The Brain-Enriched MicroRNA miR-9-3p Regulates Synaptic Plasticity and Memory.

UNLABELLED: MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression in many tissues. Although a number of brain-enriched miRNAs have been identified, only a few specific miRNAs have been revealed as critical regulators of synaptic plasticity, learning, and memory. miR-9-5p/3p are brain-enriched miRNAs known to regulate development and their changes have been implicated in several neurological disorders, yet their role in mature neurons in mice is largely unknown. Here, we report that inhibition of miR-9-3p, but not miR-9-5p, impaired hippocampal long-term potentiation (LTP) without affecting basal synaptic transmission. Moreover, inhibition of miR-9-3p in the hippocampus resulted in learning and memory deficits. Furthermore, miR-9-3p inhibition increased the expression of the LTP-related genes Dmd and SAP97, the expression levels of which are negatively correlated with LTP. These results suggest that miR-9-3p-mediated gene regulation plays important roles in synaptic plasticity and hippocampus-dependent memory. SIGNIFICANCE STATEMENT: Despite the abundant expression of the brain-specific microRNA miR-9-5p/3p in both proliferating and postmitotic neurons, most functional studies have focused on their role in neuronal development. Here, we examined the role of miR-9-5p/3p in adult brain and found that miR-9-3p, but not miR-9-5p, has a critical role in hippocampal synaptic plasticity and memory. Moreover, we identified in vivo binding targets of miR-9-3p that are involved in the regulation of long-term potentiation. Our study provides the very first evidence for the critical role of miR-9-3p in synaptic plasticity and memory in the adult mouse.

Inhibition of anterior cingulate cortex excitatory neuronal activity induces conditioned place preference in a mouse model of chronic inflammatory pain.

The anterior cingulate cortex (ACC) is known for its role in perception of nociceptive signals and the associated emotional responses. Recent optogenetic studies, involving modulation of neuronal activity in the ACC, show that the ACC can modulate mechanical hyperalgesia. In the present study, we used optogenetic techniques to selectively modulate excitatory pyramidal neurons and inhibitory interneurons in the ACC in a model of chronic inflammatory pain to assess their motivational effect in the conditioned place preference (CPP) test. Selective inhibition of pyramidal neurons induced preference during the CPP test, while activation of parvalbumin (PV)-specific neurons did not. Moreover, chemogenetic inhibition of the excitatory pyramidal neurons alleviated mechanical hyperalgesia, consistent with our previous result. Our results provide evidence for the analgesic effect of inhibition of ACC excitatory pyramidal neurons and a prospective treatment for chronic pain.

Metabotropic glutamate receptor dependent long-term depression in the cortex.

Metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD), a type of synaptic plasticity, is characterized by a reduction in the synaptic response, mainly at the excitatory synapses of the neurons. The hippocampus and the cerebellum have been the most extensively studied regions in mGluR-dependent LTD, and Group 1 mGluR has been reported to be mainly involved in this synaptic LTD at excitatory synapses. However, mGluR-dependent LTD in other brain regions may be involved in the specific behaviors or diseases. In this paper, we focus on five cortical regions and review the literature that implicates their contribution to the pathogenesis of several behaviors and specific conditions associated with mGluR-dependent LTD.

Plasticity of metabotropic glutamate receptor-dependent long-term depression in the anterior cingulate cortex after amputation.

Long-term depression (LTD) is a key form of synaptic plasticity important in learning and information storage in the brain. It has been studied in various cortical regions, including the anterior cingulate cortex (ACC). ACC is a crucial cortical region involved in such emotion-related physiological and pathological conditions as fear memory and chronic pain. In the present study, we used a multielectrode array system to map cingulate LTD in a spatiotemporal manner within the ACC. We found that low-frequency stimulation (1 Hz, 15 min) applied onto deep layer V induced LTD in layers II/III and layers V/VI. Cingulate LTD requires activation of metabotropic glutamate receptors (mGluRs), while L-type voltage-gated calcium channels and NMDA receptors also contribute to its induction. Peripheral amputation of the distal tail impaired ACC LTD, an effect that persisted for at least 2 weeks. The loss of LTD was rescued by priming ACC slices with activation of mGluR1 receptors by coapplying (RS)-3,5-dihydroxyphenylglycine and MPEP, a form of metaplasticity that involved the activation of protein kinase C. Our results provide in vitro evidence of the spatiotemporal properties of ACC LTD in adult mice. We demonstrate that tail amputation causes LTD impairment within the ACC circuit and that this can be rescued by activation of mGluR1.

Long-term potentiation of synaptic transmission in the adult mouse insular cortex: multielectrode array recordings.

The insular cortex (IC) is widely believed to be an important forebrain structure involved in cognitive and sensory processes such as memory and pain. However, little work has been performed at the cellular level to investigate the synaptic basis of IC-related brain functions. To bridge the gap, the present study was designed to characterize the basic synaptic mechanisms for insular long-term potentiation (LTP). Using a 64-channel recording system, we found that an enduring form of late-phase LTP (L-LTP) could be reliably recorded for at least 3 h in different layers of IC slices after theta burst stimulation. The induction of insular LTP is protein synthesis dependent and requires activation of both GluN2A and GluN2B subunits of the NMDA receptor, L-type voltage-gated calcium channels, and metabotropic glutamate receptor 1. The paired-pulse facilitation ratio was unaffected by insular L-LTP induction, and expression of insular L-LTP required the recruitment of postsynaptic calcium-permeable AMPA receptors. Our results provide the first in vitro report of long-term multichannel recordings of L-LTP in the IC in adult mice and suggest its potential important roles in insula-related memory and chronic pain.

N-type voltage gated calcium channels mediate excitatory synaptic transmission in the anterior cingulate cortex of adult mice.

Voltage gated calcium channels (VGCCs) are well known for its importance in synaptic transmission in the peripheral and central nervous system. However, the role of different VGCCs in the anterior cingulate cortex (ACC) has not been studied. Here, we use a multi-electrode array recording system (MED64) to study the contribution of different types of calcium channels in glutamatergic excitatory synaptic transmission in the ACC. We found that only the N-type calcium channel blocker ω-conotoxin-GVIA (ω-Ctx-GVIA) produced a great inhibition of basal synaptic transmission, especially in the superficial layer. Other calcium channel blockers that act on L-, P/Q-, R-, and T-type had no effect. We also tested the effects of several neuromodulators with or without ω-Ctx-GVIA. We found that N-type VGCC contributed partially to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid- and (R)-Baclofen-induced synaptic inhibition. By contrast, the inhibitory effects of 2-Chloroadenosine and carbamoylcholine chloride did not differ with or without ω-Ctx-GVIA, indicating that they may act through other mechanisms. Our results provide strong evidence that N-type VGCCs mediate fast synaptic transmission in the ACC.

Long-term depression of synaptic transmission in the adult mouse insular cortex in vitro.

The insular cortex (IC) is known to play important roles in higher brain functions such as memory and pain. Activity-dependent long-term depression (LTD) is a major form of synaptic plasticity related to memory and chronic pain. Previous studies of LTD have mainly focused on the hippocampus, and no study in the IC has been reported. In this study, using a 64-channel recording system, we show for the first time that repetitive low-frequency stimulation (LFS) can elicit frequency-dependent LTD of glutamate receptor-mediated excitatory synaptic transmission in both superficial and deep layers of the IC of adult mice. The induction of LTD in the IC required activation of the N-methyl-d-aspartate (NMDA) receptor, metabotropic glutamate receptor (mGluR)5, and L-type voltage-gated calcium channel. Protein phosphatase 1/2A and endocannabinoid signaling are also critical for the induction of LTD. In contrast, inhibiting protein kinase C, protein kinase A, protein kinase Mζ or calcium/calmodulin-dependent protein kinase II did not affect LFS-evoked LTD in the IC. Bath application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine produced another form of LTD in the IC, which was NMDA receptor-independent and could not be occluded by LFS-induced LTD. Our studies have characterised the basic mechanisms of LTD in the IC at the network level, and suggest that two different forms of LTD may co-exist in the same population of IC synapses.

Rapid Turnover of Cortical NCAM1 Regulates Synaptic Reorganization after Peripheral Nerve Injury.

Peripheral nerve injury can induce pathological conditions that lead to persistent sensitized nociception. Although there is evidence that plastic changes in the cortex contribute to this process, the underlying molecular mechanisms are unclear. Here, we find that activation of the anterior cingulate cortex (ACC) induced by peripheral nerve injury increases the turnover of specific synaptic proteins in a persistent manner. We demonstrate that neural cell adhesion molecule 1 (NCAM1) is one of the molecules involved and show that it mediates spine reorganization and contributes to the behavioral sensitization. We show striking parallels in the underlying mechanism with the maintenance of NMDA-receptor- and protein-synthesis-dependent long-term potentiation (LTP) in the ACC. Our results, therefore, demonstrate a synaptic mechanism for cortical reorganization and suggest potential avenues for neuropathic pain treatment.

Enhancing inhibitory synaptic function reverses spatial memory deficits in Shank2 mutant mice.

Autism spectrum disorders (ASDs) are a group of developmental disorders that cause variable and heterogeneous phenotypes across three behavioral domains such as atypical social behavior, disrupted communications, and highly restricted and repetitive behaviors. In addition to these core symptoms, other neurological abnormalities are associated with ASD, including intellectual disability (ID). However, the molecular etiology underlying these behavioral heterogeneities in ASD is unclear. Mutations in SHANK2 genes are associated with ASD and ID. Interestingly, two lines of Shank2 knockout mice (e6-7 KO and e7 KO) showed shared and distinct phenotypes. Here, we found that the expression levels of Gabra2, as well as of GABA receptor-mediated inhibitory neurotransmission, are reduced in Shank2 e6-7, but not in e7 KO mice compared with their own wild type littermates. Furthermore, treatment of Shank2 e6-7 KO mice with an allosteric modulator for the GABAA receptor reverses spatial memory deficits, indicating that reduced inhibitory neurotransmission may cause memory deficits in Shank2 e6-7 KO mice. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

PKCα-mediated phosphorylation of LSD1 is required for presynaptic plasticity and hippocampal learning and memory.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that participates in transcriptional repression or activation. Recent studies reported that LSD1 is involved in learning and memory. Although LSD1 phosphorylation by PKCα was implicated in circadian rhythmicity, the importance of LSD1 phosphorylation in learning and memory is unknown. In this study, we examined the roles of LSD1 in synaptic plasticity and memory using Lsd1 SA/SA knock-in (KI) mice, in which a PKCα phosphorylation site is mutated. Interestingly, short-term and long-term contextual fear memory as well as spatial memory were impaired in Lsd1 KI mice. In addition, short-term synaptic plasticity, such as paired pulse ratio and post-tetanic potentiation was impaired, whereas long-term synaptic plasticity, including long-term potentiation and long-term depression, was normal. Moreover, the frequency of miniature excitatory postsynaptic current was significantly increased, suggesting presynaptic dysfunction in Lsd1 KI mice. Consistent with this, RNA-seq analysis using the hippocampus of Lsd1 KI mice showed significant alterations in the expressions of presynaptic function-related genes. Intriguingly, LSD1n-SA mutant showed diminished binding to histone deacetylase 1 (HDAC1) compared to LSD1n-WT in SH-SY5Y cells. These results suggest that LSD1 is involved in the regulation of presynaptic gene expression and subsequently regulates the hippocampus-dependent memory in phosphorylation-dependent manner.

Bidirectional modulation of hyperalgesia via the specific control of excitatory and inhibitory neuronal activity in the ACC.

Neurons in the anterior cingulate cortex (ACC) are assumed to play important roles in the perception of nociceptive signals and the associated emotional responses. However, the neuronal types within the ACC that mediate these functions are poorly understood. In the present study, we used optogenetic techniques to selectively modulate excitatory pyramidal neurons and inhibitory interneurons in the ACC and to assess their ability to modulate peripheral mechanical hypersensitivity in freely moving mice. We found that selective activation of pyramidal neurons rapidly and acutely reduced nociceptive thresholds and that this effect was occluded in animals made hypersensitive using Freund's Complete Adjuvant (CFA). Conversely, inhibition of ACC pyramidal neurons rapidly and acutely reduced hypersensitivity induced by CFA treatment. A similar analgesic effect was induced by activation of parvalbumin (PV) expressing interneurons, whereas activation of somatostatin (SOM) expressing interneurons had no effect on pain thresholds. Our results provide direct evidence of the pivotal role of ACC excitatory neurons, and their regulation by PV expressing interneurons, in nociception.

Multiple repressive mechanisms in the hippocampus during memory formation.

Memory stabilization after learning requires translational and transcriptional regulations in the brain, yet the temporal molecular changes that occur after learning have not been explored at the genomic scale. We used ribosome profiling and RNA sequencing to quantify the translational status and transcript levels in the mouse hippocampus after contextual fear conditioning. We revealed three types of repressive regulations: translational suppression of ribosomal protein-coding genes in the hippocampus, learning-induced early translational repression of specific genes, and late persistent suppression of a subset of genes via inhibition of estrogen receptor 1 (ESR1/ERα) signaling. In behavioral analyses, overexpressing Nrsn1, one of the newly identified genes undergoing rapid translational repression, or activating ESR1 in the hippocampus impaired memory formation. Collectively, this study unveils the yet-unappreciated importance of gene repression mechanisms for memory formation.

Involvement of cAMP-guanine nucleotide exchange factor II in hippocampal long-term depression and behavioral flexibility.

BACKGROUND: Guanine nucleotide exchange factors (GEFs) activate small GTPases that are involved in several cellular functions. cAMP-guanine nucleotide exchange factor II (cAMP-GEF II) acts as a target for cAMP independently of protein kinase A (PKA) and functions as a GEF for Rap1 and Rap2. Although cAMP-GEF II is expressed abundantly in several brain areas including the cortex, striatum, and hippocampus, its specific function and possible role in hippocampal synaptic plasticity and cognitive processes remain elusive. Here, we investigated how cAMP-GEF II affects synaptic function and animal behavior using cAMP-GEF II knockout mice. RESULTS: We found that deletion of cAMP-GEF II induced moderate decrease in long-term potentiation, although this decrease was not statistically significant. On the other hand, it produced a significant and clear impairment in NMDA receptor-dependent long-term depression at the Schaffer collateral-CA1 synapses of hippocampus, while microscopic morphology, basal synaptic transmission, and depotentiation were normal. Behavioral testing using the Morris water maze and automated IntelliCage system showed that cAMP-GEF II deficient mice had moderately reduced behavioral flexibility in spatial learning and memory. CONCLUSIONS: We concluded that cAMP-GEF II plays a key role in hippocampal functions including behavioral flexibility in reversal learning and in mechanisms underlying induction of long-term depression.

Effects of PI3Kγ overexpression in the hippocampus on synaptic plasticity and spatial learning.

Previous studies have shown that a family of phosphoinositide 3-kinases (PI3Ks) plays pivotal roles in the brain; in particular, we previously reported that knockout of the γ isoform of PI3K (PI3Kγ) in mice impaired synaptic plasticity and reduced behavioral flexibility. To further examine the role of PI3Kγ in synaptic plasticity and hippocampus-dependent behavioral tasks we overexpressed p110γ, the catalytic subunit of PI3Kγ, in the hippocampal CA1 region. We found that the overexpression of p110γ impairs NMDA receptor-dependent long-term depression (LTD) and hippocampus-dependent spatial learning in the Morris water maze (MWM) task. In contrast, long-term potentiation (LTP) and contextual fear memory were not affected by p110γ overexpression. These results, together with the previous knockout study, suggest that a critical level of PI3Kγ in the hippocampus is required for successful induction of LTD and normal learning.