Transient cardiorespiratory events during NREM sleep: a feline model for human microarousals.

Microarousals (MAs) are brief transient events that occur during normal sleep in humans and with increased frequency in disordered sleep, especially in association with sleep apnoea. In a feline model, we discovered transient cardiorespiratory events during nonrapid eye movement (NREM) sleep that exhibited consistent features with similarities to human MAs. It was observed that MAs have two distinct phases. Phase I (MAI) is characterized by an abrupt increase in electromyogram (EMG) amplitude (> 50%), increased electrooculogram (EOG) activity and accelerated frequency of hippocampal electroencephalographic (EEG) activity. MAI lasts 4.1 +/- 0.3 s. Phase II (MAII), lasting 9.8 +/- 0.8 s, is characterized by high frequency EEG activity, but EMG, EOG and hippocampal activity remain at baseline levels. Mean inspiratory rate begins to increase 15 s before the onset of the MA, followed 10 s later by the increase in mean heart rate. Mean respiratory rate decreases sharply through MAII, and returns to baseline levels 15 s after the MA. During MAII mean heart rate decreases quickly; there is increased respiratory irregularity, followed by a prolonged ventilatory overshoot. The abrupt shift in heart rate is coincident with the change in breath timing seen during MAII. Heart rate returns to baseline levels 10 s following the MA. Integrating our findings with those described previously in humans, we propose that MAs may serve as a homeostatic mechanism which is designed to restore cardiorespiratory function allowing the continuity of sleep.

Carbachol microinjection into the caudal peribrachial area induces long-term enhancement of PGO wave activity but not REM sleep.

This study presents new findings of carbachol-induced long-term ponto-geniculo-occipital (PGO) enhancement lasting five days, but without REM sleep enhancement. A quantitative analysis of the number and types of bilateral PGO wave events during slow wave sleep with PGO activity (SP) and REM was performed in each of four cats over a period of six days following a single unilateral microinjection of carbachol nanospheres into the caudolateral peribrachial area. The results demonstrate increases in the summed total of all PGO wave events to continue for five days postcarbachol reaching a peak sixfold increase on day three in SP and REM. The tendency of PGO waves to occur in clusters of greater than three waves increased sevenfold on day three in SP and fourfold during REM. These findings indicate a dissociation of long-term PGO enhancement from long-term REM enhancement, and suggest that even a sixfold increase in PGO activity alone is not, in itself, sufficient to produce the cholinergic orchestration of REM sleep enhancement.

Effect of specific muscarinic M2 receptor antagonist on carbachol induced long-term REM sleep.

Six cats were chronically implanted with a standard set of sleep-scoring electrodes and bilateral stainless-steel guide tubes for microinjection of drugs in the peribrachial area (PBL). Pretreatment of drug injection sites in the PBL with the M2 antagonist methoctramine blocks both the immediate triggering of ponto-geniculo-occipital (PGO) waves and the later prolonged enhancement of REM sleep that is induced by carbachol. These results support the hypothesis that the carbachol effects are mediated via the M2 muscarinic receptor that is known to be present in the PBL.

Dose-related suppression of REM sleep and PGO waves by the serotonin-1 agonist eltoprazine.

Parental administration of the serotonin-1 agonist eltoprazine (0.0625 to 4.0 mg/kg [0.0002 to 0.016 mmol/kg]) in freely moving cats produced significant suppression of electrophysiologic rapid eye movement (REM) sleep signs, ponto-geniculo-occipital (PGO) activity, and REM sleep behavior. The virtual total suppression of REM sleep (0.4%, 4.0 mg/kg) and PGO wave activity (2 to 4 mg/kg) in exchange for increasing amounts of non-REM (NREM) slow-wave sleep was a dose-dependent function of the amount of eltoprazine administered. Wakefulness was unaffected by eltoprazine regardless of dose. Concurrent with this dose-dependent suppression of REM was a dose-dependent increase in electroencephalographic synchrony and mean electromyographic amplitude. Since eltoprazine was found to shift the balance between REM and NREM sleep but did not change the balance between sleep and waking, it is a potentially useful tool for the investigation of serotonergic-cholinergic interaction.

Dynamic suppression of REM sleep by parenteral administration of the serotonin-1 agonist eltoprazine.

The purpose of this study was to determine the effects of the serotonin-1 agonist eltoprazine on the control of rapid eye movement (REM) sleep. Continuous polygraph recordings were performed for 15-17 days in four adult male cats. During the first 5 control days cats received injections of 0.9% saline intraperitoneally (i.p.) twice per day (b.i.d.). Over the next 5-7 days cats received injections of 0.9% saline intraperitoneally (i.p.) twice per day (b.i.d.). Over the next 5-7 days cats received eltoprazine i.p. (1-2 mg/kg, b.i.d.). For the final 5 recovery days cats received saline alone. During the saline control period, the mean REM sleep percent was 13.8 +/- 0.91%. When eltoprazine was administered for the subsequent 5-7 days, the mean REM percent was reduced to 1.5 +/- 0.59%. During the 5-day recovery period, REM percent increased significantly (p less than 0.0001) above both control and drug injection values to a mean of 24.5 +/- 1.3% with a maximum on recovery day 1 of 28.4 +/- 2.6% (n = 4). In addition to REM suppression, eltoprazine produced other electroencephalographic changes: an increase in slow-wave sleep (S) percent without any change in overall wake (W) percent; an increase in electromyogram (EMG) amplitude; and a decrease in ponto-geniculo-occipital (PGO) wave activity. PGO wave frequency and REM% increased significantly during the recovery period. Thus our findings demonstrate REM and PGO suppression by eltoprazine and document dramatic rebound effects following its withdrawal.

Long-term enhancement of REM sleep following cholinergic stimulation.

A six day long increase in rapid eye movement (REM) sleep followed the unilateral microinjection of a single dose of the cholinergic agonist drug carbachol into the brain stem of cats. Effective drug injection sites were localized to the pontine peribrachial region containing cholinergic choline acetyltransferase (ChAT) labeled neurons. At the peak of the effect, which occurred 24-28 h post-injection, the relative amount of time devoted to REM sleep tripled, resulting in an absolute time increase from 3.12 to 11.28 h REM sleep per day. This pronounced and prolonged REM sleep increase was associated with marked enhancement of ponto-geniculo-occipital (PGO) waves and with PGO burst cell activity unilateral to the site of injection.

Automated staging of sleep in cats using neural networks.

Manual staging of sleep based on visual EEG criteria is a laborious and time-consuming task. In an effort to automate sleep staging, we have developed a neural network that 'learns' to stage sleep on the basis of wave band count data alone, in the cat. Wave band count data are collected on a microcomputer, using period-amplitude analysis. Delta waves, spindle bursts, ponto-geniculo-occipital (PGO) waves, electro-oculogram (EOG), basal electromyogram (EMG) amplitude, and movement artifact amplitude are collected, and used to 'train' the network to score sleep. These wave count data serve as the input patterns to the net, and the corresponding manually scored sleep stages serve as a 'teacher.' We demonstrate that, when used to score the states of wake, slow wave sleep (SWS), desynchronized sleep (D), and the transition period from SWS to D (SP), these neural networks agree with manual scoring an average of 93.3% for all epochs scored. Neural network programs can learn both rules and exceptions, and since the nets teach themselves these rules automatically, a minimum of human effort is required. Because programming requirements are small for neural nets, this approach is readily adaptable to microcomputer-based systems and is widely applicable to both animal and human EEG analyses. The utility of this approach for the detection and classification of a variety of clinical neurophysiological disorders is discussed.

Restricted diffusion and stability of carbachol-fluorescent nanospheres in-vivo.

Mapping neuronal populations that induce behavioral state changes after pharmacological activation requires discrete localization of drug injection sites, and is limited by widespread diffusion of molecular drugs. Nanospheres with diameters of 50-100 nm can reduce diffusion significantly because of their relatively large sizes. The cholinergic agonist carbachol was radiolabeled with methyl14C and incorporated within a latex nanosphere delivery system (LNDS). We quantitatively compared diffusion of 14C-carbachol within these nanospheres with that of free 14C-carbachol, demonstrating approximately ten-fold reduced radial diffusion by nanospheres 10 min to 24 h post-injection; approximately 90% of injected radioactivity was restricted to regions within approximately 100-150 microns and 1400-1500 microns respectively. Thus, incorporation of active agents such as drugs within nanospheres dramatically increases the precision of their delivery in-vivo (here about 1,000-fold by volume).

A cholinoceptive desynchronized sleep induction zone in the anterodorsal pontine tegmentum: locus of the sensitive region.

Carbachol, a long-acting cholinergic agonist, was microinjected (4 micrograms/250 nl per 90 s) into 90 sites within the anterodorsal pontine tegmentum of four cats and the time to onset and percentage of time spent in a desynchronized sleep-like state during 40 min postinjection were calculated. Compared with more posteroventral pontine sites, the shorter latencies and higher percentages observed confirmed earlier predictions of a sensitive cholinoceptive zone in the anterodorsal pons. In 27 trials a desynchronized sleep-like state was observed within 5 min; in 31 trials the latency was 5-10 min and in the remaining 32 trials, greater than 10 min. Plotting the desynchronized sleep-like state latency and the desynchronized sleep-like state percentage as a function of the three-dimensional coordinates revealed that injection sites with short latency (less than 5 min) and high percentage (greater than 80%) were concentrated between the coordinates of P 1.0 to 3.5 and V -3.5 to -5.5, at the lateral coordinate L 2.0. On the frontal plane, the short desynchronized sleep-like state latency and high desynchronized sleep-like state percentage sites begin in the pontine tegmental region just lateral to the ventral tegmental nucleus and extend 3 mm ventrocaudally. A regression plot of the data in sagittal plane 2.0 revealed a short latency axis, around which the short latency sites cluster, running in a slightly dorsoventral direction from about P 1.0 to V -4.0 to P 4.0 to V -5.5. This observation suggests that the sensitive zone might approximate a cylinder in shape, a hypothesis supported by the correlation of longer latencies and lower percentages at increasing radial distance from the axis. The non-linear relationship between cholinergic potency and distance from the short latency axis suggests that the desynchronized sleep-like state latency is a function of two factors; a variable diffusion-based delay of carbachol to distant neuronal populations involved in the desynchronized sleep-like state production, and a fixed recruitment-based delay following activation of neurons in the sensitive zone. Interpretation of these findings in light of earlier studies involving microstimulation of the pontine tegmentum argue in favor of a distributed network of discrete neuronal populations as the source of desynchronized sleep generation.

A cholinoceptive desynchronized sleep induction zone in the anterodorsal pontine tegmentum: spontaneous and drug-induced neuronal activity.

The effect of carbachol microapplication (4 micrograms/250 nl per 90 s) on the discharge of neurons in the anterodorsal pons of four cats was studied using a newly devised microinjector-microelectrode assembly. Neurons were classified according to the magnitude of their discharge rate increases (or decreases) in physiological desynchronized sleep as desynchronized-on (or desynchronized-off) before injecting carbachol. When carbachol produced a desynchronized sleep-like state only half (15 out of 30) of the desynchronized-on cells were activated (desynchronized-on/desynchronized sleep-like state-on) while the other half were not (desynchronized-on/desynchronized sleep-like state-not on). Compared with the non-activated cells, the desynchronized-on/desynchronized sleep-like state-on cells had three features consistent with playing an active role in desynchronized sleep generation: these cells had a higher mean discharge frequency in desynchronized sleep and higher ratio of discharge frequency in desynchronized sleep compared with wakefulness; they did not fire in phase with electromyogram excitation of neck muscles; and they were concentrated in the short latency desynchronized sleep-like state induction zone described in the companion paper. The three-way correlation between the optimal anatomical site for short latency desynchronized sleep-like state induction, the selective neuronal discharge pattern in desynchronized sleep and the cholinergic activation pattern in the desynchronized sleep-like state suggest that we may have identified a neuronal population that is cholinoceptively activated as part of the physiological mechanism of desynchronized sleep generation.

Mapping neuronal inputs to REM sleep induction sites with carbachol-fluorescent microspheres.

The cholinergic agonist carbachol was conjugated to latex microspheres that were fluorescently labeled with rhodamine and used as neuroanatomical probes that show little diffusion from their injection site and retrogradely label neurons projecting to the injection site. Microinjection of this pharmacologically active probe into the gigantocellular field of the cat pontine brain stem caused the awake cats to fall into rapid movement (REM) sleep indistinguishable from that produced by free carbachol. Three-dimensional computer reconstruction of the retrogradely labeled neurons revealed a widely distributed neuronal network in the pontine tegmentum. These pharmacologically active microspheres permit a new precision in the characterization and mapping of neurons associated with the control of behavioral state and of other cholinergic networks.

A microcomputer-based system for automated EEG collection and scoring of behavioral state in cats.

A data acquisition and analysis system based on an Apple II microcomputer has been developed for use in sleep studies in the adult cat. The system reliably counts delta, spindle, and EMG waveforms, PGO waves, and REMs using amplitude and frequency criteria. These data can be used to algorithmically score sleep-wake state with high reliability (greater than 90% agreement with manual scoring). This method allows for automatic and quantitative analysis of selected EEG waveforms and sleep-wake states with less expense, more time savings, and greater convenience than manual scoring.

Colloidal gold fluorescent microspheres: a new retrograde marker visualized by light and electron microscopy.

A new retrograde tracer, rhodamine latex microspheres, permits labeled neurons to be visualized with fluorescence light microscopy. However, their use has been limited to the light microscope. We now have developed colloidal gold fluorescent microspheres which identify retrogradely labeled neurons first by fluorescence microscopy and then by electron microscopy. This new fluorescent/EM tracer will find widespread use in the field of neuroscience to elucidate the ultrastructural integrity of neuronal networks.

Brainstem immaturity in sudden infant death syndrome: a quantitative rapid Golgi study of dendritic spines in 95 infants.

Quantitative analysis of reticular dendritic spines was performed on rapid Golgi impregnated neurons in 7 brainstem areas from 61 sudden infant death syndrome (SIDS) and 34 control infants. Throughout the first postnatal year, mean spine density in SIDS was significantly greater than the mean density in controls (P less than 0.0001). There were significantly higher values of spine density in SIDS compared to controls (P less than 0.0001) in both term and preterm infants. Within the SIDS brainstem itself, the density of dendritic spines was significantly different (P less than 0.05) between two medullary regions and between reticular and non-reticular formation areas. Among these brainstem areas in controls, there was no significant difference. Our findings indicate an immature developmental pattern of increased dendritic spine density in the SIDS brainstem which may be responsible for abnormal central respiratory and arousal control. These significant quantitative differences in spine density are considered in the present study to represent an anatomical substrate of brainstem immaturity in the multifactorial pathogenesis of SIDS.