Small RNA-mediated gene regulation during development causes long-lasting changes in cellular phenotypes. To determine whether small RNAs of the adult brain can regulate memory storage, a process ...that requires stable and long-lasting changes in the functional state of neurons, we generated small RNA libraries from the Aplysia CNS. In these libraries, we discovered an unexpectedly abundant expression of a 28 nucleotide sized class of piRNAs in brain, which had been thought to be germline specific. These piRNAs have unique biogenesis patterns, predominant nuclear localization, and robust sensitivity to serotonin, a modulatory transmitter that is important for memory. We find that the Piwi/piRNA complex facilitates serotonin-dependent methylation of a conserved CpG island in the promoter of CREB2, the major inhibitory constraint of memory in Aplysia, leading to enhanced long-term synaptic facilitation. These findings provide a small RNA-mediated gene regulatory mechanism for establishing stable long-term changes in neurons for the persistence of memory.
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▸ More than 300 genomic regions encode neuronally expressed piRNAs in Aplysia ▸ A neuronally expressed Aplysia Piwi protein interacts with piRNAs, but not miRNAs ▸ One piRNA, piR-F, silences CREB2, leading to enhanced long-term synaptic facilitation ▸ Epigenetic silencing of CREB2, a repressor of memory, offers a mechanism for memory persistence
piRNAs, which are primarily known for their role in protecting the genome of germ cells, also act to promote memory persistence in Aplysia by directing methylation and silencing of a long-term memory repressor in neurons.
Top-down prefrontal cortex inputs to the hippocampus have been hypothesized to be important in memory consolidation, retrieval, and the pathophysiology of major psychiatric diseases; however, no such ...direct projections have been identified and functionally described. Here we report the discovery of a monosynaptic prefrontal cortex (predominantly anterior cingulate) to hippocampus (CA3 to CA1 region) projection in mice, and find that optogenetic manipulation of this projection (here termed AC-CA) is capable of eliciting contextual memory retrieval. To explore the network mechanisms of this process, we developed and applied tools to observe cellular-resolution neural activity in the hippocampus while stimulating AC-CA projections during memory retrieval in mice behaving in virtual-reality environments. Using this approach, we found that learning drives the emergence of a sparse class of neurons in CA2/CA3 that are highly correlated with the local network and that lead synchronous population activity events; these neurons are then preferentially recruited by the AC-CA projection during memory retrieval. These findings reveal a sparsely implemented memory retrieval mechanism in the hippocampus that operates via direct top-down prefrontal input, with implications for the patterning and storage of salient memory representations.
In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization over large volumes has been largely restricted to labeling of proteins. But volumetric ...nucleic acid labeling may represent a far greater scientific and clinical opportunity, enabling detection of not only diverse coding RNA variants but also non-coding RNAs. Moreover, scaling immunohistochemical detection to large tissue volumes has limitations due to high cost, limited renewability/availability, and restricted multiplexing capability of antibody labels. With the goal of versatile, high-content, and scalable molecular phenotyping of intact tissues, we developed a method using carbodiimide-based chemistry to stably retain RNAs in clarified tissue, coupled with amplification tools for multiplexed detection. The resulting technology enables robust measurement of activity-dependent transcriptional signatures, cell-identity markers, and diverse non-coding RNAs in rodent and human tissue volumes. The growing set of validated probes is deposited in an online resource for nucleating related developments from across the scientific community.
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•Carbodiimide-based chemistry in CLARITY hydrogels for RNA preservation and detection•Rapid diffusion of DNA oligonucleotides for volumetric in situ hybridization•Detection of microRNAs and mRNAs in clarified mouse and human tissues•DNA-based amplification for multiplexed in situ hybridization in CLARITY
CLARITY with enhanced RNA coupling chemistry is developed for multiplexed, volumetric visualization of both long and short RNAs in a variety of intact tissues.
Learning requires the ability to link actions to outcomes. How motivation facilitates learning is not well understood. We designed a behavioral task in which mice self-initiate trials to learn ...cue-reward contingencies and found that the anterior cingulate region of the prefrontal cortex (ACC) contains motivation-related signals to maximize rewards. In particular, we found that ACC neural activity was consistently tied to trial initiations where mice seek to leave unrewarded cues to reach reward-associated cues. Notably, this neural signal persisted over consecutive unrewarded cues until reward-associated cues were reached, and was required for learning. To determine how ACC inherits this motivational signal we performed projection-specific photometry recordings from several inputs to ACC during learning. In doing so, we identified a ramp in bulk neural activity in orbitofrontal cortex (OFC)-to-ACC projections as mice received unrewarded cues, which continued ramping across consecutive unrewarded cues, and finally peaked upon reaching a reward-associated cue, thus maintaining an extended motivational state. Cellular resolution imaging of OFC confirmed these neural correlates of motivation, and further delineated separate ensembles of neurons that sequentially tiled the ramp. Together, these results identify a mechanism by which OFC maps out task structure to convey an extended motivational state to ACC to facilitate goal-directed learning.
Memory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic ...plasticity, we carried out massive parallel sequencing to profile the small RNAs of Aplysia californica. We identified 170 distinct miRNAs, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain enriched, and several of these were rapidly downregulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Further characterization of the brain-enriched miRNAs revealed that miR-124, the most abundant and well-conserved brain-specific miRNA, was exclusively present presynaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB. We therefore present direct evidence that a modulatory neurotransmitter important for learning can regulate the levels of small RNAs and present a role for miR-124 in long-term plasticity of synapses in the mature nervous system.
Piwi-interacting RNAs (piRNAs), long thought to be restricted to germline, have recently been discovered in neurons of Aplysia, with a role in the epigenetic regulation of gene expression underlying ...long-term memory. We here ask whether piwi/piRNAs are also expressed and have functional roles in the mammalian brain. Large-scale RNA sequencing and subsequent analysis of protein expression revealed the presence in brain of several piRNA biogenesis factors including a mouse piwi (Mili), as well as small RNAs, albeit at low levels, resembling conserved piRNAs in mouse testes primarily LINE1 (long interspersed nuclear element1) retrotransposon-derived. Despite the seeming low expression of these putative piRNAs, single-base pair CpG methylation analyses across the genome of Mili/piRNA-deficient (Mili
−/−) mice demonstrate that brain genomic DNA is preferentially hypomethylated within intergenic areas and LINE1 promoter areas of the genome. Furthermore, Mili mutant mice exhibit behavioral deficits such as hyperactivity and reduced anxiety. These results suggest that putative piRNAs exist in mammalian brain, and similar to the role of piRNAs in testes, they may be involved in the silencing of retrotransposons, which in brain have critical roles in contributing to genomic heterogeneity underlying adaptation, stress response, and brain pathology. We also describe the presence of another class of small RNAs in the brain, with features of endogenous siRNAs, which may have taken over the role of invertebrate piRNAs in their capacity to target both transposons, as well as protein-coding genes. Thus, RNA interference through gene and retrotransposon silencing previously encountered in Aplysia may also have potential roles in the mammalian brain.
The maintenance phase of memory-related long-term facilitation (LTF) of synapses between sensory and motor neurons of the gill-withdrawal reflex of Aplysia depends on a serotonin (5-HT)-triggered ...presynaptic upregulation of CPEB, a functional prion that regulates local protein synthesis at the synapse. The mechanisms whereby serotonin regulates CPEB levels in presynaptic sensory neurons are not known. Here, we describe a sensory neuron-specific microRNA 22 (miR-22) that has multiple binding sites on the mRNA of CPEB and inhibits it in the basal state. Serotonin triggers MAPK/Erk-dependent downregulation of miR-22, thereby upregulating the expression of CPEB, which in turn regulates, through functional CPE elements, the presynaptic expression of atypical PKC (aPKC), another candidate regulator of memory maintenance. Our findings support a model in which the neurotransmitter-triggered downregulation of miR-22 coordinates the regulation of genes contributing synergistically to the long-term maintenance of memory-related synaptic plasticity.
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•Maintenance of long-term facilitation (LTF) in Aplysia requires upregulation of CPEB•Serotonin-triggered downregulation of miR-22 permits the upregulation of CPEB in LTF•Activation of CPEB regulates the translation of target synaptic mRNAs•Atypical PKC, a CPEB target, synergistically promotes presynaptic LTF maintenance
Maintenance of memory-related long-term facilitation (LTF) of Aplysia synapses requires upregulation and prion-like activation of CPEB, a synaptic translational regulator. Fiumara et al. identify a presynaptic microRNA as a neurotransmitter-modulated regulator of CPEB levels and consequently of CPEB targets, including an atypical PKC with synergistic roles in LTF maintenance.
Optogenetic methodology enables direct targeting of specific neural circuit elements for inhibition or excitation while spanning timescales from the acute (milliseconds) to the chronic (many days or ...more). Although the impact of this temporal versatility and cellular specificity has been greater for basic science than clinical research, it is natural to ask whether the dynamic patterns of neural circuit activity discovered to be causal in adaptive or maladaptive behaviors could become targets for treatment of neuropsychiatric diseases. Here, we consider the landscape of ideas related to therapeutic targeting of circuit dynamics. Specifically, we highlight optical, ultrasonic, and magnetic concepts for the targeted control of neural activity, preclinical/clinical discovery opportunities, and recently reported optogenetically guided clinical outcomes.
Optogenetics has afforded us unprecedented new insights into diverse neural circuits and their functions. What will it take to be able to understand and treat neuropsychiatric disease in terms of circuits that doctors can manipulate?
Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by ...molecular motor kinesin. Using this strategy in Aplysia , we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence.
Memory formation involves binding of contextual features into a unitary representation
, whereas memory recall can occur using partial combinations of these contextual features. The neural basis ...underlying the relationship between a contextual memory and its constituent features is not well understood; in particular, where features are represented in the brain and how they drive recall. Here, to gain insight into this question, we developed a behavioural task in which mice use features to recall an associated contextual memory. We performed longitudinal imaging in hippocampus as mice performed this task and identified robust representations of global context but not of individual features. To identify putative brain regions that provide feature inputs to hippocampus, we inhibited cortical afferents while imaging hippocampus during behaviour. We found that whereas inhibition of entorhinal cortex led to broad silencing of hippocampus, inhibition of prefrontal anterior cingulate led to a highly specific silencing of context neurons and deficits in feature-based recall. We next developed a preparation for simultaneous imaging of anterior cingulate and hippocampus during behaviour, which revealed robust population-level representation of features in anterior cingulate, that lag hippocampus context representations during training but dynamically reorganize to lead and target recruitment of context ensembles in hippocampus during recall. Together, we provide the first mechanistic insights into where contextual features are represented in the brain, how they emerge, and how they access long-range episodic representations to drive memory recall.
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