c-Fos expression in GABAergic, serotonergic, and other neurons of the pontomedullary reticular formation and raphe after paradoxical sleep deprivation and recovery.

The brainstem contains the neural systems that are necessary for the generation of the state of paradoxical sleep (PS) and accompanying muscle atonia. Important for its initiation are the pontomesencephalic cholinergic neurons that project into the pontomedullary reticular formation and that we have recently shown increase c-Fos expression as a reflection of neural activity in association with PS rebound after deprivation in rats (Maloney et al. , 1999). As a continuation, we examined in the present study c-Fos expression in the pontomedullary reticular and raphe neurons, including importantly GABAergic neurons [immunostained for glutamic acid decarboxylase (GAD)] and serotonergic neurons [immunostained for serotonin (Ser)]. Numbers of single-labeled c-Fos+ neurons were significantly increased with PS rebound only in the pars oralis of the pontine reticular nuclei (PnO), where numbers of GAD+/c-Fos+ neurons were conversely significantly decreased. c-Fos+ neurons were positively correlated with PS, whereas GAD+/c-Fos+ neurons were negatively correlated with PS, suggesting that disinhibition of reticular neurons in the PnO from locally projecting GABAergic neurons may be important in the generation of PS. In contrast, through the caudal pons and medulla, GAD+/c-Fos+ cells were increased with PS rebound, covaried positively with PS and negatively with the electromyogram (EMG). In the raphe pallidus-obscurus, Ser+/c-Fos+ neurons were positively correlated, in a reciprocal manner to GAD+/c-Fos+ cells, with EMG, suggesting that disfacilitation by removal of a serotonergic influence and inhibition by imposition of a GABAergic influence within the lower brainstem and spinal cord may be important in the development of muscle atonia accompanying PS.

Differential c-Fos expression in cholinergic, monoaminergic, and GABAergic cell groups of the pontomesencephalic tegmentum after paradoxical sleep deprivation and recovery.

Multiple lines of evidence indicate that neurons within the pontomesencephalic tegmentum are critically involved in the generation of paradoxical sleep (PS). From single-unit recording studies, evidence suggests that unidentified but "possibly" cholinergic tegmental neurons discharge at higher rates during PS than during slow wave sleep or even waking and would thus play an active role, whereas "presumed" monoaminergic neurons cease firing during PS and would thus play a permissive role in PS generation. In the present study performed on rats, c-Fos immunostaining was used as a reflection of neuronal activity and combined with immunostaining for choline acetyltransferase (ChAT), serotonin (Ser), tyrosine hydroxylase (TH), or glutamic acid decarboxylase (GAD) for immunohistochemical identification of active neurons during PS recovery ( approximately 28% of recording time) as compared with PS deprivation (0%) and PS control (approximately 15%) conditions. With PS recovery, there was a significant increase in ChAT+/c-Fos+ cells, a significant decrease in Ser+/c-Fos+ and TH+/c-Fos+ cells, and a significant increase in GAD+/c-Fos+ cells. Across conditions, the percent PS was correlated positively with tegmental cholinergic c-Fos+ cells, negatively with raphe serotonergic and locus coeruleus noradrenergic c-Fos+ cells, and positively with codistributed and neighboring GABAergic c-Fos+ cells. These results support the hypothesis that cholinergic neurons are active, whereas monoaminergic neurons are inactive during PS. They moreover indicate that GABAergic neurons are active during PS and could thus be responsible for inhibiting neighboring monoaminergic neurons that may be essential in the generation of PS.

High-frequency gamma electroencephalogram activity in association with sleep-wake states and spontaneous behaviors in the rat.

The occurrence of high-frequency gamma activity (30-60 Hz) and its relationship to other frequency band activities were examined by spectral analysis of the electroencephalogram in association with sleep wake states and spontaneous behaviors in the rat. In the electroencephalogram, gamma wave activity was evident in unfiltered and high-frequency filtered recordings, in which it was prominent during attentive or active Wake episodes and during Paradoxical Sleep, when theta-like activity was also apparent. In amplitude spectra from these episodes, multiple peaks were evident within the gamma frequency band, indicating broad-band high-frequency activity, in association with a single low-frequency peak in the theta band. gamma peaks were attenuated during quiet Waking, in association with a low-frequency peak between theta and delta, and during Slow Wave Sleep, in association with a low-frequency peak in the delta band. In coherence spectra from ipsilateral cortical leads, peaks were also present within the gamma range and were significantly higher in Waking moving and Paradoxical Sleep than in Waking quiet and Slow Wave Sleep. In measures of frequency band amplitude, gamma activity (30.5-58.0 Hz) varied significantly across the sleep waking cycle, being similarly high during Wake and Paradoxical Sleep and lowest during Slow Wave Sleep. Across these states, gamma was negatively correlated with delta (1.5-4.0 Hz). In contrast, high beta (19.0-30.0 Hz) was significantly lower in Wake than in Slow Wave Sleep and was positively correlated with delta. gamma differed significantly across specific behaviors, being highest in Paradoxical Sleep with twitches and during Waking eating and moving behaviors, slightly lower in Waking attentive, lower in Waking grooming and as low in Waking quiet as during Slow Wave Sleep. These results indicate that the reciprocal variation of high-frequency gamma activity (and not beta) with low-frequency delta activity reflects the sleep waking cycle of the rat. Moreover, gamma activity reflects the degree of behavioral arousal, since it is high during active Waking, when the electromyogram is high, and low during quiet Waking, when the electromyogram is low. It also reflects cortical arousal, independent of motor activity, since it attains high levels in association with attentive immobility and maximal levels only during particular active behaviors (eating and moving and not grooming), and it also attains maximal levels during Paradoxical Sleep, when the nuchal electromyogram is minimal, but small twitches evidence dreaming. The co-variation of gamma and a slow oscillation in the theta band across states and behaviors suggests that a common system may modulate these fast and slow electroencephalogram rhythms, and that such modulation, potentially emanating from the basal forebrain, could predominate during certain states or behaviors, such as Paradoxical Sleep.