2+-free saline and by low Co2+ (10(-4)--5 X 10(-4) M). Active CDC are driven by a pacemaking mechanism constituted by a voltage-dependent Na+/Ca2+ channel and a Ca2+-dependent K+ channel, thus resembling that of bursting pacemakers. The pacemaking mechanism is inactive in the resting and inhibited state.

Membrane mechanism of neuroendocrine caudo-dorsal cell inhibition by the ring neuron in the pond snail Lymnaea stagnalis.

Ovulation in the pond snail Lymnaea stagnalis is controlled by the neuroendocrine caudo-dorsal cells (CDCs) in the cerebral ganglia, which release an ovulation hormone during a period of impulse activity. Firing of the single RN in the right cerebral ganglion hyperpolarizes the CDCs. This hyperpolarization is caused by the opening of potassium channels in the axons that connect both the CDC clusters. By this action, that presumably is mediated by axonal branches of the RN in the intercerebral commissure closely associated with these CDC axons, the RN decouples both the CDC clusters. Although the RN has negative feedback on the CDC, it does not control afterdischarge characteristics. The authors suggest that the RN, next to the egg-laying behavior, is involved in another behavior, not compatible with ovulation. Male reproductive activity is presented as a possible candidate for such behavior.

Quantitative analysis of eye movements during REM-sleep in developing rats.

Development of eye movements in REM-sleep was studied in rats during the second and third week of postnatal life. An increased frequency of eye movements was found after eye opening, which was due only to changes in burst activity. Since pups reared in constant darkness showed an increase in eye movement activity similar to that of those exposed to diurnal light-dark alterations, the change seems to be part of an endogenous developmental timetable rather than due to an increase of visual stimulation by eye opening.