Sleep loss and recovery after administration of drugs related to different arousal systems in rats.

Sleep is homeostatically regulated suggesting a restorative function. Sleep deprivation is compensated by an increase in length and intensity of sleep. In this study, suppression of sleep was induced pharmacologically by drugs related to different arousal systems. All drugs caused non-rapid eye movement (NREM) sleep loss followed by different compensatory processes. Apomorphine caused a strong suppression of sleep followed by an intense recovery. In the case of fluoxetine and eserine, recovery of NREM sleep was completed by the end of the light phase due to the biphasic pattern demonstrated for these drugs first in the present experiments. Yohimbine caused a long-lasting suppression of NREM sleep, indicating that either the noradrenergic system has the utmost strength among the examined systems, or that restorative functions occurring normally during NREM sleep were not blocked. Arousal systems are involved in the regulation of various wakefulness-related functions, such as locomotion and food intake. Therefore, it can be hypothesized that activation of the different systems results in qualitatively different waking states which might affect subsequent sleep differently. These differences might give some insight into the homeostatic function of sleep in which the dopaminergic and noradrenergic systems may play a more important role than previously suggested.

Basal forebrain administration of the somatostatin-analog octreotide does not affect cortical EEG in urethane anaesthetized rats.

Basal forebrain (BF) plays an important role in the regulation of cortical activation. Somatostatin (SOM) is present both in local neurons as well as in fibers in the BF. In previous studies, SOM axons were found to innervate corticopetal cholinergic cells and SOM was found to presynaptically modulate GABA and glutamate release onto cholinergic neurons in the BF. However, no systematic analysis is available about the EEG effects of SOM or its analog, octreotide (OCTR) injected directly into the BF. In the present experiments, EEG changes were examined following an OCTR injection (0.5 microliter, 500 nmol) into the BF areas containing several choline acetyl transferase-immunoreactive neurons of urethane-anaesthetized rats. Fronto-occipital EEG was recorded on both sides and relative EEG power was calculated in the delta (0-3 Hz), theta (3-9 Hz), alpha (9-16 Hz) and beta (16-48 Hz) frequency bands. OCTR injected to the BF failed to induce significant EEG changes and did not affect tail pinch-evoked cortical activation. Lack of effect may be attributed to the urethane anaesthesia as well as to the possible complex interactions between SOM and BF cholinergic and GABAergic neurons.

EEG effect of orexin A in freely moving rats.

Orexin A and orexin B are neuropeptides produced by a group of neurons located in the lateral hypothalamus which send widespread projections virtually to the whole neuraxis. Several studies indicated that orexins play a crucial role in the sleep-wake regulation and in the pathomechanism of the sleep disorder narcolepsy. As no data are available related to the EEG effects of orexin A in healthy, freely moving rats, the aim of the present experiments was to analyze EEG power changes in the generally used frequency bands after intracerebroventricular orexin A administration.Orexin A administration (0.84 and 2.8 nM/rat) differently affected fronto-occipital EEG waves in the different frequency bands recorded for 24 hours. Delta (1-4 Hz) and alpha (10-16 Hz) power decreased, while theta (4-10 Hz) and beta (16-48 Hz) power increased. Decrease of the delta power was followed by a rebound in case of the higher orexin A dose. This complex picture might be explained by the activation of several systems by the orexin A administration. Among these systems, cortical and thalamic circuits as well as the role of the neurons containing corticotrophin-releasing factor might be of significant importance.

Laminar analysis of initiation and spread of epileptiform discharges in three in vitro models.

Overexcitation of neuronal networks in some forebrain structures and pathological synchronization of neuronal activity play crucial role in epileptic seizures. Seizure activity can be elicited experimentally by different convulsants. Because of various distribution of excitatory and inhibitory connections in the neocortex there might be laminar differences in seizure sensitivity. Current source density (CSD) analysis or immunocytochemical c-Fos localization offer suitable tools to localize increased activation of neurons during seizure. In the present experiments, interictal epileptiform activity elicited by 4-aminopiridine, bicuculline or Mg(2+)-free solution was recorded with a 16-channel multielectrode assembly in different layers of the somatosensory cortex, and CSDs were calculated. Parallel c-Fos immunocytochemistry was applied. Each convulsant elicited characteristic activation pattern. 4-aminopiridine induced relatively short discharges, which were associated with a huge sink in layer V, the sink and source pattern was relatively simple. Mg(2+)-free solution elicited the longest discharges, sinks appeared typically in the supragranular layers II and III than quickly distributed toward layers V and VI. Bicuculline induced rather similar seizure pattern as Mg(2+)-free solution, but the amplitudes of field potentials were larger, while the durations shorter. The peak of c-Fos activation, however, was not parallel with the largest electrical activation. Larger amount of stained cells appeared in layers II and III in 4-aminopiridine and bicuculline, respectively. In Mg(2+)-free solution the highest c-Fos activity was detected in upper layer VI. Long-lasting cellular effects do not always correspond to the largest electrical responses, which are primarily determined by the activation of asymmetrical pyramidal neurons. Interneurons, which possess more symmetric process arborisation, play less important role in the generation of field potentials, although they may be intensively activated during seizure.

Long-term changes of activity of cortical neurons after status epilepticus induced at early developmental stages in rats.

Spontaneous activity of cortical neurons was studied under urethane anesthesia in adult rats 3 months after convulsive status epilepticus induced by lithium-pilocarpine administration at the age of 12 (SE12 group) or 25 (SE25 group) days. Whereas random firing neurons dominated in control animals (61 out of 98 cells), SE25 animals exhibited a significant increase in the incidence of bursting cells (38 out of 59 units). Similar change in SE12 animals did not reach the level of statistical significance. Status epilepticus at an early developmental stage may result in a long-lasting change in functions of surviving cortical neurons.

Nerve conduction velocity and spinal reflexes may change in rats after fumonisin B1 exposure.

Mycotoxin fumonisin B1 (FB1) a natural inhibitor of ceramide synthase contaminating mainly the corn-based food and feed may cause dysfunctions in the nervous system. In the present study peripheral neural dysfunctions were biomonitored after dietary FB1 exposure in rats. Daily oral doses of 6.2 mg/kg body weight/day FB1 were applied in rats for 2 weeks. Before and after FB1 treatment nerve conduction velocities of tibial and sciatic nerves and spinal reflexes were analyzed in vivo. Electrophysiological recordings of biphasic plantar EMG (M and H components) and evaluation of sensory and motor nerve conduction velocities were carried out. Nerve conduction velocities revealed decreasing tendencies after FB1 exposure. The flexor reflex and the H-components of the extensor reflex were significantly reduced. The proposed in vivo biomonitoring can reveal functional impairment of the peripheral nervous system caused by mycotoxin exposure. Reduction of conduction velocity and altered reflexes after FB1 exposure are suspected to be associated with modified signal transmission due to toxic systemic effects and possible changes in sphingolipid metabolism.

Effect of orexin-A on discharge rate of rat suprachiasmatic nucleus neurons in vitro.

The suprachiasmatic nuclei (SCN) constitute the principal pacemaker of the circadian timing system in mammals. The generated rhythm is forwarded mostly through projections to various hypothalamic nuclei. On the other hand, the regulated processes feedback to the SCN. One of the possible feedback pathways is the orexinergic projection from the lateral hypothalamus. Orexins are recently identified neuropeptides with an overall facilitatory effect on waking behaviors. Orexinergic fibers are widely distributed throughout the brain and are also present in the SCN. In this study we examined the effect of orexin-A on the spontaneous activity of rat SCN cell in vitro. Neurons showed 2 different firing pattern (continuous-regular, intermittent-irregular). Orexin-A increased firing rate in both cell types at 10(-8) M concentration, but caused a clear suppression of neuronal activity at 10(-7) M. Continuously firing neurons were less responsive than those firing intermittently. These results show that orexin-A may play a role in the modulation of the circadian pacemaker function. The neuropeptide might exert both direct, postsynaptic effects on SCN neurons and indirect, presynaptic effects on excitatory and inhibitory terminals. The dose-dependent modification of the firing rate indicate that the weight of these factors changes with the concentration of orexin-A.

Comparison of spontaneous and evoked epileptiform activity in three in vitro epilepsy models.

Rat neocortical slices express spontaneous epileptiform activity after incubation with GABA(A) receptor blocker bicuculline (BIC, 20 microM), with potassium channel blocker 4-aminopyridine (4-AP, 50 microM) or in Mg(2+)-free medium (LMG). Various parameters of spontaneous and evoked epileptiform discharges and their pharmacological sensitivity were analysed using extracellular field potential recordings in this comparative in vitro study. All types of convulsant solution induced spontaneous epileptiform activity, however, the analysed parameters showed that characteristics of epileptiform discharges are rather different in the three models. The longest duration of discharges was recorded in LMG, while the highest frequency of spontaneous events was detected in 4-AP. The epileptiform field responses elicited by electrical stimulation appeared in an all-or-none manner in BIC. On the contrary, in 4-AP and in LMG the amplitude of the responses increased gradually with increasing stimulation intensities. The NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (APV, 25 microM) abolished the LMG induced spontaneous epileptiform activity and significantly reduced the frequency of the epileptiform discharges in BIC and 4-AP. Blocking the AMPA type of glutamate transmission with 1-(aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466, 40 microM) rapidly abolished BIC-induced spontaneous epileptiform activity and caused a significant decrease in the frequency of 4-AP induced spontaneous epileptiform discharges. However, it had only a weak effect on the LMG-induced epileptiform activity. We conclude that the contribution of NMDA and AMPA types of glutamate receptors to the development and maintenance of epileptiform activity in cortical cell assemblies is different in the three models. There are significant alterations in contribution of NMDA and AMPA types of glutamate receptors to the above-mentioned processes in the different convulsants. In BIC the synchronisation is mainly due to the altered network properties, namely inhibition is reduced in the local circuits. Although inhibition is reduced in the local circuits, the AMPA receptor antagonist relatively easily blocked the synchronised excitation. In 4-AP, and especially in LMG, changes in the membrane characteristics of neurones play a crucial role in the increased excitability. In this case the AMPA antagonist was less effective.

Phase differences in electrical discharge rhythms between neuronal populations of the left and right suprachiasmatic nuclei.

The circadian pacemaker of the suprachiasmatic nuclei is a complex multioscillator system, which controls circadian and seasonal rhythmicity. A number of clock genes have been identified that play a key role in the generation of circadian rhythms. These clock genes are expressed in a circadian manner as has been shown in mice, rats and hamsters. The time at which their expression reaches peak values differs among the several genes. Expression profiles for a specific gene may also differ among subdivisions of the suprachiasmatic nuclei. It has been shown that mPer1 peaks slightly out of phase in the left and right suprachiasmatic nuclei and that the rhythm in c-fos expression is significantly different between the dorsomedial and ventrolateral regions. In the special case that the animal shows splitting of its locomotor activity pattern, mPer1 in the left and right suprachiasmatic nuclei appeared to oscillate in antiphase. Whether the molecular organization within the suprachiasmatic nuclei plays a role in seasonal rhythmicity, allowing animals to track daylength and become reproductive at the proper phase of the annual cycle, receives increasing interest (). The differences in peak expression times that exist between different genes, and the spatial differences in peak time for single genes, are suggestive of a genetic mechanism underlying the multioscillator structure. It is unknown, however, whether phase differences that are observed at the molecular level exist at the level of electrical activity rhythms in the suprachiasmatic nuclei in order to become potentially functional. In this study we investigated the presence of phase differences in neuronal discharge rhythms in the suprachiasmatic nuclei of the rat. To this purpose we combined simultaneous electrophysiological recordings of neuronal populations in the left and right suprachiasmatic nuclei with a detailed analysis of the phase relationship between them. The results demonstrate that neuronal subpopulations of the suprachiasmatic nuclei show phase differences both in their peak and half-maximum times of up to 4 h. We propose that these phase differences may play a role in the plasticity of the circadian timing system.

Tonic and phasic influence of basal forebrain unit activity on the cortical EEG.

Changes in arousal levels are normally accompanied by modification of gross electrical activity (EEG) in the cortex, with low amplitude fast waves characterizing high levels and large slow waves low levels of arousal. These changes in cortical EEG patterns depend mainly on two factors: on the input from the thalamus and on the state of various membrane channels in the cortical pyramidal cells, which are both regulated by ascending modulatory systems. Several lines of evidence indicate that of the activating systems the cholinergic is the most effective in activating the cortex. Its blockade with atropine induces large slow waves in the EEG, while inhibition of other systems has no such profound effect. The effect of atropine can be mimicked by lesioning the basal forebrain. Neurons in this area show very close tonic and phasic correlation with the cortical EEG, further supporting the suggestion that projections of these neurons have a special role in the regulation of cortical activity. However, there is a discrepancy between the effects of excitotoxic and selective cholinotoxic lesions of the basal forebrain. The immunohistochemical diversity of the corticopetal basal forebrain projection and the electrophysiological heterogeneity of the neurons also indicate that, in addition to cholinergic cells, other types of neurons do also participate in the regulation of cortical activity from this area. To understand the intimate details the activity of identified basal forebrain neurons must be recorded and correlated with cortical events.

EEG correlation of the discharge properties of identified neurons in the basal forebrain.

The basal forebrain (BF) is a heterogeneous structure located in the ventral aspect of the cerebral hemispheres. It contains cholinergic as well as different types of noncholinergic corticopetal neurons and interneurons, including GABAergic and peptidergic cells. The BF constitutes an extrathalamic route to the cortex, and its activity is associated with an increase in cortical release of the neurotransmitter acetylcholine, concomitant with electroencephalographic (EEG) low-voltage fast activity (LVFA). However, the specific role of the different BF cell types has largely remained unknown due to the lack of chemical identification of the recorded neurons. Here we show that the firing rate of immunocytochemically identified cholinergic and parvalbumin-containing neurons increase during cortical LVFA. In contrast, increased neuropeptide Y neuron firing is accompanied by cortical slow waves. Our results, furthermore, indicate that BF neurons posses a distinct temporal relationship to different EEG patterns and suggest a more dynamic interplay within BF as well as between BF and cortical circuitries than previously proposed.

The role of basal forebrain neurons in tonic and phasic activation of the cerebral cortex.

The basal forebrain and in particular its cholinergic projections to the cerebral cortex have long been implicated in the maintenance of cortical activation. This review summarizes evidence supporting a close link between basal forebrain neuronal activity and the cortical electroencephalogram (EEG). The anatomy of basal forebrain projections and effects of acetylcholine on cortical and thalamic neurons are discussed along with the modulatory inputs to basal forebrain neurons. As both cholinergic and GABAergic basal forebrain neurons project to the cortex, identification of the transmitter specificity of basal forebrain neurons is critical for correlating their activity with the activity of cortical neurons and the EEG. Characteristics of the different basal forebrain neurons from in vitro and in vivo studies are summarized which might make it possible to identify different neuronal types. Recent evidence suggests that basal forebrain neurons activate the cortex not only tonically, as previously shown, but also phasically. Data on basal forebrain neuronal activity are presented, clearly showing that there are strong tonic and phasic correlations between the firing of individual basal forebrain cells and the cortical activity. Close analysis of temporal correlation indicates that changes in basal forebrain neuronal activity precede those in the cortex. While correlational, these data, together with the anatomical and pharmacological findings, suggest that the basal forebrain has an important role in regulating both the tonic and the phasic functioning of the cortex.

Differential EEG effects of the anxiolytic drugs, deramciclane (EGIS-3886), ritanserin and chlordiazepoxide in rats.

The influence of serotonergic and benzodiazepine type anxiolytic drugs on the cortical activation and sleep-wakefulness cycle were compared by evaluating the effects of ritanserin and deramciclane (EGIS-3886), two 5-HT2 receptor antagonists, and chlordiazepoxide on the electroencephalogram (EEG) in freely moving rats. Following drug administration (1, 3, and 10 mg/kg, PO for all drugs), EEG was continuously sampled for 6 h and power spectra were calculated for every 5 s to assess changes in slow wave activity and sleep phases. In a separate test, anticonvulsant effects of the drugs were examined in mice. Both deramciclane and ritanserin slightly increased total time spent in deep sleep (DS) and lengthened sleep episodes. In contrast, chlordiazepoxide had a strong inhibitory action on DS, sleep time being shifted to more superficial light sleep (LS). The incidence and length of the high voltage spindle (HVS) episodes characteristic for the motionless, awake rat were increased at the highest dose of both deramciclane and ritanserin, while it was decreased by chlordiazepoxide. In mice, chlordiazepoxide had a marked anticonvulsant effect, while deramciclane was moderately effective and ritanserin ineffective. In conclusion, the 5-HT2 receptor antagonist anxiolytic drugs seem to be superior compared to the benzodiazepine type anxiolytic drug, chlordiazepoxide, as ritanserin and deramciclane improved sleep quality by increasing sleep episode length and time spent in DS, while chlordiazepoxide enhanced sleep fragmentation and decreased DS.

Light responsiveness of the suprachiasmatic nucleus: long-term multiunit and single-unit recordings in freely moving rats.

The suprachiasmatic nuclei (SCN) of the hypothalamus contain a pacemaker that generates circadian rhythms in many functions. Light is the most important stimulus that synchronizes the circadian pacemaker to the environmental cycle. In this paper we have characterized the baseline neuronal firing patterns of the SCN as well as their response to light in freely moving rats. Multiunit and single-unit recordings showed that SCN neurons increase discharge during daytime and decrease discharge at night. Discharge levels of individual neurons that were followed throughout the circadian cycle appeared in phase with the population and were characterized by low discharge rates (often below 1 Hz), with a twofold increase during the day. The effect of light on the multiunit response was dependent on the duration of light exposure and on light intensity, with light thresholds of approximately 0.1 lux. The light response level showed a strong dependency on time of day, with large responsiveness at night and low responsiveness during day. At both phases of the circadian cycle, the response level could be raised by an increase in light intensity. Single-unit measurements revealed that the time-dependent light response of SCN neurons was present also at the level of single units. The results show that the basic light response characteristics that were observed at the multiunit level result from an integrated response of similarly behaving single units. Research at the single-unit level is therefore a useful approach for investigating the basic principles of photic entrainment.

Responses of cortical EEG-related basal forebrain neurons to brainstem and sensory stimulation in urethane-anaesthetized rats.

The basal forebrain can be considered to be a rostral extension of the ascending reticular activating system. A large number of neurons in the basal forebrain have been shown to display higher firing rates when low-voltage fast activity is present in the cortical EEG as opposed to states characterized by large slow waves in both unanaesthetized and anaesthetized animals. However, a smaller number of cells with increased discharge rate during slow waves was also observed in most of these studies. While it is likely that these two types of neurons have opposite roles in the regulation of cortical activation, it is not known how they respond to inputs from the brainstem or the periphery. In the present study, extracellular recordings were made in the basal forebrain of urethane-anaesthetized rats. A total of 52 neurons were studied in which the firing rate was significantly higher during fast cortical EEG waves (F-cells), and 14 neurons in which activity was significantly greater during slow waves (S-cells). The two cell types responded differently to stimulation of the pedunculopontine tegmental nucleus (PPT) and dorsal raphe nucleus (DRN) with short (0.5-1 s) trains of pulses and to noxious sensory stimuli (tail pinch). These stimulations excited most F-cells (80-96%) and inhibited the majority of S-cells (55-67%). In the few F-cells that were inhibited by stimulation, the response varied with the background firing rate of the cell: the higher the firing rate at the time of stimulation, the higher the probability of observing an inhibitory response. In contrast, single electrical pulses delivered to the PPT and DRN excited the majority (72%) of both F- and S-cells. Previous in vitro studies have shown that the application of acetylcholine or serotonin has predominantly inhibitory effects on basal forebrain cholinergic neurons. The predominantly excitatory effect of noxious, PPT and DRN stimulation on F-cells therefore suggests that glutamatergic or other excitatory afferents play a more dominant role in regulating basal forebrain neurons. We have previously shown that F-cells are more prevalent than S-cells. In combination, these findings suggest that basal forebrain neurons, and F-cells in particular, are important in mediating the ascending excitatory drive from the brainstem to the cerebral cortex.

Phasic relationship between the activity of basal forebrain neurons and cortical EEG in urethane-anesthetized rat.

Previous studies have shown that a large number of neurons in the basal forebrain have higher firing rates when the cortical electroencephalogram (EEG) is characterized by low-voltage fast activity compared to states characterized by slow waves. A smaller number of cells with increased discharge rates during slow waves have also been observed. This putative ascending effect is thought to be tonic, but no attempt has been made to analyze a closer temporal correlation between the activity of basal forebrain neurons and the cortical EEG. Recordings were made from single units in the basal forebrain concurrently with the cortical EEG in urethane-anesthetized rats. A total of 52 neurons consistently showed higher firing during low-voltage fast activity (F-cells), whereas 14 neurons were consistently more active during cortical slow waves (S-cells). In most of the F- (90%) and S-cells (86%) the change in firing rate occurred prior to the change in the EEG. The average delay was 300-400 ms. At a deep level of anesthesia, the EEG was characterized by an alternation of flat periods and large waves. Most F-cells became active near the start of the first large wave, which is known to correspond to the onset of depolarization of cortical pyramidal neurons. In contrast, most S-cells were less active during the large waves. These data show that the activity of basal forebrain neurons is phasically correlated with the EEG in addition to the tonic correlation that has been demonstrated previously. Both types of basal forebrain neurons change their firing rate prior to the change in cortical EEG, suggesting that the basal forebrain neurons may have a regulatory influence on the EEG.

Circadian rhythm in light response in suprachiasmatic nucleus neurons of freely moving rats.

Long-term recordings of single SCN units were performed in freely moving rats simultaneously with multiunit recordings and evidence is presented for a daily change in light-responsiveness. SCN light response is high during the night and low during the day. We conclude that this difference is caused by a change in sensitivity, with higher sensitivities at night. Moreover, we demonstrate that the circadian rhythm in SCN light response is the result of the integrated behaviour of similarly behaving single SCN units.

Effects of viscerosensory stimulation on hypothalamically elicited predatory behavior in cats.

Hypnogenic (HS) or arousing (AS) stimulations of the small intestine (INT), splanchnic (SPL), and vagal (VAG) nerves were used to modify the predatory behavior (PB) elicited by stimulating the lateral hypothalamus (LHS). HS induced EEG synchronization and sleep. AS aroused the cat from slow-wave sleep. LHS induced the cat to attack an anesthetized rat and bite its neck after an exploratory activity. The following parameters of PB were determined: biting latency (BL), the interval between the beginning of LHS and the touching the rat by the cat's muzzle; exploratory time (ET), which begins with an environmental search and culminates in orienting toward the rat; attack time (AT), in which the cat stalks and bites the rat. HS, delivered for 5, 10, 15 min to INT, SPL, and VAG prior to LHS, increased BL and ET and did not affect AT. AS, delivered for 10 s to INT or VAG prior to LHS, decreased BL by reducing ET. SPL AS shortened BL by decreasing both ET and AT. The viscerosensory effects on PB were decreased by increasing the intensity of LHS; a ferocious attack with BL less than 10 s was not influenced by either HS or AS. These results indicate that the viscerosensory influence can modify PB by inhibiting or facilitating the priming events of the attack.