Effects of Chronic Sleep Restriction during Early Adolescence on the Adult Pattern of Connectivity of Mouse Secondary Motor Cortex

Cortical circuits mature in stages, from early synaptogenesis and synaptic pruning to late synaptic refinement, resulting in the adult anatomical connection matrix. Because the mature matrix is largely fixed, genetic or environmental factors interfering with its establishment can have irreversible effects. Sleep disruption is rarely considered among those factors, and previous studies have focused on very young animals and the acute effects of sleep deprivation on neuronal morphology and cortical plasticity. Adolescence is a sensitive time for brain remodeling, yet whether chronic sleep restriction (CSR) during adolescence has long-term effects on brain connectivity remains unclear. We used viral-mediated axonal labeling and serial two-photon tomography to measure brain-wide projections from secondary motor cortex (MOs), a high-order area with diffuse projections. For each MOs target, we calculated the projection fraction, a combined measure of passing fibers and axonal terminals normalized for the size of each target. We found no homogeneous differences in MOs projection fraction between mice subjected to 5 days of CSR during early adolescence (P25–P30, ≥50% decrease in daily sleep, n=14) and siblings that slept undisturbed (n=14). Machine learning algorithms, however, classified animals at significantly above chance levels, indicating that differences between the two groups exist, but are subtle and heterogeneous. Thus, sleep disruption in early adolescence may affect adult brain connectivity. However, because our method relies on a global measure of projection density and was not previously used to measure connectivity changes due to behavioral manipulations, definitive conclusions on the long-term structural effects of early CSR require additional experiments

Microstructural dynamics of motor learning and sleep-dependent consolidation: A diffusion imaging study

Memory consolidation can benefit from post-learning sleep, eventually leading to long-term microstructural brain modifications to accommodate new memory representations. Non-invasive diffusion-weighted magnetic resonance imaging (DWI) allows the observation of (micro)structural brain remodeling after time-limited motor learning. Here, we combine conventional diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) that allows modeling dendritic and axonal complexity in gray matter to investigate with improved specificity the microstructural brain mechanisms underlying time- and sleep-dependent motor memory consolidation dynamics. Sixty-one young healthy adults underwent four DWI sessions, two sequential motor trainings, and a night of total sleep deprivation or regular sleep distributed over five days. We observed rapid-motor-learning-related remodeling in occipitoparietal, temporal, and motor-related subcortical regions, reflecting temporary dynamics in learning-related neuronal brain plasticity processes. Sleep-related consolidation seems not to exert a detectable impact on diffusion parameters, at least on the timescale of a few days

Effects of Chronic Sleep Restriction during Early Adolescence on the Adult Pattern of Connectivity of Mouse Secondary Motor Cortex

Cortical circuits mature in stages, from early synaptogenesis and synaptic pruning to late synaptic refinement, resulting in the adult anatomical connection matrix. Because the mature matrix is largely fixed, genetic or environmental factors interfering with its establishment can have irreversible effects. Sleep disruption is rarely considered among those factors, and previous studies have focused on very young animals and the acute effects of sleep deprivation on neuronal morphology and cortical plasticity. Adolescence is a sensitive time for brain remodeling, yet whether chronic sleep restriction (CSR) during adolescence has long-term effects on brain connectivity remains unclear. We used viral-mediated axonal labeling and serial two-photon tomography to measure brain-wide projections from secondary motor cortex (MOs), a high-order area with diffuse projections. For each MOs target, we calculated the projection fraction, a combined measure of passing fibers and axonal terminals normalized for the size of each target. We found no homogeneous differences in MOs projection fraction between mice subjected to 5 days of CSR during early adolescence (P25–P30, ≥50% decrease in daily sleep, n=14) and siblings that slept undisturbed (n=14). Machine learning algorithms, however, classified animals at significantly above chance levels, indicating that differences between the two groups exist, but are subtle and heterogeneous. Thus, sleep disruption in early adolescence may affect adult brain connectivity. However, because our method relies on a global measure of projection density and was not previously used to measure connectivity changes due to behavioral manipulations, definitive conclusions on the long-term structural effects of early CSR require additional experiments

Sleep as a model to understand neuroplasticity and recovery after stroke : observational, perturbational and interventional approaches

Our own experiences with disturbances to sleep demonstrate its crucial role in the recovery of cognitive functions. This importance is likely enhanced in the recovery from stroke; both in terms of its physiology and cognitive abilities. Decades of experimental research have highlighted which aspects and mechanisms of sleep are likely to underlie these forms of recovery. Conversely, damage to certain areas of the brain, as well as the indirect effects of stroke, may disrupt sleep. However, only limited research has been conducted which seeks to directly explore this bidirectional link between both the macro and micro-architecture of sleep and stroke. Here we describe a series of semi-independent approaches that aim to establish this link through observational, perturbational, and interventional experiments. Our primary aim is to describe the methodology for future clinical and translational research needed to delineate competing accounts of the current data. At the observational level we suggest the use of high-density EEG recording, combined analysis of macro and micro-architecture of sleep, detailed analysis of the stroke lesion, and sensitive measures of functional recovery. The perturbational approach attempts to find the causal links between sleep and stroke. We promote the use of transcranial magnetic stimulation combined with EEG to examine the cortical dynamics of the peri-infarct stroke area. Translational research should take this a step further using optogenetic techniques targeting more specific cell populations. The interventional approach focuses on how the same clinical and translational perturbational techniques can be adapted to influence long-term recovery of function

Glucocorticoid mechanisms of functional connectivity changes in stress-related neuropsychiatric disorders

AbstractStress—especially chronic, uncontrollable stress—is an important risk factor for many neuropsychiatric disorders. The underlying mechanisms are complex and multifactorial, but they involve correlated changes in structural and functional measures of neuronal connectivity within cortical microcircuits and across neuroanatomically distributed brain networks. Here, we review evidence from animal models and human neuroimaging studies implicating stress-associated changes in functional connectivity in the pathogenesis of PTSD, depression, and other neuropsychiatric conditions. Changes in fMRI measures of corticocortical connectivity across distributed networks may be caused by specific structural alterations that have been observed in the prefrontal cortex, hippocampus, and other vulnerable brain regions. These effects are mediated in part by glucocorticoids, which are released from the adrenal gland in response to a stressor and also oscillate in synchrony with diurnal rhythms. Recent work indicates that circadian glucocorticoid oscillations act to balance synapse formation and pruning after learning and during development, and chronic stress disrupts this balance. We conclude by considering how disrupted glucocorticoid oscillations may contribute to the pathophysiology of depression and PTSD in vulnerable individuals, and how circadian rhythm disturbances may affect non-psychiatric populations, including frequent travelers, shift workers, and patients undergoing treatment for autoimmune disorders

SLEEPING WHILE AWAKE: A NEUROPHYSIOLOGICAL INVESTIGATION ON SLEEP DURING WAKEFULNESS.

Il sonno e la veglia vengono comunemente considerati come due stati distinti. L\u2019alternanza tra essi, la cui presenza \ue8 stata dimostrata in ogni specie animale studiata fino ad oggi, sembra essere una delle caratteristiche che definisce la nostra vita. Allo stesso tempo, per\uf2, le scoperte portate alla luce negli ultimi decenni hanno offuscato i confini tra questi due stati. I meccanismi del sonno hanno sempre affascinato i neurofisiologi, che infatti, nell\u2019ultimo secolo, li hanno caratterizzati in dettaglio: ora sappiamo che all\u2019attivit\ue0 del sonno sottost\ue0 una specifica attivit\ue0 neuronale chiamata slow oscillation. La slow oscillation, che \ue8 costituita da (ancora una volta) un\u2019alternanza tra periodi di attivit\ue0 e periodi di iperpolarizzazione e silenzio neuronale (OFF-periods), \ue8 la modalit\ue0 base di attivazione del cervello dormiente. Questa alternanza \ue8 dovuta alla tendenza dei neuroni surante lo stato di sonno, di passare ad un periodo silente dopo un\u2019attivazione iniziale, una tendenza a cui viene dato il nome di bistabilit\ue0 neuronale. Molti studi hanno dimostrato come la bistabilit\ue0 neuronale tipica del sonno ed i relativi OFF-periods, possano accadere anche durante la veglia in particolari condizioni patologiche, nelle transizioni del sonno e durante le deprivazioni di sonno. Per questo motivo, se accettassimo che la bistabilit\ue0 neuronale e gli OFF-periods rappresentino una caratteristica fondamentale del sonno, allora dovremmo ammettere che stiamo assistendo ad un cambio di paradigma: da una prospettiva neurofisiologica il sonno pu\uf2 intrudere nella veglia. In questa tesi ho analizzato i nuovi -fluidi- confini tra sonno e veglia e le possibili implicazioni di questi nel problema della persistenza personale attraverso il tempo. Inoltre, ho studiato le implicazioni cliniche dell\u2019intrusione di sonno nella veglia in pazienti con lesioni cerebrali focali di natura ischemica. In particolare, i miei obiettivi sono stati: 1) Dimostrare come la bistabilit\ue0 neuronale possa essere responsabile della perdita di funzione nei pazienti affetti da ischemia cerebrale e come questo potrebbe avere implicazioni nello studio della patofisiologia dell\u2019ischemia cerebrale e nella sua terapia; 2) Stabilire le basi per un modello di sonno locale presente nella vita di tutti i giorni: la sensazione di sonnolenza. Infatti, essa potrebbe riflettere la presenza di porzioni di corteccia in stato di sonno, ma durante lo stato di veglia; 3) Difendere il criterio biologico di identit\ue0, che troverebbe nell\u2019attivit\ue0 cerebrale la continuit\ue0 necessaria al mantenimento della nostra identit\ue0 nel tempo.Sleep and wakefulness are considered two mutually exclusive states. The alternation between those two states seems to be a defining characteristic of our life, a ubiquitous phenomenon demonstrated in every animal species investigated so far. However, during the last decade, advances in neurophysiology have blurred the boundaries between those states. The mechanisms of sleep have always intrigued neurophysiologists and great advances have been made over the last century in understanding them: we now know that the defining characteristic underlying sleep activity is a specific pattern of neuronal activity, namely the slow oscillation. The slow oscillation, which is characterized by the periodic alternation between periods of activity (ON-periods) and periods of hyperpolarization and neuronal silence (OFF-periods) is the default mode of activity of the sleeping cortex. This alternation is due to the tendency of neurons to fall into a silent period after an initial activation; such tendency is known as \u201cbistability\u201d. There is accumulating evidence that sleep-like bistability, and the ensuing OFF-periods, may occur locally in the awake human brain in some pathological conditions, in sleep transition, as well as after sleep deprivation. Therefore, to the extent that bistability and OFF periods represents the basic neuronal features of sleep, a paradigm shift is in place: from a neurophysiological perspective sleep can intrude into wakefulness. In this thesis, I explore the fluid boundaries between sleep and wakefulness and investigate their possible implications on the problem of personal persistence over time. Moreover, I study the clinical implications of the intrusion of sleep into wakefulness in patients with focal brain injury due to stroke. Specifically, I aim to: 1) show how the sleep-like bistability can be responsible for the loss of function in stroke patients. This may have implications for understanding the pathophysiology of stroke and helping to foster recovery; 2) establish the basis for a model of local sleep that might be present in the everyday life, id est the sensation of sleepiness. Indeed, sleepiness could reflect islands of sleep during wakefulness; 3) advocate the biological criterion of identity, in which the continuity necessary for maintaining ourselves over time could be represented by never resting activity in the brain

Circadian rhythms and sex differences set endocannabinoids to influence memory under stress

A large amount of evidence indicates that stress exposure triggers the brain processing through different specific pathways that converge in both norepinephrine- and glucocorticoids-dependent regulation of memory processes by influencing central noradrenergic mechanisms (de Quervain et al., 1998; McGaugh and Roozendaal, 2002). The amygdala has long been known to be the hub of fear memory, which is usually remembered over time (Fanselow and LeDoux, 1999; Roozendaal et al., 2009). However, when an aversive stimulus occurs, it might happen that the accuracy of such emotional memory could be distorted progressively, leading to memory generalization (Asok et al., 2019). Drugs of abuse were identified to alter the experience of reality, thus affecting memory processes (Goodman and Packard, 2016). Chapter 1 explores more in deep the role of the psychostimulants amphetamine and MDPV in the modulation of memory strength and accuracy in a previously validated model exploiting the inhibitory avoidance discrimination task, in order to assess fear memory generalization for a novel/safe, yet not identical, context that was not used to induce shocks (Atucha and Roozendaal, 2015). Previous studies indicated that both amphetamine and MDPV, through different mechanisms of action, increase brain monoamines release, particularly norepinephrine and dopamine, two neurotransmitters extensively involved in the modulation of memory (LaLumiere et al., 2005; McGaugh and Roozendaal, 2009). Therefore, Chapter 1 investigates the involvement of the noradrenergic and dopaminergic systems in mediating the amphetamine effects on memory strength and both amphetamine and MDPV effects on fear memory generalization. Extensive evidence demonstrates that norepinephrine is crucially involved in the regulation of long-term memory consolidation for emotionally arousing experiences (Ferry et al., 1999; McGaugh and Roozendaal, 2002; Roozendaal et al., 2008; Lalumiere et al., 2017; Chen et al., 2018). It is widely recognized that amphetamine enhances the consolidation of memory processing in both humans and rodents (Soetens et al., 1993; Sanday et al., 2013). Chapter 2 evaluates the influence of different intensities of stress on the amphetamine modulation of long-term memory consolidation, further characterizing the involvement of any stress-induced activation of the peripheral adrenergic response in such process. The endocannabinoid system plays a key role in the control of emotional responses to environmental challenges (Morena and Campolongo, 2014). CB1 receptors are abundantly expressed within corticolimbic regions, including the basolateral complex of the amygdala (BLA), hippocampus and mPFC (Hill et al., 2011). Glucocorticoids are stress response mediators which interact with the endocannabinoid system in the regulation of memory function (Campolongo et al., 2009; Hill et al., 2010a; Atsak et al., 2012a; Morena et al., 2016; Balsevich et al., 2017), with an emotional buffer outcome in such interaction (Morena and Campolongo, 2014). Their synthesis is characterized by a circadian release pattern, with peak levels linked to the start of the activity phase and diurnal regulation under control of the circadian clock (Dickmeis, 2009). Literature evidence indicated that the endocannabinoid signaling exhibits a circadian rhythm with variations reported in CB1 receptor expression (Rueda- Orozco et al., 2008), endocannabinoids tissue contents and in the enzymes controlling their synthesis and degradation (Valenti et al., 2004). Chapter 3 investigates how different stress intensities, soon after encoding, influence rat short-term memory in an object recognition task, whether the effects depend on circadian rhythm and if exogenous augmentation of AEA levels restores any memory impairment provoked by stress exposure. Exposure to stress alters both hippocampal anatomy and functionality (McEwen, 1999), with negative consequences on memory processes (de Kloet et al., 2018). Indeed, the hippocampus represents a key forebrain structure highly associated with emotional and recognition memory processes (Broadbent et al., 2010). According to the timing of stress exposure, stress-mediated secretion of glucocorticoids alters hippocampal functions and plasticity (Kim et al., 2015), thus affecting hippocampal-dependent memories in rodents and humans (Donley et al., 2005). Furthermore, previous findings from our laboratory have demonstrated the involvement of the 2-AG signaling in counteracting the stress-mediated impairments on memory function (Morena et al., 2014, 2015; Ratano et al., 2018). By adding on Chapter 3 findings, Chapter 4 highlights that stress impairing effects on short-term recognition memory depend on time-of-day in a stress intensity-dependent fashion and examines if different stress intensities affect the hippocampal endocannabinoid system components, whether the effects are time-of- day-dependent, and if boosting 2-AG signaling ameliorates memory performance. Excessive fear and anxiety are hallmarks of a variety of disabling psychiatric disorders (Myers and Davis, 2007). The neurocircuitry of fear memory involves the BLA as the key region modulating the acquisition, retrieval and extinction of fear response (Johansen et al., 2011; Adolphs, 2013; Herry and Johansen, 2014; Zelikowsky et al., 2014), by receiving inputs from somatosensory cortex, thalamus, and hippocampus that encodes contextual information and compares current contextual cues to previously encoded memories (Maren and Quirk, 2004). Chapter 5 evaluates whether the endocannabinoids AEA and 2-AG, in the BLA or the CA1 region of the dorsal hippocampus, differentially regulate fear memory retrieval depending on the environment-associated emotional arousal, if these outcomes are mediated by indirect activation of CB1 and/or CB2 receptors, and whether the BLA-dorsal CA1 interplay plays any role in such effects. Women are twice as likely as men to develop PTSD making the search for biological mechanisms underlying these gender disparities especially crucial (Breslau, 2009). One striking feature of PTSD is the alteration in the ability to extinguish fear responses to trauma-associated cues (Yehuda et al., 2015). In male rodents, the endocannabinoid system can modulate fear extinction and has been suggested as a therapeutic target for PTSD (Morena et al., 2018; Segev et al., 2018). Chapter 6 investigates whether exogenous augmentation of the endocannabinoids AEA and 2-AG in male and female rats affect fear expression and extinction, which is the role of CB1 and transient potential receptor of vanilloid type-1 channel (TRPV1) receptors in such mediation, and how the endocannabinoid machinery within the amygdala, PFC and periaqueductal grey (PAG) is influenced post-extinction. Chapter 7 provides a review of the existing literature regarding the effects of time-of- day on memory function, shedding light on the underlying mechanisms of contrasting results by portraying how stress-dependent modulation of memory is influenced by circadian rhythms. Chapter 7 also focuses on the interaction between the endocannabinoid system and the level of stress associated to the experimental context / previous aversive experiences and capitalizes on our recent findings that a manipulation of the endocannabinoid system might be capable to effectively modulate the circadian- dependent effects of stress on memory and to prevent its detrimental effects on memory function