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ACD can configure probes for the various manual and automated assays for INS for RNAscope Assay, or for Basescope Assay compatible for your species of interest.

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Daily changes in light influence mood via inhibitory networks within the thalamic perihabenular nucleus

Science advances

2022 Jun 10

Weil, T;Daly, KM;Yarur Castillo, H;Thomsen, MB;Wang, H;Mercau, ME;Hattar, S;Tejeda, H;Fernandez, DC;
PMID: 35687680 | DOI: 10.1126/sciadv.abn3567

Exposure to irregular lighting schedules leads to deficits in affective behaviors. The retino-recipient perihabenular nucleus (PHb) of the dorsal thalamus has been shown to mediate these effects in mice. However, the mechanisms of how light information is processed within the PHb remains unknown. Here, we show that the PHb contains a distinct cluster of GABAergic neurons that receive direct retinal input. These neurons are part of a larger inhibitory network composed of the thalamic reticular nucleus and zona incerta, known to modulate thalamocortical communication. In addition, PHbGABA neurons locally modulate excitatory-relay neurons, which project to limbic centers. Chronic exposure to irregular light-dark cycles alters photo-responsiveness and synaptic output of PHbGABA neurons, disrupting daily oscillations of genes associated with inhibitory and excitatory PHb signaling. Consequently, selective and chronic PHbGABA manipulation results in mood alterations that mimic those caused by irregular light exposure. Together, light-mediated disruption of PHb inhibitory networks underlies mood deficits.
Central Control Circuit for Context-Dependent Micturition

Cell.

2016 Sep 22

Hou XH, Hyun M, Taranda J, Huang KW, Todd E, Feng D, Atwater E, Croney D, Zeidel ML, Osten P, Sabatini BL.
PMID: 27662084 | DOI: 10.1016/j.cell.2016.08.073

Urine release (micturition) serves an essential physiological function as well as a critical role in social communication in many animals. Here, we show a combined effect of olfaction and social hierarchy on micturition patterns in adult male mice, confirming the existence of a micturition control center that integrates pro- and anti-micturition cues. Furthermore, we demonstrate that a cluster of neurons expressing corticotropin-releasing hormone (Crh) in the pontine micturition center (PMC) is electrophysiologically distinct from their Crh-negative neighbors and sends glutamatergic projections to the spinal cord. The activity of PMC Crh-expressing neurons correlates with and is sufficient to drive bladder contraction, and when silenced impairs micturition behavior. These neurons receive convergent input from widespread higher brain areas that are capable of carrying diverse pro- and anti-micturition signals, and whose activity modulates hierarchy-dependent micturition. Taken together, our results indicate that PMC Crh-expressing neurons are likely the integration center for context-dependent micturition behavior.

Cellular senescence drives age-dependent hepatic steatosis.

Nat Commun.

2017 Jun 13

Ogrodnik M, Miwa S, Tchkonia T, Tiniakos D, Wilson CL, Lahat A, Day CP, Burt A, Palmer A, Anstee QM, Grellscheid SN, Hoeijmakers JHJ, Barnhoorn S, Mann DA, Bird TG, Vermeij WP, Kirkland JL, Passos JF, von Zglinicki T, Jurk D.
PMID: 28608850 | DOI: 10.1038/ncomms15691

The incidence of non-alcoholic fatty liver disease (NAFLD) increases with age. Cellular senescence refers to a state of irreversible cell-cycle arrest combined with the secretion of proinflammatory cytokines and mitochondrial dysfunction. Senescent cells contribute to age-related tissue degeneration. Here we show that the accumulation of senescent cells promotes hepatic fat accumulation and steatosis. We report a close correlation between hepatic fat accumulation and markers of hepatocyte senescence. The elimination of senescent cells by suicide gene-meditated ablation of p16Ink4a-expressing senescent cells in INK-ATTAC mice or by treatment with a combination of the senolytic drugs dasatinib and quercetin (D+Q) reduces overall hepatic steatosis. Conversely, inducing hepatocyte senescence promotes fat accumulation in vitro and in vivo. Mechanistically, we show that mitochondria in senescent cells lose the ability to metabolize fatty acids efficiently. Our study demonstrates that cellular senescence drives hepatic steatosis and elimination of senescent cells may be a novel therapeutic strategy to reduce steatosis.

Engram-specific transcriptome profiling of contextual memory consolidation.

Nat Commun

2019 May 20

Rao-Ruiz P, Couey JJ, Marcelo IM, Bouwkamp CG, Slump DE, Matos MR, van der Loo RJ, Martins GJ, van den Hout M, van IJcken WF, Costa RM, van den Oever MC, Kushner SA.
PMID: 31110186 | DOI: 10.1038/s41467-019-09960-x

Sparse populations of neurons in the dentate gyrus (DG) of the hippocampus are causally implicated in the encoding of contextual fear memories. However, engram-specific molecular mechanisms underlying memory consolidation remain largely unknown. Here we perform unbiased RNA sequencing of DG engram neurons 24 h after contextual fear conditioning to identify transcriptome changes specific to memory consolidation. DG engram neurons exhibit a highly distinct pattern of gene expression, in which CREB-dependent transcription features prominently (P = 6.2 × 10-13), including Atf3 (P = 2.4 × 10-41), Penk (P = 1.3 × 10-15), and Kcnq3 (P = 3.1 × 10-12). Moreover, we validate the functional relevance of the RNAseq findings by establishing the causal requirement of intact CREB function specifically within the DG engram during memory consolidation, and identify a novel group of CREB target genes involved in the encoding of long-term memory

Adnp-mutant mice with cognitive inflexibility, CaMKIIα hyperactivity, and synaptic plasticity deficits

Molecular psychiatry

2023 Jun 26

Cho, H;Yoo, T;Moon, H;Kang, H;Yang, Y;Kang, M;Yang, E;Lee, D;Hwang, D;Kim, H;Kim, D;Kim, JY;Kim, E;
PMID: 37365244 | DOI: 10.1038/s41380-023-02129-5

ADNP syndrome, involving the ADNP transcription factor of the SWI/SNF chromatin-remodeling complex, is characterized by developmental delay, intellectual disability, and autism spectrum disorders (ASD). Although Adnp-haploinsufficient (Adnp-HT) mice display various phenotypic deficits, whether these mice display abnormal synaptic functions remain poorly understood. Here, we report synaptic plasticity deficits associated with cognitive inflexibility and CaMKIIα hyperactivity in Adnp-HT mice. These mice show impaired and inflexible contextual learning and memory, additional to social deficits, long after the juvenile-stage decrease of ADNP protein levels to ~10% of the newborn level. The adult Adnp-HT hippocampus shows hyperphosphorylated CaMKIIα and its substrates, including SynGAP1, and excessive long-term potentiation that is normalized by CaMKIIα inhibition. Therefore, Adnp haploinsufficiency in mice leads to cognitive inflexibility involving CaMKIIα hyperphosphorylation and excessive LTP in adults long after its marked expressional decrease in juveniles.
Neuronal cell types, projections, and spatial organization of the central amygdala

iScience

2022 Dec 22

O'Leary, TP;Kendrick, RM;Bristow, BN;Sullivan, KE;Wang, L;Clements, J;Lemire, AL;Cembrowski, MS;
PMID: 36425768 | DOI: 10.1016/j.isci.2022.105497

The central amygdala (CEA) has been richly studied for interpreting function and behavior according to specific cell types and circuits. Such work has typically defined molecular cell types by classical inhibitory marker genes; consequently, whether marker-gene-defined cell types exhaustively cover the CEA and co-vary with connectivity remains unresolved. Here, we combined single-cell RNA sequencing, multiplexed fluorescent in situ hybridization, immunohistochemistry, and long-range projection mapping to derive a "bottom-up" understanding of CEA cell types. In doing so, we identify two major cell types, encompassing one-third of all CEA neurons, that have gone unresolved in previous studies. In spatially mapping these novel types, we identify a non-canonical CEA subdomain associated with Nr2f2 expression and uncover an Isl1-expressing medial cell type that accounts for many long-range CEA projections. Our results reveal new CEA organizational principles across cell types and spatial scales and provide a framework for future work examining cell-type-specific behavior and function.
NG2 glia-derived GABA release tunes inhibitory synapses and contributes to stress-induced anxiety

Nature communications

2021 Sep 30

Zhang, X;Liu, Y;Hong, X;Li, X;Meshul, CK;Moore, C;Yang, Y;Han, Y;Li, WG;Qi, X;Lou, H;Duan, S;Xu, TL;Tong, X;
PMID: 34593806 | DOI: 10.1038/s41467-021-25956-y

NG2 glia, also known as oligodendrocyte precursor cells (OPCs), play an important role in proliferation and give rise to myelinating oligodendrocytes during early brain development. In contrast to other glial cell types, the most intriguing aspect of NG2 glia is their ability to directly sense synaptic inputs from neurons. However, whether this synaptic interaction is bidirectional or unidirectional, or its physiological relevance has not yet been clarified. Here, we report that NG2 glia form synaptic complexes with hippocampal interneurons and that selective photostimulation of NG2 glia (expressing channelrhodopsin-2) functionally drives GABA release and enhances inhibitory synaptic transmission onto proximal interneurons in a microcircuit. The mechanism involves GAD67 biosynthesis and VAMP-2 containing vesicular exocytosis. Further, behavioral assays demonstrate that NG2 glia photoactivation triggers anxiety-like behavior in vivo and contributes to chronic social defeat stress.
Onset of differentiation is post-transcriptionally controlled in adult neural stem cells

Nature

2019 Jan 30

Baser A, Skabkin M, Kleber S, Dang Y, Gülcüler Balta GS, Kalamakis G, Göpferich M, Ibañez DC, Schefzik R, Lopez AS, Bobadilla EL, Schultz C, Fischer B, Martin-Villalba A.
PMID: 30700908 | DOI: 10.1038/s41586-019-0888-x

Whether post-transcriptional regulation of gene expression controls differentiation of stem cells for tissue renewal remains unknown. Quiescent stem cells exhibit a low level of protein synthesis1, which is key to maintaining the pool of fully functional stem cells, not only in the brain but also in the bone marrow and hair follicles2-6. Neurons also maintain a subset of messenger RNAs in a translationally silent state, which react 'on demand' to intracellular and extracellular signals. This uncoupling of general availability of mRNA from translation into protein facilitates immediate responses to environmental changes and avoids excess production of proteins, which is the most energy-consuming process within the cell. However, when post-transcriptional regulation is acquired and how protein synthesis changes along the different steps of maturation are not known. Here we show that protein synthesis undergoes highly dynamic changes when stem cells differentiate to neurons in vivo. Examination of individual transcripts using RiboTag mouse models reveals that whereas stem cells translate abundant transcripts with little discrimination, translation becomes increasingly regulated with the onset of differentiation. The generation of neurogenic progeny involves translational repression of a subset of mRNAs, including mRNAs that encode the stem cell identity factors SOX2 and PAX6, and components of the translation machinery, which are enriched in a pyrimidine-rich motif. The decrease of mTORC1 activity as stem cells exit the cell cycle selectively blocks translation of these transcripts. Our results reveal a control mechanism by which the cell cycle is coupled to post-transcriptional repression of key stem cell identity factors, thereby promoting exit from stemness.

The Claustrum Supports Resilience to Distraction

Curr Biol.

2018 Aug 16

Atlan G, Terem A, Peretz-Rivlin N, Sehrawat K, Gonzales BJ, Pozner G, Tasaka G, Goll Y, Refaeli R, Zviran O, Lim BK, Groysman M, Goshen I, Mizrahi A, Nelken I, Citri A.
PMID: 30122531 | DOI: 10.1016/j.cub.2018.06.068

A barrage of information constantly assaults our senses, of which only a fraction is relevant at any given point in time. However, the neural circuitry supporting the suppression of irrelevant sensory distractors is not completely understood. The claustrum, a circuit hub with vast cortical connectivity, is an intriguing brain structure, whose restrictive anatomy, thin and elongated, has precluded functional investigation. Here, we describe the use of Egr2-CRE mice to access genetically defined claustral neurons. Utilizing conditional viruses for anterograde axonal labeling and retrograde trans-synaptic tracing, we validated this transgenic model for accessing the claustrum and extended the known repertoire of claustral input/output connectivity. Addressing the function of the claustrum, we inactivated CLEgr2+ neurons, chronically as well as acutely, in mice performing an automated two-alternative forced-choice behavioral task. Strikingly, inhibition of CLEgr2+ neurons did not significantly impact task performance under varying delay times and cue durations, but revealed a selective role for the claustrum in supporting performance in the presence of an irrelevant auditory distractor. Further investigation of behavior, in the naturalistic maternal pup-retrieval task, replicated the result of sensitization to an auditory distractor following inhibition of CLEgr2+ neurons. Initiating investigation into the underlying mechanism, we found that activation of CLEgr2+ neurons modulated cortical sensory processing, suppressing tone representation in the auditory cortex. This functional study, utilizing selective genetic access, implicates the claustrum in supporting resilienceto distraction, a fundamental aspect of attention.

Incisionless targeted adeno-associated viral vector delivery to the brain by focused ultrasound-mediated intranasal administration

EBioMedicine

2022 Sep 21

Ye, D;Yuan, J;Yang, Y;Yue, Y;Hu, Z;Fadera, S;Chen, H;
PMID: 36152518 | DOI: 10.1016/j.ebiom.2022.104277

Adeno-associated viral (AAV) vectors are currently the leading platform for gene therapy with the potential to treat a variety of central nervous system (CNS) diseases. There are numerous methods for delivering AAVs to the CNS, such as direct intracranial injection (DI), intranasal delivery (IN), and intravenous injection with focused ultrasound-induced blood-brain barrier disruption (FUS-BBBD). However, non-invasive and efficient delivery of AAVs to the brain with minimal systemic toxicity remain the major challenge. This study aims to investigate the potential of focused ultrasound-mediated intranasal delivery (FUSIN) in AAV delivery to brain.Mice were intranasally administered with AAV5 encoding enhanced green fluorescence protein (AAV5-EGFP) followed by FUS sonication in the presence of systemically injected microbubbles. Mouse brains and other major organs were harvested for immunohistological staining, PCR quantification, and in situ hybridization. The AAV delivery outcomes were compared with those of DI, FUS-BBBD, and IN delivery.FUSIN achieved safe and efficient delivery of AAV5-EGFP to spatially targeted brain locations, including a superficial brain site (cortex) and a deep brain region (brainstem). FUSIN achieved comparable delivery outcomes as the established DI, and displayed 414.9-fold and 2073.7-fold higher delivery efficiency than FUS-BBBD and IN. FUSIN was associated with minimal biodistribution in peripheral organs, which was comparable to that of DI.Our results suggest that FUSIN is a promising technique for non-invasive, efficient, safe, and spatially targeted AAV delivery to the brain.National Institutes of Health (NIH) grants R01EB027223, R01EB030102, R01MH116981, and UG3MH126861.
Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos

BMC Biol. 2014 Aug 15;12(1):55.

Gross-Thebing T, Paksa A, Raz E.
PMID: 25124741

Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos
Angiotensin Type-2 Receptors Influence the Activity of Vasopressin Neurons in the Paraventricular Nucleus of the Hypothalamus in Male Mice.

Endocrinology.

2016 Jun 06

de Kloet AD, Pitra S, Wang L, Hiller H, Pioquinto DJ, Smith JA, Sumners C, Stern JE, Krause EG.
PMID: 27267713 | DOI: -

It is known that angiotensin-II acts at its type-1 receptor to stimulate vasopressin (AVP) secretion, which may contribute to angiotensin-II-induced hypertension. Less well-known, is the impact angiotensin type-2 receptor (AT2R) activation on these processes. Studies conducted in a transgenic AT2R enhanced green fluorescent protein (eGFP) reporter mouse revealed that although AT2R are not themselves localized to AVP neurons within the paraventricular nucleus of the hypothalamus (PVN), they are localized to neurons that extend processes into the PVN. In the present set of studies, we set out to characterize the origin, phenotype and function of nerve terminals within the PVN that arise from AT2R-eGFP-positive neurons and synapse onto AVP neurons. Initial experiments combined genetic and neuroanatomical techniques to determine that gamma-aminobutyric acid (GABA)ergic neurons derived from the peri-PVN area containing AT2R make appositions onto AVP neurons within the PVN, thereby positioning AT2R to negatively regulate neuroendocrine secretion. Subsequent patch-clamp electrophysiological experiments revealed that selective activation of AT2R in the peri-PVN area using Compound 21 facilitates inhibitory (i.e., GABAergic) neurotransmission and leads to reduced activity of AVP neurons within the PVN. Final experiments determined the functional impact of AT2R activation by testing the effects of Compound 21 on plasma AVP levels. Collectively, these experiments revealed that AT2R expressing neurons make GABAergic synapses onto AVP neurons that inhibit AVP neuronal activity and suppress baseline systemic AVP levels. These findings have direct implications in the targeting of AT2R for disorders of AVP secretion and also for the alleviation of high blood pressure.

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Description
sense
Example: Hs-LAG3-sense
Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron#
Example: Mm-Htt-intron2
Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan
Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)
A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp
Example: Hs-PDGFB-No-XMm
Does not cross detect with the species (Sp)
XSp
Example: Rn-Pde9a-XMm
designed to cross detect with the species (Sp)
O#
Example: Mm-Islr-O1
Alternative design targeting different regions of the same transcript or isoforms
CDS
Example: Hs-SLC31A-CDS
Probe targets the protein-coding sequence only
EnEmProbe targets exons n and m
En-EmProbe targets region from exon n to exon m
Retired Nomenclature
tvn
Example: Hs-LEPR-tv1
Designed to target transcript variant n
ORF
Example: Hs-ACVRL1-ORF
Probe targets open reading frame
UTR
Example: Hs-HTT-UTR-C3
Probe targets the untranslated region (non-protein-coding region) only
5UTR
Example: Hs-GNRHR-5UTR
Probe targets the 5' untranslated region only
3UTR
Example: Rn-Npy1r-3UTR
Probe targets the 3' untranslated region only
Pan
Example: Pool
A mixture of multiple probe sets targeting multiple genes or transcripts

Enabling research, drug development (CDx) and diagnostics

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