Lesions of the pedunculopontine tegmental nucleus reduce paradoxical sleep (PS) propensity: evidence from a short-term PS deprivation study in rats.

Cholinergic neurons in the mesopontine tegmentum are thought to play a critical role in the generation of paradoxical sleep (PS). However, no study has yet examined whether lesions of these neurons cause deficits of PS in the rat. We describe here the effects of lesions of the pedunculopontine tegmental nucleus (PPT) on spontaneous PS and on PS propensity, expressed during and after a short period of PS deprivation. Lesions were induced by bilateral injections of ibotenate. PS deprivation was performed manually by gently waking rats each time they showed polygraphic signs of PS. Two weeks after lesions, an 8-h baseline recording was performed; the following day, rats were PS deprived for 6 h and polygraphic recordings were then continued for 2 h, to examine recovery sleep. The same protocol was repeated 1 week later. Compared with controls and with rats with limited PPT lesions, rats bearing > 60% NADPH-diaphorase-positive cell loss within the PPT showed unaffected PS under baseline conditions. However, they made fewer attempts to enter PS during deprivation and they exhibited an attenuated rebound increase in PS time after deprivation. The number of PS attempts and the magnitude of PS rebound were negatively correlated with the percent loss of diaphorase-positive neurons within the PPT. Thus, PS propensity that accumulated as a result of PS deprivation was reduced after extensive PPT lesions. In summary, although spontaneous PS was found to be unaltered, the PS deprivation procedure used in this study demonstrated the dysfunctioning of PS caused by PPT lesions.

Diversity of receptive field changes in auditory cortex during natural sleep.

Twenty years ago, the study by Livingstone and Hubel [(1981) Nature, 291, 554] was viewed as a first step toward understanding how changes in state of vigilance affect sensory processing. Since then, however, very few attempts have been made to progress in this direction. In the present study, 56 cells were recorded in the auditory cortex of adult, undrugged guinea pigs, and the frequency tuning curves were tested during continuous and stable periods of wakefulness and of slow-wave sleep (SWS). Twelve cells were also tested during paradoxical sleep. Over the whole cell population, the response latency, the frequency selectivity and the size of the suprathreshold receptive field were not significantly modified during SWS compared with waking. However, this lack of global effects resulted from the heterogeneity of response changes displayed by cortical cells. During SWS, the receptive field size varied as a function of the changes in evoked responses: it was unchanged for the cells whose evoked responses were not modified (38% of the cells), reduced for the cells whose responses were decreased (48%) and enlarged for the cells whose responses were increased (14%). This profile of changes differs from the prevalent receptive field shrinkage that was observed in the auditory thalamus during SWS [Edeline et al. (2000), J. Neurophysiol., 84, 934]. It also contrasts with the receptive field enlargement that was described under anaesthesia when the EEG spontaneously shifted from a desynchronized to a synchronized pattern [Wörgötter et al. (1998), Nature, 396, 165]. Reasons for these differences are discussed.

Auditory thalamus neurons during sleep: changes in frequency selectivity, threshold, and receptive field size.

The present study describes how the frequency receptive fields (RF) of auditory thalamus neurons are modified when the state of vigilance of an unanesthetized animal naturally fluctuates among wakefulness (W), slow-wave sleep (SWS), and paradoxical sleep (PS). Systematic quantification of several RF parameters-including strength of the evoked responses, response latency, acoustic threshold, shape of rate-level function, frequency selectivity, and RF size-was performed while undrugged, restrained guinea pigs presented spontaneous alternances of W, SWS, and PS. Data are from 102 cells recorded during W and SWS and from 53 cells recorded during W, SWS, and PS. During SWS, thalamic cells behaved as an homogeneous population: as compared with W, most of them (97/102 cells) exhibited decreased evoked spike rates. The frequency selectivity was enhanced and the RF size was reduced. In contrast during PS, two populations of cells were identified: one (32/53 cells) showed the same pattern of changes as during SWS, whereas the other (21/53 cells) expressed values of evoked spike rates and RF properties that did not significantly differ from those in W. These two populations were equally distributed in the different anatomical divisions of the auditory thalamus. Last, during both SWS and PS, the responses latency was longer and the acoustic threshold was higher than in W but the proportion of monotonic versus nonmonotonic rate-level functions was unchanged. During both SWS and PS, no relationship was found between the changes in burst percentage and the changes of the RF properties. These results point out the dual aspect of sensory processing during sleep. On the one hand, they show that the auditory messages sent by thalamic cells to cortical neurons are reduced both in terms of firing rate at a given frequency and in terms of frequency range. On the other hand, the fact that the frequency selectivity and the rate-level function are preserved suggests that the messages sent to cortical cells are not deprived of informative content, and that the analysis of complex acoustic sounds should remain possible. This can explain why, although attenuated, reactivity to biologically relevant stimuli is possible during sleep.

Expression in paradoxical sleep of a conditioned heart rate response.

After a session of habituation to a tone, awake rats underwent two conditioning sessions during which the tone was paired with footshock. The tone alone was presented during paradoxical sleep (PS) following each session; it never awakened the animal. Heart rate (HR) was recorded at each tone presentation in wakefulness and PS. During conditioning in wakefulness, tone presentation elicited HR accelerative responses. Tone-evoked HR accelerations were also detected in PS following the two conditioning sessions. Such changes were not observed in rats that received unpaired presentations of tone and footshock. These results demonstrate that an HR conditioned response acquired in wakefulness can be expressed during PS.

Chronic, low-level exposure to the cholinesterase inhibitor DFP. I. Time course of neurochemical changes in the rat pontomesencephalic tegmentum.

Rats were repeatedly administered with a low dose of diisopropylfluorosphosphate (DFP; 0.2 mg/kg/day, SC, for 9 or 21 days), an irreversible cholinesterase (ChE) inhibitor. Control rats received a daily injection of oil vehicle. Neurochemical changes occurring in the pontomesencephalic tegmentum (PMT), a brain stem region critically involved in behavioral state control, were evaluated at various times of treatment and after DFP withdrawal. First, enzyme assay revealed a profile of ChE inhibition in the whole PMT which looked like that observed in the striatum; both the inhibition and recovery proceeded more slowly than they did in the plasma. Second, quantitative histochemistry indicated that ChE activity in the mesopontine cholinergic nuclei and the pontine reticular formation progressively decreased across the first days of DFP exposure, to reach an asymptotic level of inhibition after 6 days (74-82% inhibition). The inhibition was less pronounced in the locus coeruleus (49%). Third, [3H]QNB autoradiography showed that muscarinic receptor density was unchanged in any of the PMT areas selected. These results are discussed regarding the question of regional variation in susceptibility to anti-ChE agents. To what extent behavioral state alterations occur concomitantly with ChE activity changes is assessed in the companion article.

Chronic, low-level exposure to the cholinesterase inhibitor DFP. II. Time course of behavioral state changes in rats.

Rats were repeatedly administered with low doses of diisopropylfluorophosphate (DFP; 0.2 mg/kg/day, SC), an irreversible cholinesterase (ChE) inhibitor. Control rats received a daily injection of oil vehicle or of saline. Recordings of the sleep-wake states were obtained in the 6 h following 1, 3, 6, 9, 13, 17, and 21 injections, as well as 2, 4, and 19 days after 9-day treatment. DFP administration increased waking at the expense of slow-wave sleep (SWS), but not of paradoxical sleep (PS); as a result, the PS/SWS ratio was strongly enhanced. These changes developed across days, were maximal after six to nine injections, and were then maintained at that level until cessation of treatment. This time course of behavioral state alterations paralleled the time course of ChE inhibition in the mesopontine cholinergic nuclei and the pontine reticular formation described in the companion article. In contrast, after DFP withdrawal, behavioral states returned to control values more rapidly (in 2-4 days) than did ChE activity. These results are discussed regarding the promoting role of cholinergic neurotransmission in brain-activated states.

Neuronal plasticity induced by fear conditioning is expressed during paradoxical sleep: evidence from simultaneous recordings in the lateral amygdala and the medial geniculate in rats.

The lateral amygdala (LA) and its afferent connections from the medial geniculate (MG) play a pivotal role in auditory fear conditioning. The authors evaluated whether those neurons could express in paradoxical sleep (PS) physiological plasticity acquired in waking. After a habituation session, rats received tone-footshock pairings in 3 sessions. After each session, the tone alone was presented during PS episodes. Multiunit activity was simultaneously recorded in the LA and the medial part of the MG. Both in LA and MG, conditioned responses emerged rapidly (within 5 trials), were expressed with short latency (<20 ms), and were maintained in PS after training. Such changes were not observed in pseudoconditioned rats. These results are discussed regarding the question of the primary sites of plasticity in auditory fear conditioning and regarding the functional significance of preserved expression in PS of learning-induced neuronal plasticity.

Pontine microinjection of carbachol does not reliably enhance paradoxical sleep in rats.

It has been repeatedly shown in cats that acute administration of carbachol into the pontine reticular formation (PRF) readily evokes a state that closely mimics natural paradoxical sleep (PS). Surprisingly, there are few corresponding studies in rats. In order to further characterize the effects of pontine carbachol in rats, 151 injections of different doses (from 3 micrograms to 0.005 microgram in 0.1 microliter saline) of carbachol were made at different sites within the PRF of 70 rats. Sleep-waking states obtained in the 4 hours following carbachol administration were compared to control values, obtained both under baseline condition (no injection) and following pontine injection of 0.1 microliter saline. On the one hand, from the whole set of carbachol injections, it appeared that: 1) most injections (112/151) did not significantly alter the sleep-wake states; 2) when carbachol was effective, it induced either increased PS (20 injections) or increased waking (19 injections); and 3) effective injection sites were intermingled with noneffective sites. Dose- or site-dependency effects can account in part, but not totally, for these discordant results. On the other hand, in accordance with previous rat studies, we found that: 1) the PRF medial and ventral to the motor trigeminal nucleus was the most effective region for carbachol to increase PS; 2) carbachol-induced PS enhancement was of moderate magnitude (+60% above control saline level over the 4-hour recording time); 3) latency to onset of the first PS episode was not shortened; and 4) only the number of PS episodes was increased, their duration was not prolonged. These characteristics of carbachol-induced PS enhancement strongly differ, both in terms of magnitude and timing, from those described in cats. We suggest that the less reliable and weaker effects of pontine carbachol injection in rats compared to cats can be due to methodological problems inherent in the intracerebral microinjection technique and also to species-related differences in the mechanisms controlling the PS state.

Processing of learned information in paradoxical sleep: relevance for memory.

After a short review of the post-learning paradoxical sleep (PS) deprivation effects and of the PS changes induced by learning, we present a set of electrophysiological and behavioural experiments showing that: (1) processing of relevant information is possible during PS; (2) new associations can be formed during PS; (3) previously learned information can be reprocessed during PS; and (4) the effects of information processed during PS can be transferred to the awake state and be expressed in behaviour. Altogether, these results support the idea that dynamic processes occurring during post-learning PS can contribute to the effectiveness of memory processing and facilitate memory retrieval in wakefulness.

Non-awaking basal forebrain stimulation enhances auditory cortex responsiveness during slow-wave sleep.

Unilateral basal forebrain (BF) stimulations were delivered during slow-wave sleep (SWS) while multi-unit recordings were performed bilaterally in the auditory cortex. Ten tone presentations were followed by 10 pairing trials between BF stimulation and tone. Non-awaking BF stimulations facilitated the tone-evoked responses ipsilaterally only. Atropine blocked the facilitation of the ipsilateral evoked responses observed after pairing in wakefulness. Thus, non-awaking cholinergic input can enhance cortical responsiveness during SWS.

Transient and prolonged facilitation of tone-evoked responses induced by basal forebrain stimulations in the rat auditory cortex.

We investigated the relationships between cortical arousal and cholinergic facilitation of evoked responses in the auditory cortex. The basal forebrain (BF) was stimulated unilaterally, while cluster recordings were obtained simultaneously from both auditory cortices in urethane-anesthetized rats. The global electroencephalogram (EEG; large frontoparietal derivation) and the local EEG (from the auditory cortex) were recorded. The BF was stimulated at two intensities, a lower one which did not desynchronize the EEG and a higher one which did. Twenty pairing trials were delivered, during which a tone was presented 50 ms after the end of the BF stimulation. At low intensity, the pairing procedure led to a transient increase in the ipsilateral tone-evoked responses. At high intensity, the pairing increased the ipsilateral evoked responses up to 15 min after pairing. Such effects were not observed for the contralateral recordings. Systemic atropine injection prevented the facilitations observed ipsilaterally. BF stimulations alone did not induce any increased evoked response either at low or at high intensity. These results show (1) that a tone, presented while the cortex is activated by cholinergic neurons of the BF, evokes enhanced cortical responses, and (2) that the duration of this facilitation is dependent on the stimulation intensity. These results are discussed in the context of neural mechanisms involved in general arousal and cortical plasticity.

Learning-induced plasticity in the medial geniculate nucleus is expressed during paradoxical sleep.

Fear conditioning to an acoustic stimulus produces increases in tone-evoked discharges of neurons in the medial division of the medial geniculate nucleus (MG). This study examined the responses of MG neurons to a conditioned tone presented in paradoxical sleep (PS). After 1 session of habituation to a tone, awake rats underwent conditioning in 3 sessions during which the tone was used as the conditioned stimulus preceding a footshock. Control rats received unpaired presentations of tone and shock. The same tone, which never awakened the animal, was presented during PS following each daily session. Responses of MG neurons to the tone in PS were increased after conditioning. This enhancement was as large as that in waking and was manifested earlier after tone onset than in waking. No change appeared after pseudoconditioning. These results demonstrate that associatively induced plasticity in the MG can be expressed during PS.

Basal forebrain stimulation facilitates tone-evoked responses in the auditory cortex of awake rat.

The effects of unilateral basal forebrain stimulation on the tone-evoked responses recorded in the auditory cortex ipsilateral and contralateral to the stimulation site, were investigated in fully awake rats. After 10 tone alone presentations, 20 pairing trials were given during which the basal forebrain stimulation was followed by the tone 30 ms later. Ten test-tones were presented immediately, 15 min and 1 h after pairing. Immediately after pairing, the short-latency "on" and "off" tone-evoked responses were enhanced in the ipsilateral but not in the contralateral cortex. This enhancement did not persist 15 min later. Systemic atropine injection prevented the ipsilateral facilitation. The responses to the tone were not modified when tested after 20 basal forebrain stimulations delivered in the absence of the tone. These results are the first demonstration in awake animals that an activation of the auditory cortex by cholinergic neurons of the basal forebrain is able to facilitate cortical responsiveness. A temporal contiguity between the cholinergic activation and the neuronal discharges elicited by the sensory stimulus is required for the facilitation to take place. The results are compared to previous ones obtained in anesthetized animals, and the functional role of cholinergic activation from the basal forebrain in cortical processing is discussed.

Improvement of learning by mesencephalic reticular stimulation during postlearning paradoxical sleep.

Evidences have been given which suggest that a newly formed memory trace is processed during paradoxical sleep (PS) following learning. The present experiments were aimed at testing the hypothesis that during postlearning PS the new memory trace is in a similar state as immediately after acquisition. For this purpose, a mild electrical stimulation of the mesencephalic reticular formation (MRF)--known to enhance retention performance when delivered just after learning--was administered during postlearning PS phases. Wistar rats were trained to run in a six-unit spatial discrimination maze for food reward. After each daily trial, extradural cortical electrodes (ECoG) activity was monitored polygraphically for 4 h. Half of the animals received nonawakening MRF stimulations during the first six phases of PS. Control rats received no stimulation. The learning results showed a marked improvement in performance, in terms of error number reduction, in the stimulated group. Results of a second experiment confirmed the facilitative effect of MRF stimulations given during postlearning PS. Moreover, they emphasized the specific role of PS, by showing that the same stimulations were ineffective when delivered, at the same time intervals after training, during six periods of waking or six periods of slow-wave sleep. These results lend support to the idea of a reactivation of the new memory trace during PS. They suggest that dynamic processes, similar to those immediately following acquisition or exposure to a reactivating treatment (i.e., a reminder), take place during postlearning PS.

Impairment of learning by cueing during postlearning slow-wave sleep in rats.

Rats were trained in a two-way shuttle avoidance task, 20 trials per day for 3 consecutive days. The conditioned stimulus preceding a footshock was an ear shock in expt. 1, it was a tone in expt. 2. In both experiments, experimental animals received non-awakening ear shocks during 6 periods of slow-wave sleep (SWS) following each conditioning session. Control animals received no ear shock during SWS. Results showed that ear shocks delivered during SWS impaired subsequent learning performances only in expt. 1, i.e. when they were relevant to the learning task and could act as a reminder treatment.

Is increase in post-learning paradoxical sleep modified by cueing?

Rats were submitted to three sessions of an active avoidance conditioning. The conditioned stimulus (CS) was either an ear shock or a tone. Sleep was measured after each session. Experimental animals received non-awakening ear shocks during post-learning paradoxical sleep (PS). When ear shocks were the CS and were subsequently used as cues during PS, PS increases were observed, but they displayed 2 unusual characteristics: they were due to longer average size of PS phases rather than to higher number of phases as usually; PS did not return to baseline level even when learning seemed to be achieved. By contrast, when ear shocks were not associated with conditioning, animals shocked during PS exhibited the usual PS increases.

Improvement of learning by cueing during postlearning paradoxical sleep.

Rats were submitted to an active avoidance conditioning in a shuttle box with slight ear shocks used as conditioned stimulus (CS) preceding a foot shock. Three conditioning sessions were performed with a 24 h intersession interval. Animals were divided into 3 groups. After each session, the first group received the CS as cue during the first 6 phases of paradoxical sleep (PS) following learning. The second group received the CS as cue during 6 periods of wakefulness. The third group received no cue. Animals cued during PS showed a significant improvement in performances. The effect of cueing during the awake state appeared to be marginal compared to the clear-cut effects when the cue was presented during PS. A control experiment showed that the same ear shocks presented during PS were ineffective when they were not associated with the learning task (in this experiment a tone was used as CS during conditioning). These results are discussed in terms of memory reactivation during postlearning paradoxical sleep.

Reminder abolishes impairment of learning induced by paradoxical sleep retardation.

Rats were submitted to one daily trial in a relatively complex maze. When sleep was delayed (by the water tank technique) for 180 min after each trial, learning was impaired. A reminder treatment (90 sec exposure to contextual cues) immediately before each trial, counteracted the effects of sleep deprivation. The reminder did not in itself contain sufficient information to facilitate performance of non sleep delayed animals. These results suggest that a retrieval failure is involved in memory impairment caused by post-learning paradoxical sleep deprivation.