Sleep loss drives brain region- and cell type-specific alterations in ribosome-associated transcripts involved in synaptic plasticity and cellular timekeeping

AbstractSleep and sleep loss are thought to impact synaptic plasticity, and recent studies have shown that sleep and sleep deprivation (SD) differentially affect gene transcription and protein translation in the mammalian forebrain. However, much less is known regarding how sleep and SD affect these processes in different microcircuit elements within the hippocampus and neocortex - for example, in inhibitory vs. excitatory neurons. Here we use translating ribosome affinity purification (TRAP) and in situ hybridization to characterize the effects of sleep vs. SD on abundance of ribosome-associated transcripts in Camk2a-expressing (Camk2a+) pyramidal neurons and parvalbumin-expressing (PV+) interneurons in mouse hippocampus and neocortex. We find that while both Camk2a+ neurons and PV+ interneurons in neocortex show concurrent SD-driven increases in ribosome-associated transcripts for activity-regulated effectors of plasticity and transcriptional regulation, these transcripts are minimally affected by SD in hippocampus. Similarly we find that while SD alters several ribosome-associated transcripts involved in cellular timekeeping in neocortical Camk2a+ and PV+ neurons, effects on circadian clock transcripts in hippocampus are minimal, and restricted to Camk2a+ neurons. Taken together, our results indicate that SD effects on transcripts destined for translation are both cell type- and brain region-specific, and that these effects are substantially more pronounced in the neocortex than the hippocampus. We conclude that SD-driven alterations in the strength of synapses, excitatory-inhibitory balance, and cellular timekeeping are likely more heterogeneous than previously appreciated.Significance StatementSleep loss-driven changes in transcript and protein abundance have been used as a means to better understand the function of sleep for the brain. Here we use translating ribosome affinity purification (TRAP) to characterize changes in abundance of ribosome-associated transcripts in excitatory and inhibitory neurons in mouse hippocampus and neocortex after a brief period of sleep or sleep loss. We show that these changes are not uniform, but are generally more pronounced in excitatory neurons than inhibitory neurons, and more pronounced in neocortex than in hippocampus.

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Sleep loss drives brain region- and cell type-specific alterations in ribosome-associated transcripts involved in synaptic plasticity and cellular timekeeping

Journal of Neuroscience ◽

10.1523/jneurosci.1883-20.2021 ◽

2021 ◽

pp. JN-RM-1883-20

Author(s):

Carlos Puentes-Mestril ◽

James Delorme ◽

Lijing Wang ◽

Marcus Donnelly ◽

Donald Popke ◽

...

Keyword(s):

Synaptic Plasticity ◽

Brain Region ◽

Sleep Loss ◽

Cell Type ◽

Cell Type Specific

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Neuron-specific spinal cord translatomes reveal a neuropeptide code for mouse dorsal horn excitatory neurons

Scientific Reports ◽

10.1038/s41598-021-84667-y ◽

2021 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Rebecca Rani Das Gupta ◽

Louis Scheurer ◽

Pawel Pelczar ◽

Hendrik Wildner ◽

Hanns Ulrich Zeilhofer

Keyword(s):

Spinal Cord ◽

Dorsal Horn ◽

Functional Organization ◽

Affinity Purification ◽

Expression Patterns ◽

Inhibitory Neurons ◽

Dorsal Horn Neurons ◽

Expression Of Genes ◽

Excitatory Neurons ◽

Genes Encoding

AbstractThe spinal dorsal horn harbors a sophisticated and heterogeneous network of excitatory and inhibitory neurons that process peripheral signals encoding different sensory modalities. Although it has long been recognized that this network is crucial both for the separation and the integration of sensory signals of different modalities, a systematic unbiased approach to the use of specific neuromodulatory systems is still missing. Here, we have used the translating ribosome affinity purification (TRAP) technique to map the translatomes of excitatory glutamatergic (vGluT2+) and inhibitory GABA and/or glycinergic (vGAT+ or Gad67+) neurons of the mouse spinal cord. Our analyses demonstrate that inhibitory and excitatory neurons are not only set apart, as expected, by the expression of genes related to the production, release or re-uptake of their principal neurotransmitters and by genes encoding for transcription factors, but also by a differential engagement of neuromodulator, especially neuropeptide, signaling pathways. Subsequent multiplex in situ hybridization revealed eleven neuropeptide genes that are strongly enriched in excitatory dorsal horn neurons and display largely non-overlapping expression patterns closely adhering to the laminar and presumably also functional organization of the spinal cord grey matter.

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Behavioral-state modulation of inhibition is context-dependent and cell type specific in mouse visual cortex

eLife ◽

10.7554/elife.14985 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 107

Author(s):

Janelle MP Pakan ◽

Scott C Lowe ◽

Evelyn Dylda ◽

Sander W Keemink ◽

Stephen P Currie ◽

...

Keyword(s):

Visual Cortex ◽

Pyramidal Neurons ◽

Visual Stimulation ◽

Inhibitory Neurons ◽

Behavioral State ◽

Visual Responses ◽

Cell Type ◽

Sensory Stimuli ◽

Context Dependent

Cortical responses to sensory stimuli are modulated by behavioral state. In the primary visual cortex (V1), visual responses of pyramidal neurons increase during locomotion. This response gain was suggested to be mediated through inhibitory neurons, resulting in the disinhibition of pyramidal neurons. Using in vivo two-photon calcium imaging in layers 2/3 and 4 in mouse V1, we reveal that locomotion increases the activity of vasoactive intestinal peptide (VIP), somatostatin (SST) and parvalbumin (PV)-positive interneurons during visual stimulation, challenging the disinhibition model. In darkness, while most VIP and PV neurons remained locomotion responsive, SST and excitatory neurons were largely non-responsive. Context-dependent locomotion responses were found in each cell type, with the highest proportion among SST neurons. These findings establish that modulation of neuronal activity by locomotion is context-dependent and contest the generality of a disinhibitory circuit for gain control of sensory responses by behavioral state.

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Early role for a Na+,K+-ATPase (ATP1A3) in brain development

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2023333118 ◽

2021 ◽

Vol 118 (25) ◽

pp. e2023333118

Author(s):

Richard S. Smith ◽

Marta Florio ◽

Shyam K. Akula ◽

Jennifer E. Neil ◽

Yidi Wang ◽

...

Keyword(s):

Single Cell ◽

Expression Patterns ◽

Inhibitory Neurons ◽

Cell Type ◽

Animal Cells ◽

Concentration Gradients ◽

Α Subunit ◽

Early Postnatal Development ◽

Excitatory Neurons ◽

A Cell

Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function catalyzed by the Na+,K+-ATPase α-subunit. Here, we describe ATP1A3 variants encoding dysfunctional α3-subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we built an ATP1A3 transcriptional atlas of fetal cortical development using mRNA in situ hybridization and transcriptomic profiling of ∼125,000 individual cells with single-cell RNA sequencing (Drop-seq) from 11 areas of the midgestational human neocortex. We found that fetal expression of ATP1A3 is most abundant to a subset of excitatory neurons carrying transcriptional signatures of the developing subplate, yet also maintains expression in nonneuronal cell populations. Moving forward a year in human development, we profiled ∼52,000 nuclei from four areas of an infant neocortex and show that ATP1A3 expression persists throughout early postnatal development, most predominantly in inhibitory neurons, including parvalbumin interneurons in the frontal cortex. Finally, we discovered the heteromeric Na+,K+-ATPase pump complex may form nonredundant cell-type–specific α-β isoform combinations, including α3-β1 in excitatory neurons and α3-β2 in inhibitory neurons. Together, the developmental malformation phenotype of affected individuals and single-cell ATP1A3 expression patterns point to a key role for α3 in human cortex development, as well as a cell-type basis for pre- and postnatal ATP1A3-associated diseases.

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Sleep loss drives acetylcholine- and somatostatin interneuron–mediated gating of hippocampal activity to inhibit memory consolidation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019318118 ◽

2021 ◽

Vol 118 (32) ◽

pp. e2019318118

Author(s):

James Delorme ◽

Lijing Wang ◽

Femke Roig Kuhn ◽

Varna Kodoth ◽

Jingqun Ma ◽

...

Keyword(s):

Cell Population ◽

Granule Cells ◽

Dorsal Hippocampus ◽

Affinity Purification ◽

Enrichment Analysis ◽

Sleep Loss ◽

Contextual Fear Conditioning ◽

Cell Type ◽

Contextual Fear ◽

Cell Type Specific

Sleep loss disrupts consolidation of hippocampus-dependent memory. To characterize effects of learning and sleep loss, we quantified activity-dependent phosphorylation of ribosomal protein S6 (pS6) across the dorsal hippocampus of mice. We find that pS6 is enhanced in dentate gyrus (DG) following single-trial contextual fear conditioning (CFC) but is reduced throughout the hippocampus after brief sleep deprivation (SD; which disrupts contextual fear memory [CFM] consolidation). To characterize neuronal populations affected by SD, we used translating ribosome affinity purification sequencing to identify cell type–specific transcripts on pS6 ribosomes (pS6-TRAP). Cell type–specific enrichment analysis revealed that SD selectively activated hippocampal somatostatin-expressing (Sst+) interneurons and cholinergic and orexinergic hippocampal inputs. To understand the functional consequences of SD-elevated Sst+ interneuron activity, we used pharmacogenetics to activate or inhibit hippocampal Sst+ interneurons or cholinergic input from the medial septum. The activation of either cell population was sufficient to disrupt sleep-dependent CFM consolidation by gating activity in granule cells. The inhibition of either cell population during sleep promoted CFM consolidation and increased S6 phosphorylation among DG granule cells, suggesting their disinhibition by these manipulations. The inhibition of either population across post-CFC SD was insufficient to fully rescue CFM deficits, suggesting that additional features of sleeping brain activity are required for consolidation. Together, our data suggest that state-dependent gating of DG activity may be mediated by cholinergic input and local Sst+ interneurons. This mechanism could act as a sleep loss–driven inhibitory gate on hippocampal information processing.

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Somatostatin and parvalbumin inhibitory synapses onto hippocampal pyramidal neurons are regulated by distinct mechanisms

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1719523115 ◽

2018 ◽

Vol 115 (3) ◽

pp. 589-594 ◽

Cited By ~ 26

Author(s):

Meryl E. Horn ◽

Roger A. Nicoll

Keyword(s):

Calcium Channels ◽

Brain Function ◽

Pyramidal Neurons ◽

Neuronal Firing ◽

Inhibitory Neurons ◽

Inhibitory Synapses ◽

Hippocampal Pyramidal Neurons ◽

Excitatory Neurons ◽

Glutamatergic Signaling ◽

Different Populations

Excitation–inhibition balance is critical for optimal brain function, yet the mechanisms underlying the tuning of inhibition from different populations of inhibitory neurons are unclear. Here, we found evidence for two distinct pathways through which excitatory neurons cell-autonomously modulate inhibitory synapses. Synapses from parvalbumin-expressing interneurons onto hippocampal pyramidal neurons are regulated by neuronal firing, signaling through L-type calcium channels. Synapses from somatostatin-expressing interneurons are regulated by NMDA receptors, signaling through R-type calcium channels. Thus, excitatory neurons can cell-autonomously regulate their inhibition onto different subcellular compartments through their input (glutamatergic signaling) and their output (firing). Separately, while somatostatin and parvalbumin synapses onto excitatory neurons are both dependent on a common set of postsynaptic proteins, including gephyrin, collybistin, and neuroligin-2, decreasing neuroligin-3 expression selectively decreases inhibition from somatostatin interneurons, and overexpression of neuroligin-3 selectively enhances somatostatin inhibition. These results provide evidence that excitatory neurons can selectively regulate two distinct sets of inhibitory synapses.

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Distinct in vivo dynamics of excitatory synapses onto cortical pyramidal neurons and inhibitory interneurons

10.1101/2021.04.04.438418 ◽

2021 ◽

Author(s):

Joshua B. Melander ◽

Aran Nayebi ◽

Bart C. Jongbloets ◽

Dale A. Fortin ◽

Maozhen Qin ◽

...

Keyword(s):

Pyramidal Neurons ◽

Synaptic Weight ◽

Barrel Cortex ◽

Inhibitory Neurons ◽

Inhibitory Interneurons ◽

Excitatory Synapses ◽

Cell Type ◽

Cortical Function ◽

Cell Type Specific

SUMMARYCortical function relies on the balanced activation of excitatory and inhibitory neurons. However, little is known about the organization and dynamics of shaft excitatory synapses onto cortical inhibitory interneurons, which cannot be easily identified morphologically. Here, we fluorescently visualize the excitatory postsynaptic marker PSD-95 at endogenous levels as a proxy for excitatory synapses onto layer 2/3 pyramidal neurons and parvalbumin-positive (PV+) inhibitory interneurons in the mouse barrel cortex. Longitudinal in vivo imaging reveals that, while synaptic weights in both neuronal types are log-normally distributed, synapses onto PV+ neurons are less heterogeneous and more stable. Markov-model analyses suggest that the synaptic weight distribution is set intrinsically by ongoing cell type-specific dynamics, and substantial changes are due to accumulated gradual changes. Synaptic weight dynamics are multiplicative, i.e., changes scale with weights, though PV+ synapses also exhibit an additive component. These results reveal that cell type-specific processes govern cortical synaptic strengths and dynamics.

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028 Sleep loss disrupts hippocampal memory consolidation via an acetylcholine- and somatostatin interneuron-mediated inhibitory gate

SLEEP ◽

10.1093/sleep/zsab072.027 ◽

2021 ◽

Vol 44 (Supplement_2) ◽

pp. A12-A13

Author(s):

James Delorme ◽

Lijing Wang ◽

Femke Kuhn ◽

Varna Kodoth ◽

Jingqun Ma ◽

...

Keyword(s):

Memory Consolidation ◽

Dorsal Hippocampus ◽

Affinity Purification ◽

Ribosomal Subunit ◽

Sleep Loss ◽

Contextual Fear Conditioning ◽

Cell Type ◽

Single Trial ◽

Contextual Fear ◽

Cell Type Specific

Abstract Introduction Sleep loss profoundly disrupts consolidation of hippocampus-dependent memory. To better characterize effects of learning and sleep loss on the hippocampal circuit, we quantified activity-dependent phosphorylation of ribosomal subunit S6 (pS6) across the dorsal hippocampus of mice. Methods We first measured pS6 throughout the hippocampus after learning (single trial contextual fear conditioning; CFC), and after subsequent sleep or sleep deprivation (SD). To characterize cell populations with activity affected by SD, we used translating ribosome affinity purification (TRAP)-seq to identify cell type-specific transcripts on pS6 ribosomes after SD vs. sleep. We next used pharmacogenetics to mimic the effects of SD, selectively activating hippocampal Sst+ interneurons or cholinergic inputs to hippocampus from the medial septum (MS) while mice slept in the hours following CFC. We also inhibited these neuronal populations to assess effects on memory consolidation. Results We find that pS6 in enhanced in the dentate gyrus (DG) following single-trial CFC, but is reduced throughout the hippocampus after brief SD – a manipulation which disrupts contextual fear memory (CFM) consolidation. Cell type-specific enrichment analysis (CSEA) of these transcripts revealed that hippocampal somatostatin-expressing (Sst+) interneurons, and cholinergic and orexinergic inputs to hippocampus, are selectively activated after SD. We used TRAP targeted to hippocampal Sst+ interneurons to identify cellular mechanisms mediating SD-driven Sst+ interneuron activation. . We find that activation of Sst+ interneurons is sufficient to disrupt CFM consolidation, by gating activity in surrounding pyramidal neurons, while inhibition of Sst+ interneurons enhances memory consolidation. Similarly, pharmacogenetic activation of cholinergic input to hippocampus from the MS disrupted CFM. Inhibition of MS cholinergic neurons promoted CFM consolidation and disinhibited neurons in the DG, increasing pS6 expression among DG granule cells. Conclusion Our data suggest that state-dependent gating of DG activity during SD is mediated by cholinergic input. Together these data provide evidence for an inhibitory gate on hippocampal information processing, which is activated by sleep loss. Support (if any) R01-NS118440 to SJA from NINDS, DP2-MH104119 to SJA from the NIH Director’s Office, and a Human Frontiers Science Program Young Investigator Award

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Cerebellar nuclei evolved by repeatedly duplicating a conserved cell type set

10.1101/2020.06.25.170118 ◽

2020 ◽

Cited By ~ 1

Author(s):

Justus M Kebschull ◽

Noam Ringach ◽

Ethan B Richman ◽

Drew Friedmann ◽

Sai Saroja Kolluru ◽

...

Keyword(s):

Brain Region ◽

Cell Types ◽

Cerebellar Nuclei ◽

Inhibitory Neurons ◽

Cell Type ◽

Cell Class ◽

Single Nucleus ◽

Entire Cell ◽

Region Evolution

AbstractHow have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell type resolution in the cerebellar nuclei (CN), the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole-CNS projection tracing in mice, we identified a conserved cell type set containing two classes of region-specific excitatory neurons and three classes of region-invariant inhibitory neurons. This set constitutes an archetypal CN that was repeatedly duplicated to form new regions. 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.

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The eIF4E homolog 4EHP (eIF4E2) regulates hippocampal long-term depression and impacts social behavior

Molecular Autism ◽

10.1186/s13229-020-00394-7 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Shane Wiebe ◽

Xiang Qi Meng ◽

Sung-Hoon Kim ◽

Xu Zhang ◽

Jean-Claude Lacaille ◽

...

Keyword(s):

Protein Synthesis ◽

Synaptic Plasticity ◽

Social Behavior ◽

Translational Control ◽

Affinity Purification ◽

Social Behaviors ◽

Autism Spectrum ◽

Repetitive Behaviors ◽

Physical Interaction ◽

Excitatory Neurons

Abstract Background The regulation of protein synthesis is a critical step in gene expression, and its dysfunction is implicated in autism spectrum disorder (ASD). The eIF4E homologous protein (4EHP, also termed eIF4E2) binds to the mRNA 5′ cap to repress translation. The stability of 4EHP is maintained through physical interaction with GRB10 interacting GYF protein 2 (GIGYF2). Gene-disruptive mutations in GIGYF2 are linked to ASD, but causality is lacking. We hypothesized that GIGYF2 mutations cause ASD by disrupting 4EHP function. Methods Since homozygous deletion of either gene is lethal, we generated a cell-type-specific knockout model where Eif4e2 (the gene encoding 4EHP) is deleted in excitatory neurons of the forebrain (4EHP-eKO). In this model, we investigated ASD-associated synaptic plasticity dysfunction, ASD-like behaviors, and global translational control. We also utilized mice lacking one copy of Gigyf2, Eif4e2 or co-deletion of one copy of each gene to further investigate ASD-like behaviors. Results 4EHP is expressed in excitatory neurons and synaptosomes, and its amount increases during development. 4EHP-eKO mice display exaggerated mGluR-LTD, a phenotype frequently observed in mouse models of ASD. Consistent with synaptic plasticity dysfunction, the mice displayed social behavior impairments without being confounded by deficits in olfaction, anxiety, locomotion, or motor ability. Repetitive behaviors and vocal communication were not affected by loss of 4EHP in excitatory neurons. Heterozygous deletion of either Gigyf2, Eif4e2, or both genes in mice did not result in ASD-like behaviors (i.e. decreases in social behavior or increases in marble burying). Interestingly, exaggerated mGluR-LTD and impaired social behaviors were not attributed to changes in hippocampal global protein synthesis, which suggests that 4EHP and GIGYF2 regulate the translation of specific mRNAs to mediate these effects. Limitations This study did not identify which genes are translationally regulated by 4EHP and GIGYF2. Identification of mistranslated genes in 4EHP-eKO mice might provide a mechanistic explanation for the observed impairment in social behavior and exaggerated LTD. Future experiments employing affinity purification of translating ribosomes and mRNA sequencing in 4EHP-eKO mice will address this relevant issue. Conclusions Together these results demonstrate an important role of 4EHP in regulating hippocampal plasticity and ASD-associated social behaviors, consistent with the link between mutations in GIGYF2 and ASD.

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