Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues

Kumar, Vivek; Krolewski, David M.; Hebda-Bauer, Elaine K.; Parsegian, Aram; Martin, Brian; Foltz, Matthew; Akil, Huda; Watson, Stanley J.

doi:10.1007/s00429-020-02194-4

Cited by 10 publications

(13 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Split-initiator DNA probes ( version 2.0) were designed in our lab (Choi et al, 2018; Kumar et al, 2021) ( SI Appendix , Table S9) and synthesized by Integrated DNA Technologies (Coralville, Iowa, USA). DNA hairpins conjugated with AlexaFluor-546 (AF-546), AF-594 and AF-647 were purchased from Molecular Instruments, Inc. (Los Angeles, California, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Fresh-frozen brains were cryosectioned to get 12 sections (20 µm thickness) between ‘Bregma -2.92 mm to -3.40 mm’ for vHC and between ‘Bregma -2.18 mm to -3.40 mm’ for amygdala and used for HCR FISH. We optimized the HCR FISH method as described previously (Choi et al, 2014, 2018; Kumar et al 2021). Briefly, sections were immersion fixed in 4% paraformaldehyde-saline, washed with 5x sodium chloride/sodium citrate/0.01% tween-20 (SSCTw) buffer for 3 times, 5 min each, then acetylated in 0.1M triethanolamine, pH 8.0 with 0.25% vol/vol acetic anhydride solution for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…We optimized the HCR FISH method as described previously (Choi et al, 2014(Choi et al, , 2018Kumar et al 2021). Briefly, sections were immersion fixed in 4% paraformaldehyde-saline, washed with 5x sodium chloride/sodium citrate/0.01% tween-20 (SSCTw) buffer for 3 times, 5 min each, then acetylated in 0.1M triethanolamine, pH 8.0 with 0.25% vol/vol acetic anhydride solution for 10 min.…”

Section: Fluorescence In Situ Hybridization Chain Reaction (Hcr Fish)...mentioning

confidence: 99%

See 2 more Smart Citations

High emotional reactivity is associated with activation of a molecularly distinct hippocampal-amygdala circuit modulated by the glucocorticoid receptor

Wei

Kumar

Moore

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Emotions are characterized not only by their valence but also by whether they are stable or labile. Yet, we do not understand the molecular or circuit mechanisms that control the dynamic nature of emotionality. We have shown that glucocorticoid receptor overexpression in the forebrain (GRov) leads to a highly reactive mouse with increased anxiety behavior coupled with greater swings in emotional responses. This phenotype is established early in development and persists into adulthood. However, the neural circuitry mediating this lifelong emotional lability remains unknown. In the present study, optogenetic stimulation in ventral dentate gyrus (vDG) of GRov mice led to a greater range and a prolonged duration of anxiety behavior. cFos expression showed that the amplified behavioral response to vDG activation in GRov mice is coupled to increased neuronal activity in specific brain regions. Relative to wild type mice, GRov mice displayed glutamatergic/GABAergic activation imbalance in ventral CA1 (vCA1) and selectively increased glutamatergic activation in the basal posterior amygdaloid complex. Fluorescence in situ hybridization chain reaction studies showed that forebrain GR overexpression led to increased activation of molecularly distinct subpopulations of neurons within the hippocampus and the posterior basolateral amygdala (pBLA). Increased cFos labeling was observed in the Calbindin 1 (Calbn1+) glutamatergic neurons in vCA1 and in the DARPP-32/Ppp1r1b+ glutamatergic neurons in pBLA. We propose that a molecularly distinct hippocampal-amygdala circuit is shaped by stress early in life and tunes the dynamics of emotional responses.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Fluorescence In Situ Hybridization Chain Reaction (Hcr Fish)...mentioning

confidence: 99%

See 1 more Smart Citation

High emotional reactivity is associated with activation of a molecularly distinct hippocampal-amygdala circuit modulated by the glucocorticoid receptor

Wei

Kumar

Moore

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…We designed the Split-initiator DNA probes ( version 2.0) (Choi et al, 2018; Kumar et al, 2021) (Table S1B and S1C) and the probes were synthesized by Integrated DNA Technologies (Coralville, Iowa, USA). We purchased DNA hairpins conjugated with B3 AlexaFluor-546 (AF-546) and B2 AF-647 from Molecular Instruments (Los Angeles, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Section: Fluorescence In Situ Hybridization Chain Reaction (Hcr Fish)...mentioning

confidence: 99%

Effect of selective lesions of nucleus accumbens μ-opioid receptor-expressing cells on heroin self-administration in male and female rats: a study with novelOprm1-Creknock-in rats

Bossert¹,

Mejías-Aponte²,

Saunders

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

The brain μ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-basedOprm1-Cre knock-in transgenic rat that provides cell-type specific genetic access to MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used theOprm1-Cre knock-in rats to study the role of nucleus accumbens (NAc) MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and fluorescence in situ hybridization chain reaction (HCR-FISH), we found no differences inOprm1expression in NAc, dorsal striatum (DS), and dorsal hippocampus, or MOR receptor density (except DS) or function betweenOprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly co-expressed with Oprm1 (95-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells and report sex-specific effects: the lesions decreased acquisition of heroin self-administration in maleOprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in femaleOprm1-Cre rats. The validation of anOprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study with these rats suggests a sex-specific role of NAc MOR-expressing cells in heroin self-administration.

show abstract

Whole-brain mapping reveals the divergent impact of ketamine on the dopamine system

Datta,

Chen,

Chauhan

et al. 2023

Cell Reports

View full text Add to dashboard Cite

Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues

Cited by 10 publications

References 38 publications

High emotional reactivity is associated with activation of a molecularly distinct hippocampal-amygdala circuit modulated by the glucocorticoid receptor

High emotional reactivity is associated with activation of a molecularly distinct hippocampal-amygdala circuit modulated by the glucocorticoid receptor

Effect of selective lesions of nucleus accumbens μ-opioid receptor-expressing cells on heroin self-administration in male and female rats: a study with novelOprm1-Creknock-in rats

Whole-brain mapping reveals the divergent impact of ketamine on the dopamine system

Contact Info

Product

Resources

About