ABSTRACT. Falling asleep at the wrong time can place an individual at risk of immediate physical harm. However, not sleeping degrades cognition and adaptive behavior. To understand how animals match sleep need with environmental demands, we used live-brain imaging to examine the physiological response properties of the dorsal Fan Shaped Body (dFB) following interventions that modify sleep (sleep deprivation, starvation, time-restricted feeding, memory consolidation). We report that dFB neurons change their physiological response-properties to Dopamine (DA) and Allatostatin-A (AstA) in response to different types of waking. That is, dFB neurons are not simply passive components of a hard-wired circuit. Rather, the dFB neurons intrinsically regulate their response to the activity from upstream circuits. Finally, we show that the dFB appears to contain a memory trace of prior exposure to metabolic challenges induced by starvation or time-restricted feeding. Together these data highlight that the sleep homeostat is plastic and suggests an underlying mechanism

Paul Shaw Washington University School of Medicine is St. Louis

ABSTRACT. Sleep loss and sleep disruptions result in cellular stress and induction of adaptive signaling mechanisms such as the proteostasis pathway the unfolded protein response (UPR). Little is known about the role of proteostasis signaling pathways in regulating sleep. Previous work from our lab has demonstrated that the UPR chaperone, BiP promotes recovery sleep in the fly. However, the involvement of other UPR molecules in regulating sleep has until now remained unexplored. Here, we present data from pharmacological and genetic studies on the role of the UPR sensors protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1 (IRE1) in regulating sleep and wake behavior in Drosophila melanogaster. Further, the maintenance of proteostasis is critical for the proper functioning of cells, and therefore an organism as a whole. With age, the molecular mechanisms that relieve cellular stress and assist in the proper folding of proteins become less efficient. In addition to this disruption of proteostasis, both sleep quality and cognition are impaired with age, Using a mouse model of aging we present data examining the relationship between cellular stress, sleep quality, and cognition and test if reducing cellular stress via increasing protein chaperone levels improves sleep quality and memory.

Nirinjini Naidoo Chronobiology and Sleep Institute, Division of Sleep Medicine, Perelman School of Medicine, University of Pennsylvania

ABSTRACT. Sleep is essential for the optimal functioning of numerous biological systems and its chronic disruption has important negative health consequences. Sleep regulatory processes strive to maintain an adequate sleep-wake balance both during everyday life as well as after challenging this balance by e.g. sleep deprivation. While the mechanisms behind sleep regulation remain elusive, genetic factors are known to play important roles and identifying the underlying genes and molecular pathways might yield important insights to the processes that make us sleepy. We used a systems genetics approach and assembled an extensive multi-scale dataset from 33 recombinant inbred mouse lines from the BXD/RwwJ panel. First, all mice were interrogated for sleep-wake behavior, EEG activity, and locomotor activity during baseline, sleep deprivation, and subsequent recovery, yielding a sleep-wake phenome comprised of 341 sleep-wake related end phenotypes. Second, as intermediary phenotypes, a targeted plasma metabolome, and cortical and liver transcriptomes were obtained under baseline and sleep-deprivation conditions. We recently extended this dataset with the cortical epigenome reporting on chromatin accessibility under these two conditions using ATAC-sequencing. The results pointed to profound effects of a single, short (6h) sleep deprivation pervasively altering the cortical and liver transcriptome (78 and 60% changed of all expressed genes) and blood metabolome (60% of the 124 metabolites quantified), and remodeled 25K chromatin regions. A strong genetic contribution was evident at all phenotypic levels including the response to sleep deprivation. We found a bi-directional relationship between fatty acid processing and NREM sleep homeostasis involving the enzyme Acot11 and between activity of EEG slow-waves and chromatin dynamics through Wrn DNA-helicase.

Authors: M. Jan, S. Diessler, C.E.V. Neves, Y. Emmenegger, S. Jimenez, P. Franken University of Lausanne, CH

ABSTRACT. Monogenic Mendelian inheritance has only been identified in a small number of human pedigrees with phenotypes so specific that they can be distinguished outside of the laboratory (above all advanced sleep phase disorder, ASPD). Human sleep genetics faces the challenge that the majority of sleep-related traits are highly polygenic, whilst the expense and time required for the exquisitely specific laboratory methods that exist for monitoring the physiology of human sleep limits the size of the datasets that can be acquired. This talk will review the range of measures that are available for data collection from human participants in the field, both direct measures (actigraphy and home polysomnography, PSG) and questionnaire data estimating preferred and actual sleep timing, sleep quality, and specific sleep disorders. Basic measures already analysed in large population studies will be presented, and more sophisticated ones with the potential for introduction to larger cohort studies will be introduced. This includes a new study applying, for the first time, the Insomnia Severity Index in general populations, and a novel association with the CORO7 locus.

M von Schantz1, 2, 3

1Faculty of Health and Life Sciences, Northumbria University, Newcastle Upon Tyne, UK 2Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK 3Medical Research Council/Wits Rural Public Health and Health Transitions Research Unit (Agincourt), School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

ABSTRACT. Growing evidence indicates that neurons are particularly reliant on the spatial and temporal regulation of mRNA translation for their survival and function; and mutations in numerous components of the translational machinery have been linked to neurological disorders. Although the mammalian genome contains hundreds of nuclear-encoded transfer RNA (tRNA) genes, surprisingly no disease-linked mutations in these genes have been reported as of yet. We recently identified the first tissue-specific mammalian tRNA gene, n-Tr20. n-Tr20 is one of 5 isodecoders in the nuclear-encoded tRNAArg UCU family and, in contrast to the other of this family, is specifically expressed in the nervous system. Loss of n-Tr20 in mice dramatically reduced the tRNAArgUCU pool in the nervous system, resulting in ribosome stalling on the cognate AGA codons, and activation of the integrated stress response (ISR). Here we present evidence from an allelic series of mutations in n-Tr20 that loss of function of this tRNA can induce alterations in neuronal function. These changes are accompanied by widespread transcriptional reprogramming. Our work highlights the exquisite sensitivity of the nervous system to even subtle disruption of cellular homeostasis, and raises the possibility that the regulation of tRNA expression may play a critical role in complex neuronal processes.

Susan L. Ackerman members Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA USA

ABSTRACT. In this symposium the cerebellar nuclei receive the attention they rightfully deserve. As essentially the sole recipient of the output of the cerebellar cortex, they are at a key position to mediate in delivering the processing power of the cerebellar cortex to the rest of the brain. As has become abundantly clear, the cerebellum, and therefore also the cerebellar nuclei, not only are vital in developing, maintaining and improving motor skills, but also have similar functions in cognition, affection and autonomic processes.

In this first vignette, to set the stage for this symposium, the basic anatomy of the cerebellar nuclei within the central nervous system in general and within the cerebellum in particular will be discussed. First, we need to realize that the enormous variety in cerebellar afferent information not only targets the cerebellar cortex but also directly affects the cerebellar nuclei. Second, cerebellar functionality is based on highly organized connections, characterized by parallel series of interconnected parts that are centered around the cerebellar nuclei and which are known as modules. Yet, within this apparent uniform modular circuitry, remarkable differences in molecular, genetic and physiological properties have been noted. If, indeed, the modules, with the cerebellar nuclei at their center, form the structural basis for cerebellar function, the question should be addressed how this uniform structural basis, with all its intrinsic differences, develops and is maintained. Hence, in the following contributions the evolution, development, molecular blueprint and function of the cerebellar nuclei will be further explored.

Tom J.H. Ruigrok1 1Department of Neuroscience, Erasmus Medical Center Rotterdam, the Netherlands Funding support: Ministry of Health, Welfare and Sport, THE NETHERLANDS

ABSTRACT. The extant cerebellar nuclei of jawed vertebrates likely originate from a single ancestral nucleus. Here we investigate how additional cerebellar nuclei were gained over evolutionary time in the avian and mammalian lineages at cell-type resolution. We applied single-nucleus RNA sequencing in chickens, mice, and humans, STARmap spatial transcriptomic analysis in chicken and mice, and whole-CNS projection mapping in mice. Our work revealed a conserved cell type set containing three classes of region-invariant inhibitory neurons and two classes of region-specific excitatory neurons. This cell type set forms an archetypal cerebellar nucleus that was repeatedly duplicated to create new regions, and thus cerebellar output channels. In excitatory neurons, duplication was accompanied by divergence in gene expression and shifts in projection patterns. By contrast, inhibitory neurons maintained their gene expression signatures. Interestingly, the excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human Lateral CN. Our data provide the first characterization of CN transcriptomic cell types in three species and suggest a model of brain region evolution by duplication and divergence of entire cell type sets. We are now working to extend these findings to tetrapods and to query connectomic cell types using cellular barcoding techniques MAPseq and BARseq in different species.

JM Kebschull Johns Hopkins University

ABSTRACT. The development and evolution of the cerebellar nuclei provide an intriguing model for the emergence and adaptation of complex circuits in the vertebrate brain. We have focused on the temporal patterning of cell production in development as a substrate for adaptation. This approach stems from the insight that projection neurons that establish connections between the cerebellum and other brain regions are allocated as a series of discrete temporal cohorts. These temporal cohorts establish that scaffold around which cerebellar nuclei are assembled in development. Their mode of production offers a model for the addition of new connectional modules through heterochronic adaptation of patterning at their common point of the origin, the embryonic rhombic lip. Our search for temporal regulators of this process followed a path from evolution to autistic spectrum disorder (ASD). Using a single cell RNA sequencing approach, we found that thyroid hormone signalling acts a key regulator of gene expression in developing nuclei and within the cerebellum at stages that are far earlier than conventional critical periods for thyroid hormone activity in the developing brain. Discrete disruption of thyroid signalling in the embryonic chick and mouse lead respectively, to disrupted nucleus formation and cognitive deficits in adult mice consistent with ASD.

MRC Centre for Neurodevelopmental Disorders, King’s College London, UK

ABSTRACT. The cerebellum is critical for fast and accurate movements, but how it supports such behavioral improvement remains debated. Leading hypotheses propose that the cerebellar cortex computes forward models, predictions of sensory consequences of movements, that are used to enhance motor control. Alternatively, models of associative learning that link sensory events with anticipatory commands account for a variety of other cerebellum-dependent behaviors. In this talk I will present data that unify these disparate hypotheses, demonstrating anticipatory control signals in the cerebellar nuclei and how they may be generated by associative learning in upstream cerebellar cortical circuitry. We find that in mice performing a skilled reach task, many cerebellar nuclear neurons residing in the interposed nucleus, burst as the limb decelerates to a target. This bursting activity scales with the rate of deceleration, and optogenetic perturbation of these cells bidirectionally scales deceleration supporting causality. We hypothesized that this bursting activity is akin to a conditioned response in delay eyelid conditioning, which predicts that inputs to the cerebellum can be flexibly associated with anticipatory control to enhance reach endpoint accuracy. To test this, we performed optogenetic perturbations of cerebellar inputs, in closed loop with reaching movements. Light stimulation effectively skewed reach kinematics, however, kinematic effects were quickly adapted, restoring accurate endpoints. Removal of light led to opposing after-effects on reach kinematics, supporting a model wherein cues from cerebellar inputs are integrated into a control policy in Purkinje cells to achieve expert performance by bypassing slow sensory guidance of movement with learned anticipatory control. Overall, our work supports the view that the cerebellum enhances motor control and coordination by generating anticipatory control signals through learned associations of early phases of movement with appropriate late-phase commands.

Abigail Person University of Colorado, USA