Potential neural substrates underlying circadian and olfactory disruptions in preclinical Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia, with over 45 million patients worldwide, and poses significant economic and emotional burdens to both patients and caregivers, significantly raising the number of those affected. Unfortunately, much of the existing research on the disease only addresses a small subset of associated symptomologies and pathologies. In this review, we propose to target the earliest stages of the disease, when symptomology first arises. In these stages, before the onset of hallmark symptoms of AD such as cognitive impairments and memory loss, circadian and olfactory disruptions arise and are detectable. Functional similarities between circadian and olfactory systems provide a basis upon which to seek out common mechanisms in AD which may target them early on in the disease. Existing studies of interactions between these systems, while intriguing, leave open the question of the neural substrates underlying them. Potential substrates for such interactions are proposed in this review, such as indirect projections that may functionally connect the two systems and dopaminergic signaling. These substrates may have significant implications for mechanisms underlying disruptions to circadian and olfactory function in early stages of AD. In this review, we propose early detection of AD using a combination of circadian and olfactory deficits and subsequent early treatment of these deficits may provide profound benefits to both patients and caregivers. Additionally, we suggest that targeting research toward the intersection of these two systems in AD could uncover mechanisms underlying the broader set of symptoms and pathologies that currently elude researchers.

A brainstem to circadian system circuit links Tau pathology to sundowning-related disturbances in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) patients exhibit progressive disruption of entrained circadian rhythms and an aberrant circadian input pathway may underlie such dysfunction. Here we examine AD-related pathology and circadian dysfunction in the APPSwe-Tau (TAPP) model of AD. We show these mice exhibit phase delayed body temperature and locomotor activity with increases around the active-to-rest phase transition. Similar AD-related disruptions are associated with sundowning, characterized by late afternoon and early evening agitation and aggression, and we show TAPP mice exhibit increased aggression around this transition. We show such circadian dysfunction and aggression coincide with hyperphosphorylated Tau (pTau) development in lateral parabrachial (LPB) neurons, with these disturbances appearing earlier in females. Finally, we show LPB neurons, including those expressing dynorphin (LPBdyn), project to circadian structures and are affected by pTau, and LPBdyn ablations partially recapitulate the hyperthermia of TAPP mice. Altogether we link pTau in a brainstem circadian input pathway to AD-related disturbances relevant to sundowning.

Voluntary Wheel Running Exercise Does Not Attenuate Circadian and Cardiac Dysfunction Caused by Conditional Deletion of Bmal1.

Circadian misalignment occurs with age, jet lag, and shift work, leading to maladaptive health outcomes including cardiovascular diseases. Despite the strong link between circadian disruption and heart disease, the cardiac circadian clock is poorly understood, prohibiting identification of therapies to restore the broken clock. Exercise is the most cardioprotective intervention identified to date and has been suggested to reset the circadian clock in other peripheral tissues. Here, we tested the hypothesis that conditional deletion of core circadian gene Bmal1 would disrupt cardiac circadian rhythm and function and that this disruption would be ameliorated by exercise. To test this hypothesis, we generated a transgenic mouse with spatial and temporal deletion of Bmal1 only in adult cardiac myocytes (Bmal1 cardiac knockout [cKO]). Bmal1 cKO mice demonstrated cardiac hypertrophy and fibrosis concomitant with impaired systolic function. This pathological cardiac remodeling was not rescued by wheel running. While the molecular mechanisms responsible for the profound cardiac remodeling are unclear, it does not appear to involve activation of the mammalian target of rapamycin (mTOR) signaling or changes in metabolic gene expression. Interestingly, cardiac deletion of Bmal1 disrupted systemic rhythms as evidenced by changes in the onset and phasing of activity in relationship to the light/dark cycle and by decreased periodogram power as measured by core temperature, suggesting cardiac clocks can regulate systemic circadian output. Together, we suggest a critical role for cardiac Bmal1 in regulating both cardiac and systemic circadian rhythm and function. Ongoing experiments will determine how disruption of the circadian clock causes cardiac remodeling in an effort to identify therapeutics to attenuate the maladaptive outcomes of a broken cardiac circadian clock.

Median preoptic GABA and glutamate neurons exert differential control over sleep behavior.

Previous studies suggest that the median preoptic nucleus (MnPO) of the hypothalamus plays an important role in regulating the wake-sleep cycle and, in particular, homeostatic sleep drive. However, the precise cellular phenotypes, targets, and central mechanisms by which the MnPO neurons regulate the wake-sleep cycle remain unknown. Both excitatory and inhibitory MnPO neurons innervate brain regions implicated in sleep promotion and maintenance, suggesting that both cell types may participate in sleep control. Using genetically targeted approaches, we investigated the role of the MnPO GABAergic (MnPOVgat) and glutamatergic (MnPOVglut2) neurons in modulating wake-sleep behavior of mice. We found that both neuron populations differentially participate in wake-sleep control, with MnPOVgat neurons being involved in sleep homeostasis and MnPOVglut2 neurons facilitating sleep during allostatic (stressful) challenges.

Systems and Circuits Linking Chronic Pain and Circadian Rhythms.

Research over the last 20 years regarding the link between circadian rhythms and chronic pain pathology has suggested interconnected mechanisms that are not fully understood. Strong evidence for a bidirectional relationship between circadian function and pain has been revealed through inflammatory and immune studies as well as neuropathic ones. However, one limitation of many of these studies is a focus on only a few molecules or cell types, often within only one region of the brain or spinal cord, rather than systems-level interactions. To address this, our review will examine the circadian system as a whole, from the intracellular genetic machinery that controls its timing mechanism to its input and output circuits, and how chronic pain, whether inflammatory or neuropathic, may mediate or be driven by changes in these processes. We will investigate how rhythms of circadian clock gene expression and behavior, immune cells, cytokines, chemokines, intracellular signaling, and glial cells affect and are affected by chronic pain in animal models and human pathologies. We will also discuss key areas in both circadian rhythms and chronic pain that are sexually dimorphic. Understanding the overlapping mechanisms and complex interplay between pain and circadian mediators, the various nuclei they affect, and how they differ between sexes, will be crucial to move forward in developing treatments for chronic pain and for determining how and when they will achieve their maximum efficacy.

Potential Pathways for Circadian Dysfunction and Sundowning-Related Behavioral Aggression in Alzheimer's Disease and Related Dementias.

Patients with Alzheimer's disease (AD) and related dementias are commonly reported to exhibit aggressive behavior and other emotional behavioral disturbances, which create a tremendous caretaker burden. There has been an abundance of work highlighting the importance of circadian function on mood and emotional behavioral regulation, and recent evidence demonstrates that a specific hypothalamic pathway links the circadian system to neurons that modulate aggressive behavior, regulating the propensity for aggression across the day. Such shared circuitry may have important ramifications for clarifying the complex interactions underlying "sundowning syndrome," a poorly understood (and even controversial) clinical phenomenon in AD and dementia patients that is characterized by agitation, aggression, and delirium during the late afternoon and early evening hours. The goal of this review is to highlight the potential output and input pathways of the circadian system that may underlie circadian dysfunction and behavioral aggression associated with sundowning syndrome, and to discuss possible ways these pathways might inform specific interventions for treatment. Moreover, the apparent bidirectional relationship between chronic disruptions of circadian and sleep-wake regulation and the pathology and symptoms of AD suggest that understanding the role of these circuits in such neurobehavioral pathologies could lead to better diagnostic or even preventive measures.

Suprachiasmatic VIP neurons are required for normal circadian rhythmicity and comprised of molecularly distinct subpopulations.

The hypothalamic suprachiasmatic (SCN) clock contains several neurochemically defined cell groups that contribute to the genesis of circadian rhythms. Using cell-specific and genetically targeted approaches we have confirmed an indispensable role for vasoactive intestinal polypeptide-expressing SCN (SCNVIP) neurons, including their molecular clock, in generating the mammalian locomotor activity (LMA) circadian rhythm. Optogenetic-assisted circuit mapping revealed functional, di-synaptic connectivity between SCNVIP neurons and dorsomedial hypothalamic neurons, providing a circuit substrate by which SCNVIP neurons may regulate LMA rhythms. In vivo photometry revealed that while SCNVIP neurons are acutely responsive to light, their activity is otherwise behavioral state invariant. Single-nuclei RNA-sequencing revealed that SCNVIP neurons comprise two transcriptionally distinct subtypes, including putative pacemaker and non-pacemaker populations. Altogether, our work establishes necessity of SCNVIP neurons for the LMA circadian rhythm, elucidates organization of circadian outflow from and modulatory input to SCNVIP cells, and demonstrates a subpopulation-level molecular heterogeneity that suggests distinct functions for specific SCNVIP subtypes.

Role of serotonergic dorsal raphe neurons in hypercapnia-induced arousals.

During obstructive sleep apnea, elevation of CO2 during apneas contributes to awakening and restoring airway patency. We previously found that glutamatergic neurons in the external lateral parabrachial nucleus (PBel) containing calcitonin gene related peptide (PBelCGRP neurons) are critical for causing arousal during hypercapnia. However, others found that genetic deletion of serotonin (5HT) neurons in the brainstem also prevented arousal from hypercapnia. To examine interactions between the two systems, we showed that dorsal raphe (DR) 5HT neurons selectively targeted the PBel. Either genetically directed deletion or acute optogenetic silencing of DRSert neurons dramatically increased the latency of mice to arouse during hypercapnia, as did silencing DRSert terminals in the PBel. This effect was mediated by 5HT2a receptors which are expressed by PBelCGRP neurons. Our results indicate that the serotonergic input from the DR to the PBel via 5HT2a receptors is critical for modulating the sensitivity of the PBelCGRP neurons that cause arousal to rising levels of blood CO2.

Selective activation of serotoninergic dorsal raphe neurons facilitates sleep through anxiolysis.

A role for the brain's serotoninergic (5HT) system in the regulation of sleep and wakefulness has been long suggested. Yet, previous studies employing pharmacological, lesion and genetically driven approaches have produced inconsistent findings, leaving 5HT's role in sleep-wake regulation incompletely understood. Here we sought to define the specific contribution of 5HT neurons within the dorsal raphe nucleus (DRN5HT) to sleep and arousal control. To do this, we employed a chemogenetic strategy to selectively and acutely activate DRN5HT neurons and monitored sleep-wake using electroencephalogram recordings. We additionally assessed indices of anxiety using the open field and elevated plus maze behavioral tests and employed telemetric-based recordings to test effects of acute DRN5HT activation on body temperature and locomotor activity. Our findings indicate that the DRN5HT cell population may not modulate sleep-wake per se, but rather that its activation has apparent anxiolytic properties, suggesting the more nuanced view that DRN5HT neurons are sleep permissive under circumstances that produce anxiety or stress.

A time to fight: Circadian control of aggression and associated autonomic support.

The central circadian clock, located in the suprachiasmatic nucleus of the mammalian hypothalamus (SCN), regulates daily behavioral rhythms including the temporal propensity for aggressive behavior. Such aggression propensity rhythms are regulated by a functional circuit from the SCN to neurons that drive attack behavior in the ventromedial hypothalamus (VMH), via a relay in the subparaventricular zone (SPZ). In addition to this pathway, the SCN also regulates sleep-wake and locomotor activity rhythms, via the SPZ, in a circuit to the dorsomedial hypothalamus (DMH), a structure that is also known to play a key role in autonomic function and the sympathetic "fight-or-flight" response (which prepares the body for action in stressful situations such as an agonistic encounter). While the autonomic nervous system is known to be under pronounced circadian control, it is less apparent how such autonomic rhythms and their underlying circuitry may support the temporal propensity for aggressive behavior. Additionally, it is unclear how circadian and autonomic dysfunction may contribute to aberrant social and emotional behavior, such as agitation and aggression. Here we review the literature concerning interactions between the circadian and autonomic systems and aggression, and we discuss the implications of these relationships for human neural and behavioral pathologies.

Newly identified sleep-wake and circadian circuits as potential therapeutic targets.

Optogenetics and chemogenetics are powerful tools, allowing the specific activation or inhibition of targeted neuronal subpopulations. Application of these techniques to sleep and circadian research has resulted in the unveiling of several neuronal populations that are involved in sleep-wake control, and allowed a comprehensive interrogation of the circuitry through which these nodes are coordinated to orchestrate the sleep-wake cycle. In this review, we discuss six recently described sleep-wake and circadian circuits that show promise as therapeutic targets for sleep medicine. The parafacial zone (PZ) and the ventral tegmental area (VTA) are potential druggable targets for the treatment of insomnia. The brainstem circuit underlying rapid eye movement sleep behavior disorder (RBD) offers new possibilities for treating RBD and neurodegenerative synucleinopathies, whereas the parabrachial nucleus, as a nexus linking arousal state control and breathing, is a promising target for developing treatments for sleep apnea. Therapies that act upon the hypothalamic circuitry underlying the circadian regulation of aggression or the photic regulation of arousal and mood pathway carry enormous potential for helping to reduce the socioeconomic burden of neuropsychiatric and neurodegenerative disorders on society. Intriguingly, the development of chemogenetics as a therapeutic strategy is now well underway and such an approach has the capacity to lead to more focused and less invasive therapies for treating sleep-wake disorders and related comorbidities.

A hypothalamic circuit for the circadian control of aggression.

'Sundowning' in dementia and Alzheimer's disease is characterized by early-evening agitation and aggression. While such periodicity suggests a circadian origin, whether the circadian clock directly regulates aggressive behavior is unknown. We demonstrate that a daily rhythm in aggression propensity in male mice is gated by GABAergic subparaventricular zone (SPZGABA) neurons, the major postsynaptic targets of the central circadian clock, the suprachiasmatic nucleus. Optogenetic mapping revealed that SPZGABA neurons receive input from vasoactive intestinal polypeptide suprachiasmatic nucleus neurons and innervate neurons in the ventrolateral part of the ventromedial hypothalamus (VMH), which is known to regulate aggression. Additionally, VMH-projecting dorsal SPZ neurons are more active during early day than early night, and acute chemogenetic inhibition of SPZGABA transmission phase-dependently increases aggression. Finally, SPZGABA-recipient central VMH neurons directly innervate ventrolateral VMH neurons, and activation of this intra-VMH circuit drove attack behavior. Altogether, we reveal a functional polysynaptic circuit by which the suprachiasmatic nucleus clock regulates aggression.

The development of sleep-wake rhythms and the search for elemental circuits in the infant brain.

Despite the predominance of sleep in early infancy, developmental science has yet to play a major role in shaping concepts and theories about sleep and its associated ultradian and circadian rhythms. Here we argue that developmental analyses help us to elucidate the relative contributions of the brainstem and forebrain to sleep-wake control and to dissect the neural components of sleep-wake rhythms. Developmental analysis also makes it clear that sleep-wake processes in infants are the foundation for those of adults. For example, the infant brainstem alone contains a fundamental sleep-wake circuit that is sufficient to produce transitions among wakefulness, quiet sleep, and active sleep. In addition, consistent with the requirements of a "flip-flop" model of sleep-wake processes, this brainstem circuit supports rapid transitions between states. Later in development, strengthening bidirectional interactions between the brainstem and forebrain contribute to the consolidation of sleep and wake bouts, the elaboration of sleep homeostatic processes, and the emergence of diurnal or nocturnal circadian rhythms. The developmental perspective promoted here critically constrains theories of sleep-wake control and provides a needed framework for the creation of fully realized computational models. Finally, with a better understanding of how this system is constructed developmentally, we will gain insight into the processes that govern its disintegration due to aging and disease.

Development of SCN connectivity and the circadian control of arousal: a diminishing role for humoral factors?

The suprachiasmatic nucleus (SCN) is part of a wake-promoting circuit comprising the dorsomedial hypothalamus (DMH) and locus coeruleus (LC). Although widely considered a "master clock," the SCN of adult rats is also sensitive to feedback regarding an animal's behavioral state. Interestingly, in rats at postnatal day (P)2, repeated arousing stimulation does not increase neural activation in the SCN, despite doing so in the LC and DMH. Here we show that, by P8, the SCN is activated by arousing stimulation and that selective destruction of LC terminals with DSP-4 blocks this activational effect. We next show that bidirectional projections among the SCN, DMH, and LC are nearly absent at P2 but present at P8. Despite the relative lack of SCN connectivity with downstream structures at P2, day-night differences in sleep-wake activity are observed, suggesting that the SCN modulates behavior at this age via humoral factors. To test this hypothesis, we lesioned the SCN at P1 and recorded sleep-wake behavior at P2: Day-night differences in sleep and wake were eliminated. We next performed precollicular transections at P2 and P8 that isolate the SCN and DMH from the brainstem and found that day-night differences in sleep-wake behavior were retained at P2 but eliminated at P8. Finally, the SCN or DMH was lesioned at P8: When recorded at P21, rats with either lesion exhibited similarly fragmented wake bouts and no evidence of circadian modulation of wakefulness. These results suggest an age-related decline in the SCN's humoral influence on sleep-wake behavior that coincides with the emergence of bidirectional connectivity among the SCN, DMH, and LC.

Distinct retinohypothalamic innervation patterns predict the developmental emergence of species-typical circadian phase preference in nocturnal Norway rats and diurnal nile grass rats.

How does the brain develop differently to support nocturnality in some mammals, but diurnality in others? To answer this question, one might look to the suprachiasmatic nucleus (SCN), which is entrained by light via the retinohypothalamic tract (RHT). However, because the SCN is more active during the day in all mammals studied thus far, it alone cannot determine circadian phase preference. In adult Norway rats (Rattus norvegicus), which are nocturnal, the RHT also projects to the ventral subparaventricular zone (vSPVZ), an adjacent region that expresses an in-phase pattern of SCN-vSPVZ neuronal activity. In contrast, in adult Nile grass rats (Arvicanthis niloticus), which are diurnal, an anti-phase pattern of SCN-vSPVZ neuronal activity is expressed. We hypothesized that these species differences result in part from a weak or absent RHT-to-vSPVZ projection in grass rats. Here, using a developmental comparative approach, we assessed species differences in behavior, hypothalamic activity, and RHT anatomy. We report that a robust retina-to-vSPVZ projection develops in Norway rats around the end of the second postnatal week when nocturnal wakefulness and the in-phase pattern of neuronal activity emerge. In grass rats, however, such a projection does not develop and the emergence of the anti-phase pattern during the second postnatal week is accompanied by increased diurnal wakefulness. When considered within the context of previously published reports on RHT projections in a variety of species, the current findings suggest that how and when the retina connects to the hypothalamus differentially shapes brain and behavior to produce animals that occupy opposing temporal niches.

Comparison of area 17 cellular composition in laboratory and wild-caught rats including diurnal and nocturnal species.

In this study we examine the size of primary sensory areas in the neocortex and the cellular composition of area 17/V1 in three rodent groups: laboratory nocturnal Norway rats (Long-Evans; Rattus norvegicus), wild-caught nocturnal Norway rats (R. norvegicus), and laboratory diurnal Nile grass rats (Arvicanthis niloticus). Specifically, we used areal measures of myeloarchitecture of the primary sensory areas to compare area size and the isotropic fractionator method to estimate the number of neurons and nonneurons in area 17 in each species. Our results demonstrate that the percentage of cortex devoted to area 17 is significantly greater and the percentage of cortex devoted to S1 is significantly smaller in the diurnal Nile grass rat compared with the nocturnal Norway rat groups. Further, the laboratory rodent groups have a greater percentage of cortex devoted to auditory cortex compared with the wild-caught group. We also demonstrate that wild-caught rats have a greater density of neurons in area 17 compared to laboratory-reared animals. However, there were no other clear cellular composition differences in area 17 or differences in the percentage of brain weight devoted to area 17 between nocturnal and diurnal rats. Thus, there are differences in primary sensory area size between diurnal versus nocturnal and laboratory versus wild-caught rat groups and cellular density between wild-caught and laboratory rat groups. Our results demonstrate that the differences in the size and cellular composition of cortical areas do not fit with what would be expected based on brain scaling differences alone, and have a consistent relationship with lifestyle and sensory morphology.

Brainstem and hypothalamic regulation of sleep pressure and rebound in newborn rats.

Sleep pressure and rebound comprise the two compensatory or "homeostatic" responses to sleep deprivation. Although sleep pressure is expressed by infant rats as early as postnatal day (P)5, sleep rebound does not appear to emerge until after P11. We reexamined the developmental expression of these sleep-regulatory processes in P2 and P8 rats by depriving them of sleep for 30 min using a cold, arousing stimulus delivered to a cold-sensitive region of the snout. This method effectively increased sleep pressure over the 30-min period (i.e., increases in the number of arousing stimuli presented over time). Moreover, sleep rebound (i.e., increased sleep during the recovery period) is demonstrated for the first time at these ages. Next, we showed that precollicular transections in P2 rats prevent sleep rebound without affecting sleep pressure, suggesting that the brainstem is sufficient to support sleep pressure, but sleep rebound depends on neural mechanisms that lie rostral to the transection. Finally, again in P2 rats, we used c-fos immunohistochemistry to examine neural activation throughout the neuraxis during sleep deprivation and recovery. Sleep deprivation and rebound were accompanied by significant increases in neural activation in both brainstem and hypothalamic nuclei, including the ventrolateral preoptic area and median preoptic nucleus. This early developmental expression of sleep pressure and rebound and the apparent involvement of brainstem and hypothalamic structures in their expression further solidify the notion that sleep-wake processes in newborns-defined at these ages without reference to state-dependent EEG activity-provide the foundation on which the more familiar processes of adults are built.

The development of day-night differences in sleep and wakefulness in norway rats and the effect of bilateral enucleation.

The suprachiasmatic nucleus exhibits circadian rhythmicity in fetal and infant rats, but little is known about the consequences of this rhythmicity for infant behavior. Here, in experiment 1, the authors measured sleep and wakefulness in rats during the day and night in postnatal day (P)2, P8, P15, and P21 subjects. As early as P2, day-night differences in sleep-wake activity were detected. Nocturnal wakefulness began to emerge around P15 and was reliably expressed by P21. The authors hypothesized that the process of photic entrainment over the 1st postnatal week, which depends on the development of connectivity between the retinohypothalamic tract (RHT) and the SCN, influences the later emergence of nocturnal wakefulness. To test this hypothesis, in experiment 2 infant rats were enucleated bilaterally at P3 and P11, that is, before and after photic entrainment. Whereas pups enucleated at P11 and tested at P21 exhibited increased wakefulness at night, identical to sham controls, pups enucleated at P3 and tested at P21 exhibited the opposite pattern of increased wakefulness during the day. Pups tested at P28 and P35 exhibited this same pattern of increased daytime wakefulness. All together, these results suggest that prenatal and postnatal experience modulates the development of species-typical circadian sleep-wake patterns. Moreover, the authors suggest that visual system stimulation, via the RHT's connections with the SCN, exerts an organizational influence on the developing circadian system and, consequently, contributes to the emergence of nocturnality in this species.