Pontine control of rapid eye movement sleep and fear memory

Aims

We often experience dreams of strong irrational and negative emotional contents with postural muscle paralysis during rapid eye movement (REM) sleep, but how REM sleep is generated and its function remain unclear. In this study, we investigate whether the dorsal pontine sub-laterodorsal tegmental nucleus (SLD) is necessary and sufficient for REM sleep and whether REM sleep elimination alters fear memory.

Methods

To investigate whether activation of SLD neurons is sufficient for REM sleep induction, we expressed channelrhodopsin-2 (ChR2) in SLD neurons by bilaterally injecting AAV1-hSyn-ChR2-YFP in rats. We next selectively ablated either glutamatergic or GABAergic neurons from the SLD in mice in order to identify the neuronal subset crucial for REM sleep. We finally  investigated the role of REM sleep in consolidation of fear memory using rat model with complete SLD lesions.

Results

We demonstrate the sufficiency of the SLD for REM sleep by showing that photo-activation of ChR2 transfected SLD neurons selectively promotes transitions from non-REM (NREM) sleep to REM sleep in rats. Diphtheria toxin-A (DTA) induced lesions of the SLD in rats or specific deletion of SLD glutamatergic neurons but not GABAergic neurons in mice completely abolish REM sleep, demonstrating the necessity of SLD glutamatergic neurons for REM sleep. We then show that REM sleep elimination by SLD lesions in rats significantly enhances contextual and cued fear memory consolidation by 2.5 and 1.0 folds, respectively, for at least 9 months. Conversely, fear conditioning and fear memory trigger doubled amounts of REM sleep in the following night, and chemo-activation of SLD neurons projecting to the medial septum (MS) selectively enhances hippocampal theta activity in REM sleep; this stimulation immediately after fear acquisition reduces contextual and cued fear memory consolidation by 60% and 30%, respectively.

Conclusion

SLD glutamatergic neurons generate REM sleep and REM sleep and SLD via the hippocampus particularly down-regulate contextual fear memory.     

Combined blockade of cholinergic receptors shifts the brain from stimulus encoding to memory consolidation

High central nervous system levels of acetylcholine (ACh) are commonly regarded as crucial for learning and memory, and a decline in cholinergic neurotransmission is associated with Alzheimer's dementia. However, recent findings revealed exceptions to this rule: The low ACh tone characterizing slow wave sleep (SWS) has proven necessary for consolidation of hippocampus-dependent declarative memories during this sleep stage. Such observations, together with recent models of a hippocampal-neocortical dialogue underlying systems memory consolidation, suggest that high levels of ACh support memory encoding, whereas low levels facilitate consolidation. We tested this hypothesis in human subjects by blocking cholinergic neurotransmission during wakefulness, starting 30 min after learning. Subjects received the muscarinic antagonist scopolamine (4 microg/kg bodyweight intravenously) and the nicotinic antagonist mecamylamine (5 mg orally). Compared to placebo, combined muscarinic and nicotinic receptor blockade significantly improved consolidation of declarative memories tested 10 hr later, but simultaneously impaired acquisition of similar material. Consolidation of procedural memories, which are not dependent on hippocampal functioning, was unaffected. Neither scopolamine nor mecamylamine alone enhanced declarative memory consolidation. Our findings support the notion that ACh acts as a switch between modes of acquisition and consolidation. We propose that the natural shift in central nervous system cholinergic tone from high levels during wakefulness to minimal levels during SWS optimizes declarative memory consolidation during a period with no need for new memory encoding.     

Somatostatin Neurons of the Bed Nucleus of Stria Terminalis Enhance Associative Fear Memory Consolidation in Mice

Excessive fear learning and generalized, extinction-resistant fear memories are core symptoms of anxiety and trauma-related disorders. Despite significant evidence from clinical studies reporting hyperactivity of the bed nucleus of stria terminalis (BNST) under these conditions, the role of BNST in fear learning and expression is still not clarified. Here, we tested how BNST modulates fear learning in male mice using a chemogenetic approach. Activation of GABAergic neurons of BNST during fear conditioning or memory consolidation resulted in enhanced cue-related fear recall. Importantly, BNST activation had no acute impact on fear expression during conditioning or recalls, but it enhanced cue-related fear recall subsequently, potentially via altered activity of downstream regions. Enhanced fear memory consolidation could be replicated by selectively activating somatostatin (SOM), but not corticotropin-releasing factor (CRF), neurons of the BNST, which was accompanied by increased fear generalization. Our findings suggest the significant modulation of fear memory strength by specific circuits of the BNST.SIGNIFICANCE STATEMENT The bed nucleus of stria terminalis (BNST) mediates different defensive behaviors, and its connections implicate its integrative modulatory role in fear memory formation; however, the involvement of BNST in fear learning has yet to be elucidated in detail. Our data highlight that BNST stimulation enhances fear memory formation without direct effects on fear expression. Our study identified somatostatin (SOM) cells within the extended amygdala as specific neurons promoting fear memory formation. These data underline the importance of anxiety circuits in maladaptive fear memory formation, indicating elevated BNST activity as a potential vulnerability factor to anxiety and trauma-related disorders.     

The whats and whens of sleep-dependent memory consolidation

Sleep benefits memory consolidation. The reviewed studies indicate that this consolidating effect is not revealed under all circumstances but is linked to specific psychological conditions. Specifically, we discuss to what extent memory consolidation during sleep depends on the type of learning materials, type of learning and retrieval test, different features of sleep and the subject population. Post-learning sleep enhances consolidation of declarative, procedural and emotional memories. The enhancement is greater for weakly than strongly encoded associations and more consistent for explicitly than implicitly encoded memories. Memories associated with expected reward gain preferentially access to sleep-dependent consolidation. For declarative memories, sleep benefits are more consistently revealed with recall than recognition procedures at retrieval testing. Slow wave sleep (SWS) particularly enhances declarative memories whereas rapid eye movement (REM) sleep preferentially supports procedural and emotional memory aspects. Declarative memory profits already from rather short sleep periods (1–2 h). Procedural memory profits seem more dose-dependent on the amount of sleep following the day after learning. Children's sleep with high amounts of SWS distinctly enhances declarative memories whereas elderly and psychiatric patients with disturbed sleep show impaired sleep-associated consolidation often of declarative memories. Based on the constellation of psychological conditions identified we hypothesize that access to sleep-dependent consolidation requires memories to be encoded under control of prefrontal-hippocampal circuitry, with the same circuitry controlling subsequent consolidation during sleep.     

Delayed changes of sleep duration after rewarded olfactory discrimination learning in the rat

The aim of this experiment was to determine if a task of associative olfactory learning, based on the ethological repertory of rats and learnt rapidly in 5 successive trials, could modify slow wave sleep (SWS) and/or paradoxical sleep (PS) duration after learning and/or after a retrieval–reactivation test 24 h later. Somnopolygraphic recordings were performed for 20 h per day on trained and control (submitted to a pseudo-learning test) rats. SWS and PS durations were analyzed per 20 h and per 4 h time-periods. Compared to control rats, after learning, trained rats showed a significant increase in SWS duration counterbalanced by a significant decrease in wake duration focused on the 5–8 h post-training time-window and a significant decrease in PS duration during the 17–20 h post-training time-window. After the retrieval–reactivation test trained rats only showed a decreased PS duration compared to control rats submitted to a pseudo-retrieval test. Thus, a rather simple learning task succeeded in eliciting an increase in SWS duration in a limited time-window. As the learning task used can be compared to human associate-paired learning, this result sustains the hypothesis of a link between declarative memory and SWS. In control rats, changes in PS duration might be linked to odorized-environment exposure.     

Evidence for 2-stage models of sleep and memory: Learning-dependent changes in spindles and theta in rats

What processes are involved in the formation of enduring memory traces? Sleep has been proposed to play a role in memory consolidation and the present study provides evidence to support 2-stage models of sleep and memory including both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Previous research has shown REM sleep increases following avoidance learning and memory is impaired if REM deprivation occurs during these post-training periods indicating that REM sleep may have a role in memory consolidation processes. These discrete post-training periods have been termed REM sleep windows (RSWs). It is not known whether the electroencephalogram has unique characteristics during the RSW. Further investigation of the RSW was one of the primary goals of this study. We investigated the epidural-recorded electrophysiological learning-related changes following avoidance training in rats. Theta power increased in the learning group during the RSW, suggesting that theta is involved in memory consolidation during this period. Sleep spindles subsequently increased in slow wave sleep (SWS). The results suggest that both NREM and REM sleep are involved in sleep-dependent memory consolidation, and provide support for existing 2-stage models. Perhaps first theta increases to organize and consolidate material via hippocampal–neocortical dialogue, followed by subsequent refinement in the cortex by spindles during SWS.     

Effects of cortisol suppression on sleep-associated consolidation of neutral and emotional memory.

BACKGROUND: Previous research indicates that hippocampus-dependent declarative memory benefits from early nocturnal sleep, when slow-wave sleep (SWS) prevails and cortisol release is minimal, whereas amygdala-dependent emotional memory is enhanced through late sleep, when rapid eye movement (REM) sleep predominates. The role of the strong cortisol rise accompanying late sleep for emotional memory consolidation has not yet been investigated. METHODS: Effects of the cortisol synthesis inhibitor metyrapone on sleep-associated consolidation of memory for neutral and emotional texts were investigated in a randomized, double-blind, placebo-controlled study in 14 healthy men. Learning took place immediately before treatment, which was followed by 8 hours of sleep. Retrieval was tested at 11 am the next morning. RESULTS: Metyrapone suppressed cortisol during sleep and blocked particularly the late-night rise in cortisol. It reduced SWS and concomitantly impaired the consolidation of neutral texts. Emotional texts were spared from this impairing influence, however. Metyrapone even amplified emotional enhancement in text recall indicating amygdala-dependent memory. CONCLUSIONS: Cortisol blockade during sleep impairs hippocampus-dependent declarative memory formation but enhances amygdala-dependent emotional memory formation. The natural cortisol rise during late sleep may thus protect from overshooting emotional memory formation, a mechanism possibly pertinent to the development of posttraumatic stress disorder.     

Dissociable learning-dependent changes in REM and non-REM sleep in declarative and procedural memory systems

Sleep spindles and rapid eye movements have been found to increase following an intense period of learning on a combination of procedural memory tasks. It is not clear whether these changes are task specific, or the result of learning in general. The current study investigated changes in spindles, rapid eye movements, K-complexes and EEG spectral power following learning in good sleepers randomly assigned to one of four learning conditions: Pursuit Rotor (n=9), Mirror Tracing (n=9), Paired Associates (n=9), and non-learning controls (n=9). Following Pursuit Rotor learning, there was an increase in the duration of Stage 2 sleep, spindle density (number of spindles/min), average spindle duration, and an increase in low frequency sigma power (12-14Hz) at occipital regions during SWS and at frontal regions during Stage 2 sleep in the second half of the night. These findings are consistent with previous findings that Pursuit Rotor learning is consolidated during Stage 2 sleep, and provide additional data to suggest that spindles across all non-REM stages may be a mechanism for brain plasticity. Following Paired Associates learning, theta power increased significantly at central regions during REM sleep. This study provides the first evidence that REM sleep theta activity is involved in declarative memory consolidation. Together, these findings support the hypothesis that brain plasticity during sleep does not involve a unitary process; that is, different types of learning have unique sleep-related memory consolidation mechanisms that act in dissociable brain regions at different times throughout the night.     

Differential Effects of Non-REM and REM Sleep on Memory Consolidation?

Sleep benefits memory consolidation. Previous theoretical accounts have proposed a differential role of slow-wave sleep (SWS), rapid-eye-movement (REM) sleep, and stage N2 sleep for different types of memories. For example the dual process hypothesis proposes that SWS is beneficial for declarative memories, whereas REM sleep is important for consolidation of non-declarative, procedural and emotional memories. In fact, numerous recent studies do provide further support for the crucial role of SWS (or non-REM sleep) in declarative memory consolidation. However, recent evidence for the benefit of REM sleep for non-declarative memories is rather scarce. In contrast, several recent studies have related consolidation of procedural memories (and some also emotional memories) to SWS (or non-REM sleep)-dependent consolidation processes. We will review this recent evidence, and propose future research questions to advance our understanding of the role of different sleep stages for memory consolidation.     

Sleep stage II contributes to the consolidation of declarative memories

Various studies suggest that non-rapid eye movement (NREM) sleep, especially slow-wave sleep (SWS), is vital to the consolidation of declarative memories. However, sleep stage 2 (S2), which is the other NREM sleep stage besides SWS, has gained only little attention. The current study investigated whether S2 during an afternoon nap contributes to the consolidation of declarative memories. Participants learned associations between faces and cities prior to a brief nap. A cued recall test was administered before and following the nap. Spindle, delta and slow oscillation activity was recorded during S2 in the nap following learning and in a control nap. Increases in spindle activity, delta activity, and slow oscillation activity in S2 in the nap following learning compared to the control nap were associated with enhanced retention of face-city associations. Furthermore, spindles tended to occur more frequently during up-states than down-states within slow oscillations during S2 following learning versus S2 of the control nap. These findings suggest that spindles, delta waves, and slow oscillations might promote memory consolidation not only during SWS, as shown earlier, but also during S2.     

Impaired Declarative Memory Consolidation During Sleep in Patients With Primary Insomnia: Influence of Sleep Architecture and Nocturnal Cortisol Release

BACKGROUND: A central cognitive function of sleep is to consolidate newly acquired memories for long-term storage. Here, we investigated whether the overnight consolidation of declarative memory in patients with chronic sleep disturbances is impaired, owing to less slow wave sleep (SWS) and an increased cortisol release. METHODS: Polysomnographic recordings, serum cortisol concentrations, and overnight memory consolidation in 16 patients with primary insomnia were compared with those of 13 healthy control subjects. RESULTS: Patients displayed distinctly less overnight consolidation of declarative memory (p < .05), which was significantly correlated with SWS in the control subjects (r = .69) but with rapid eye movement (REM) sleep in the patients (r = .56), who had a diminished amount of SWS (p < .05). Increased cortisol levels in the middle of the night were associated with impaired retrieval of declarative memory after sleep for both control subjects (r = -.52) and patients (r = -.46). CONCLUSIONS: Primary insomnia is associated with a diminished sleep-related consolidation of declarative memory. Efficient overnight consolidation of declarative memory is associated with high amounts of SWS and low serum cortisol levels during the early part of the night. Where SWS is decreased, REM sleep might play a partly compensatory role in the consolidation of declarative memory.     

Rapid synaptic potentiation within the anterior cingulate cortex mediates trace fear learning

Although the cortex has been extensively studied in long-term memory storage, less emphasis has been placed on immediate cortical contributions to fear memory formation. AMPA receptor plasticity is strongly implicated in learning and memory, and studies have identified calcium permeable AMPA receptors (CP-AMPARs) as mediators of synaptic strengthening. Trace fear learning engages the anterior cingulate cortex (ACC), but whether plastic events occur within the ACC in response to trace fear learning, and whether GluN2B subunits are required remains unknown. Here we show that the ACC is necessary for trace fear learning, and shows a rapid 20% upregulation of membrane AMPA receptor GluA1 subunits that is evident immediately after conditioning. Inhibition of NMDA receptor GluN2B subunits during training prevented the upregulation, and disrupted trace fear memory retrieval 48 h later. Furthermore, intra-ACC injections of the CP-AMPAR channel antagonist, 1-naphthylacetyl spermine (NASPM) immediately following trace fear conditioning blocked 24 h fear memory retrieval. Accordingly, whole cell patch clamp recordings from c-fos positive and c-fos negative neurons within the ACC in response to trace fear learning revealed an increased sensitivity to NASPM in recently activated neurons that was reversed by reconsolidation update extinction. Our results suggest that trace fear learning is mediated through rapid GluN2B dependent trafficking of CP-AMPARs, and present in vivo evidence that CP-AMPAR activity within the ACC immediately after conditioning is necessary for subsequent memory consolidation processes.     

Sleep-dependent surges in growth hormone do not contribute to sleep-dependent memory consolidation

In the search for the mechanisms that mediate the effects of sleep on the consolidation of memories, growth hormone (GH) recently became of interest, because in humans it is released mainly during slow-wave sleep (SWS), a period of enhanced declarative memory consolidation. In addition, recent studies showed that GH is involved in proper memory function in GH deficient and elderly humans and this effect has been linked to regulatory influences of GH on hippocampal NMDA receptors. Here, we blocked GH secretion by intravenous infusion of somatostatin in healthy young subjects during the first 3 h of sleep, which contain mainly SWS. Declarative and procedural memory consolidation was tested across this period, using a word pair association task and a mirror tracing task, respectively. Although GH was effectively suppressed, memory performance as well as sleep were entirely unaffected by this suppression. Whereas GH may in the long run generally support brain systems required for maintaining proper memory function, our data exclude a necessary contribution of the nocturnal surge in pituitary GH secretion to the acute processing and formation of specific memories during sleep.     

Sleep-dependent memory consolidation – What can be learnt from children?

Extensive research has been accumulated demonstrating that sleep is essential for processes of memory consolidation in adults. In children and infants, a great capacity to learn and to memorize coincides with longer and more intense sleep. Here, we review the available data on the influence of sleep on memory consolidation in healthy children and infants, as well as in children with attention-deficit/hyperactivity disorder (ADHD) as a model of prefrontal impairment, and consider possible mechanisms underlying age-dependent differences. Findings indicate a major role of slow wave sleep (SWS) for processes of memory consolidation during early development. Importantly, longer and deeper SWS during childhood appears to produce a distinctly superior strengthening of hippocampus-dependent declarative memories, but concurrently prevents an immediate benefit from sleep for procedural memories, as typically observed in adults. Studies of ADHD children point toward an essential contribution of prefrontal cortex to the preferential consolidation of declarative memory during SWS. Developmental studies of sleep represent a particularly promising approach for characterizing the supra-ordinate control of memory consolidation during sleep by prefrontal-hippocampal circuitry underlying the encoding of declarative memory.     

Dissociated theta phase synchronization in amygdalo- hippocampal circuits during various stages of fear memory

The amygdala and the hippocampus are critically involved in the formation and retention of fear memories. However, their precise contribution to, and their interplay during, fear memory formation are not fully understood. In the present study we investigated network activities in the amygdalo-hippocampal system of freely behaving mice at different stages of fear memory consolidation and retention. Our data show enhanced theta phase synchronization in this pathway during the retrieval of fear memory at long-term (24 h post-training), but not short-term (2 min, 30 min and 2 h post-training) stages, following both contextual and auditory cued conditioning. However, retrieval of remotely conditioned fear (30 days post-training) failed to induce an increase in synchronization despite there still being memory retention. Thus, our data indicate that the amygdalo-hippocampal interaction reflects a dynamic interaction of ensemble activities related to various stages of fear memory consolidation and/or retention, and support the notion that recent and remote memories are organized through different network principles.     

Sex and modulatory menstrual cycle effects on sleep related memory consolidation

The benefit of sleep in general for memory consolidation is well known. The relevance of sleep characteristics and the influence of hormones are not well studied. We explored the effects of a nap on memory consolidation of motor (finger-tapping-task) and verbal (associated-word-pairs) tasks in following settings: A: young, healthy males and females during early-follicular phase (n=40) and B: females during mid-luteal and early-follicular phase in the menstrual cycle (n=15). We found a sex and in women a menstrual cycle effect on memory performance following a nap. Men performed significantly better after a nap and women did so only in the mid-luteal phase of their menstrual cycle. Only the men and the women in their mid-luteal phase experienced a significant increase in spindle activity after learning. Furthermore, in women estrogen correlated significantly with the offline change in declarative learning and progesterone with motor learning. The ratio of the 2nd and 4th digit, which has been associated to fetal sex hormones and cognitive sex differences, significantly predicted the average performance of the female subjects in the learning tasks. Our results demonstrate that sleep-related memory consolidation has a higher complexity and more influencing factors than previously assumed. There is a sex and menstrual cycle effect, which seems to be mediated by female hormones and sleep spindles. Further, contrary to previous reports, consolidation of a simple motor task can be induced by a 45 min NREM sleep nap, thus not dependent on REM sleep.     

Dissociated Role of Thalamic and Cortical Input to the Lateral Amygdala for Consolidation of Long-Term Fear Memory

Post-encoding coordinated reactivation of memory traces distributed throughout interconnected brain regions is thought to be critical for consolidation of memories. However, little is known about the role of neural circuit pathways during post-learning periods for consolidation of memories. To investigate this question, we optogenetically silenced the inputs from both auditory cortex and thalamus in the lateral amygdala (LA) for 15 min immediately following auditory fear conditioning (FC) and examined its effect on fear memory formation in mice of both sexes. Optogenetic inhibition of both inputs disrupted long-term fear memory formation tested 24 h after FC. This effect was specific such that the same inhibition did not affect short-term memory and context-dependent memory. Moreover, long-term memory was intact if the inputs were inhibited at much later time points after FC (3 h or 1 d after FC), indicating that optical inhibition for 15 min itself does not produce any nonspecific deleterious effect on fear memory retrieval. Selective inhibition of thalamic input was sufficient to impair consolidation of auditory fear memory. In contrast, selective inhibition of cortical input disrupted remote fear memory without affecting recent memory. These results reveal a dissociated role of thalamic and cortical input to the LA during early post-learning periods for consolidation of long-term fear memory.SIGNIFICANCE STATEMENT Coordinated communications between brain regions are thought to be essential during post-learning periods for consolidation of memories. However, the role of specific neural circuit pathways in this process has been scarcely explored. Using a precise optogenetic inhibition of auditory input pathways, either thalamic or cortical or both, to the LA during post-training periods, we here show that thalamic input is required for consolidation of both recent and remote fear memory, whereas cortical input is crucial for consolidation of remote fear memory. These results reveal a dissociated role of auditory input pathways to the LA for consolidation of long-term fear memory.     

Coupling between the prelimbic cortex,nucleus reuniens,and hippocampus during NREM sleep remains stable under cognitive and homeostatic demands

The interplay between the medial prefrontal cortex and hippocampus during non-rapid eye movement (NREM) sleep contributes to the consolidation of contextual memories. To assess the role of the thalamic nucleus reuniens (Nre) in this interaction, we investigated the coupling of neuro-oscillatory activities among prelimbic cortex, Nre, and hippocampus across sleep states and their role in the consolidation of contextual memories using multi-site electrophysiological recordings and optogenetic manipulations. We showed that ripples are time-locked to the Up state of cortical slow waves, the transition from UP to DOWN state in thalamic slow waves, the troughs of cortical spindles, and the peaks of thalamic spindles during spontaneous sleep, rebound sleep and sleep following a fear conditioning task. In addition, spiking activity in Nre increased before hippocampal ripples, and the phase-locking of hippocampal ripples and thalamic spindles during NREM sleep was stronger after acquisition of a fear memory. We showed that optogenetic inhibition of Nre neurons reduced phase-locking of ripples to cortical slow waves in the ventral hippocampus whilst their activation altered the preferred phase of ripples to slow waves in ventral and dorsal hippocampi. However, none of these optogenetic manipulations of Nre during sleep after acquisition of fear conditioning did alter sleep-dependent memory consolidation. Collectively, these results showed that Nre is central in modulating hippocampus and cortical rhythms during NREM sleep.     

Activation of cortical interneurons during sleep: an anatomical link to homeostatic sleep regulation?

Although slow wave activity in the EEG has been linked to homeostatic sleep regulation, the neurobiological substrate of sleep homeostasis is not well understood. Whereas cortical neurons typically exhibit reduced discharge rates during slow wave sleep (SWS), a subpopulation of GABAergic interneurons, which express the enzyme neuronal nitric oxide synthase (nNOS), has recently been found to be activated during SWS. The extent of activation of these nNOS neurons is proportional to homeostatic sleep 'drive'. These cells are an exception among cortical interneurons in that they are projection neurons. We propose that cortical nNOS neurons are positioned to influence neuronal activity across widespread brain areas. They could thus provide a long-sought anatomical link for understanding homeostatic sleep regulation.     

Effects of pentylenetetrazol-induced brief convulsive seizures on spatial memory and fear memory

Previous studies have demonstrated that in the pentylenetetrazol (PTZ) kindling model, recurrent seizures either impair or have no effect on learning and memory. However, the effects of brief seizures on learning and memory remain unknown. Here, we found that a single injection of a convulsive dose of PTZ (50 mg/kg, ip) induced brief seizures in Sprague–Dawley rats. Administration of PTZ before training impaired the acquisition of spatial memory in the Morris water maze (MWM) and fear memory in contextual fear conditioning. However, the administration of PTZ immediately after training did not affect memory consolidation in either task. These findings suggest that brief seizures have different effects on acquisition and consolidation of spatial and fear memory.