Journal: eLife

Article Title: Analyzing the brainstem circuits for respiratory chemosensitivity in freely moving mice

doi: 10.7554/elife.70671

Figure Lengend Snippet: Figure 2. Excitatory chemosensory responses of RTN neurons in awake mice. (A) AAV9-Syn-GCaMP6s injection into the RTN. (B) Micrograph of lens placement and viral transduction of neurons (green) relative to the facial nucleus (ChAT +neurons red). (C) Venn diagram of cell counts of GCaMP6s transduced (green), and ChAT+ (red), neurons under the GRIN lens (1 representative section from each of 4 mice). (D) Neurons transduced

Article Snippet: DOI: https://doi.org/10.7554/eLife.70671 22 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody (donkey polyclonal) anti- goat IgG (H+L) Alexa Fluor 680 Jackson Immunoresearch RRID:AB_2340432 (1:250 dilution) Recombinant DNA reagent AAV- 9: pGP- AAV- syn- GCaMP6sWPRE.4.641 Addgene, Watertown, MA, USA BS1- NOSAAV9 Dilution 1:10 Recombinant DNA reagent AAV- DJ: pAAV- SERT- GCaMP6s This paper Raphe neuronal specific virus with calcium indicator Dilution 1:10 Recombinant DNA reagent AAV- 9: pGP- AAV- syn- GCaMP6fWPRE.24.693 Addgene, Watertown, MA, USA BS3- NXFAAV9 Dilution 1:10 Recombinant DNA reagent pAAV.Syn.GCaMP6s.WPRE.

Techniques: Injection, Transduction

Journal: eLife

Article Title: Analyzing the brainstem circuits for respiratory chemosensitivity in freely moving mice

doi: 10.7554/elife.70671

Figure Lengend Snippet: Figure 3. Sniff-coding, and CO2-inhibited RTN neuronal responses in awake mice. (A) Recording of mouse WBP in response to hypercapnia time- matched with Inscopix recorded GCaMP6 signals. CO2-Inhibited (blue) and sniff-coding (eggplant purple) calcium traces correspond to the neurons shown in panel F. (B) Expanded recordings from (A), the start of the Ca2+ transient is shown by the dotted line. (C) Average waveform of RTN inhibited neurons in response to hypercapnia. (D) Sniff-correlation histogram and (E) Spike triggered average (eggplant purple line) of all Ca2+ events (grey lines) temporally correlated to the beginning of sniff activity (dotted verticle line). (F) GCaMP6s fluorescence of transduced RTN neurons in freely behaving mice. Individual regions of interest (ROIs) drawn around CO2-inhibited (blue) and sniff coactivated (eggplant purple) neurons. (Scalebar- 20 μm).

Article Snippet: DOI: https://doi.org/10.7554/eLife.70671 22 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody (donkey polyclonal) anti- goat IgG (H+L) Alexa Fluor 680 Jackson Immunoresearch RRID:AB_2340432 (1:250 dilution) Recombinant DNA reagent AAV- 9: pGP- AAV- syn- GCaMP6sWPRE.4.641 Addgene, Watertown, MA, USA BS1- NOSAAV9 Dilution 1:10 Recombinant DNA reagent AAV- DJ: pAAV- SERT- GCaMP6s This paper Raphe neuronal specific virus with calcium indicator Dilution 1:10 Recombinant DNA reagent AAV- 9: pGP- AAV- syn- GCaMP6fWPRE.24.693 Addgene, Watertown, MA, USA BS3- NXFAAV9 Dilution 1:10 Recombinant DNA reagent pAAV.Syn.GCaMP6s.WPRE.

Techniques: Activity Assay, Fluorescence

Journal: eLife

Article Title: Analyzing the brainstem circuits for respiratory chemosensitivity in freely moving mice

doi: 10.7554/elife.70671

Figure Lengend Snippet: Figure 5. CO2-excitated medullary raphe neurons in awake mice. (A) AAV injection into the Raphe. (B) Micrograph of lens placement and AAV9-Syn- GCaMP6s viral transduction of neurons (green) relative to the Raphe (TPH +neurons red). (C) GCaMP6s fluorescence signal from AAV9-Syn-GCaMP6s transduced raphe neurons in freely behaving mice (Scale bar- 50 μm). (D) Venn diagram of cell counts of AAV9-Syn-GCaMP6s transduced neurons (green) under the GRIN lens with TPH +neurons (red) in the raphe (1 representative section from each of 4 mice). (E) Micrograph of lens placement and AAV9-SERT-GCaMP6s viral transduction of neurons (green) relative to the Raphe (TPH +neurons red). (F) GCaMP6s fluorescence signal from AAV9- SERT-GCaMP6s transduced raphe neurons in freely behaving mice (Scale bar- 50 μm). (G) WBP recording in response to hypercapnia time-matched with Inscopix recorded GCaMP6 signals. GCaMP fluorescence of raphe excitatory graded (EG), and excitatory adapting (EA) neurons in response to hypercapnia. (H–I) Average waveform of raphe EG neuronal responses to hypercapnia aligned to the WBP trace in G. Abbreviations defined in Figure 2.

Article Snippet: DOI: https://doi.org/10.7554/eLife.70671 22 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody (donkey polyclonal) anti- goat IgG (H+L) Alexa Fluor 680 Jackson Immunoresearch RRID:AB_2340432 (1:250 dilution) Recombinant DNA reagent AAV- 9: pGP- AAV- syn- GCaMP6sWPRE.4.641 Addgene, Watertown, MA, USA BS1- NOSAAV9 Dilution 1:10 Recombinant DNA reagent AAV- DJ: pAAV- SERT- GCaMP6s This paper Raphe neuronal specific virus with calcium indicator Dilution 1:10 Recombinant DNA reagent AAV- 9: pGP- AAV- syn- GCaMP6fWPRE.24.693 Addgene, Watertown, MA, USA BS3- NXFAAV9 Dilution 1:10 Recombinant DNA reagent pAAV.Syn.GCaMP6s.WPRE.

Techniques: Injection, Transduction, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: Effect of in utero opioid exposure on SERT and ABCB1 expression in human placental membranous vesicles. ( A ) Downregulation of SERT levels in qWestern blot analysis of protein lysates, comparing 20 first and second trimester opioid-exposed human placentas with 20 unexposed controls individually matched for fetal sex, GA, and maternal age. ( B ) Reduced levels of the drug transporter ABCB1 in opioid-exposed placentas. Inside out placental brush border membrane vesicles were used to measure and quantify the ABCB1 expression. In both ( A , B ), Grb2 served as a loading control. Data are presented as fold change (* for p < 0.05, *** for p < 0.001).

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: In Utero, Expressing, Membrane, Control

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: SERT is modified by maternal opioid exposure. ( A ) Immunoblot detection of SERT in placental vesicles of first and second trimester fetuses, using a rabbit polyclonal anti-serotonin transporter antibody specific for 70 kDa SERT. Grb2 served as a loading control. Subjects who admitted to chronic opioid use were compared to fetal sex-, GA-, and maternal age-matched controls. Left panel , lanes 1–4 are unexposed controls. Lanes 5–8 are subjects not exposed to opioids or EtOH but who were being treated for depression with SSRIs. Right panel , 12 opiod-exposed cases not exposed to other drugs. ( B ) Alteration of the SERT protein band pattern is seen only in opioid-exposed cases (lanes 4–10), not with other drug exposures—Adderall (lanes 1–3) and Keppra (lanes 11–12). The cleaved forms of SERT were observed only in opiate cases (lanes 4–10, top panel). ( C ) The smaller of the double-bands between 34 and 32 kDA representing cleaved forms of SERT (clSERT) in the opioid-exposed cases may represent a hypo-phosphorylated SERT fragment (see ).

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: Modification, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: Cleaved SERT isoforms in opioid-exposed cases are within the central domain . ( A ) The immunoblot used for was used for detection of the central domain and C-terminal of SERT in placental vesicles from first and second trimester human pregnancies. Western immunoblots demonstrate expression of SERT isoforms in vesicles from 12-opioid exposed placentas at two GAs. Post-translational modification of SERT was studied by treatment with phosphatase inhibitors (Lane 5 vs. 6). This resulted in enhancement of the lower of the opioid-induced bands at 32–34 kDa, suggesting that most of this smaller clSERT is unphosphorylated. Antibodies to the N-terminal of SERT did not recognize either of the smaller (32 and 34 kDa) bands, while an antibody to the C-terminal region of SERT recognized both bands (upper panel), identical to the bands identified by antibodies to the central region of SERT (lower panel). SDS-PAGE stained with Coomassie blue was used to assure equal protein loading. The actin band (42 kDa) is labeled. ( B ) An antibody specific for the macrophage marker CD163 was used as a negative control, in combination with an antibody to GAPDH as a positive control, to confirm the purity of placental vesicles, the lack of contamination by other sources of SERT, and the specificity of the SERT double bands in panels ( A , B ) Note the absence of a band at 70 kDa.

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: Western Blot, Expressing, Modification, SDS Page, Staining, Labeling, Marker, Negative Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: SERT sequencing in opioid cases. The Edman degradation experiment was performed to get the N-terminal sequence of truncated SERT protein double-band versions in opioid-exposed placental vesicles. Two SERT fragments were present in the opioid samples. The sequences were compared with two 5HT transporter isoforms downloaded from Unipro. Sequence alignment was performed using ClustalW2 on two isoforms of SERT fragments on SLC6A4 (Synonyms:HTT, SERT; Organism Homo sapiens (Human), Taxonomic identifier9606 [NCBI]). The first four amino acids of the Central region of SERT were detected in two potential truncated bands corresponding to the sequence of SERT. In combination with the western blot results, we confirm the presence of a truncated SERT protein. Truncated proteins were located using the sequence alignment tool. Version 1 (the upper band) encompasses the amino acids 183–227 (out of 672) of human SERT. Truncated Version 2 (lower band) encompasses amino acids 224–276 of human SERT, which includes the phosphorylation site Thr276.

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: Sequencing, Western Blot, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression

doi: 10.3390/ijms252111570

Figure Lengend Snippet: Prenatal alcohol exposure-associated downregulation of SERT and ABCB1 in placenta vesicles, placenta-derived exosomes, and fetal brain-derived exosomes. ( A ) EtOH exposure is associated with downregulation of total SERT in right side out (brush border membranous) placental vesicles (IOV) and placenta-derived exosomes from maternal blood (PE; bars 3–4) from human placenta tissues (n = 20) obtained from first and second trimester pregnancies. Analysis was completed by ELISA using a SERT ELISA kit, and results were obtained in picograms/microliters according to SERT standards. Bars represent fold changes between SERT levels in vesicles from EtOH-exposed cases compared to unexposed controls. ( B ) A similar reduction in expression of ABCB1 was seen in inside-out vesicles IOV. ( C ) Downregulation of SERT in fetal brain-derived exosomes. All assays were completed in triplicate (catalog #HS0096, detection range is within 0, 0.5, 1.0, 2.5, 5.0, 10 ng/mL, or picogram/microliter, and sensitivity is 0.1 ng/mL). ( *** for p < 0.001).

Article Snippet: SLC6A4 antibody to C-terminus SERT (OAEB02575) was from Aviva Systems Biology, Corp. (San Diego, CA, USA).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing