Journal: Nature Communications

Article Title: Astrocytic metabolic control of orexinergic activity in the lateral hypothalamus regulates sleep and wake architecture

doi: 10.1038/s41467-024-50166-7

Figure Lengend Snippet: a Schematic of stereotaxic injections of either Cre-encoding PhP.eB virus (aMCT4 KD mice) or control PhP.eB virus (control mice) in the LH of MCT4 f/f mice and immunofluorescence micrograph showing eGFP expression (green) near Orexin-A (magenta) positive cells 12 weeks after virus injection. Scale bar = 100 µm. Representative hypnograms of Control mouse and aMCT4 KD mouse during 24 h EEG recordings ( b ) or same aMCT4 KD before and after L-lactate infusion ( g ) (W – wake; N – NREM and R – REM). Quantification of the average percentage of time spent in wake, NREM or REM sleep during the 24 h recording phase in aMCT4 KD mice compared to control mice ( c ) or in aMCT4 KD mice before and lactate infusion ( h ). Average of the number of wake, NREM and REM episodes during the 24 h recording phase in aMCT4 KD mice compared to control mice ( d ) or in aMCT4 KD mice before and after lactate infusion ( i ). Average duration of the wake, NREM and REM episodes during the 24 h recording phase in aMCT4 KD mice compared to control mice ( e ) or in aMCT4 KD mice before and lactate infusion ( j ) ( c , d , e : red, aMCT4 KD n = 12; gray, controls n = 15, two-tailed unpaired t test; ZT12-18: %wake ** p = 0.005, %NREM p = 0.006; episode number wake * p = 0.033, episode number NREM * p = 0.040, episode duration wake * p = 0.048; h , i , j : n = 5, two-tailed paired t test; ZT12-18 %wake and %NREM ** p = 0.003; ZT18-24 %wake * p = 0.039, %NREM * p = 0.043; ZT12-18 episode number NREM ** p = 0.005). ZT = Zeitgeber Time. Pooled data are shown as mean ± SEM. f Experimental design and timeline for L-lactate infusion; 24 h EEG recordings were analyzed over time in the same aMCT4 KD mouse. Source data are provided as a file.

Article Snippet: MCT4 f/f or MCT1 f/f mice received either AAV-PhP.eB-GFAP (0.7)-EGFP-T2A-iCre virus (aMCT4 KD or aMCT1 KD mice) or AAV-PhP.eB-GFAP (0.7)-EGFP virus (control mice) (#VB1131 Vector Biolabs) or vehicle only (control mice) (ACSF, #3525 Tocris). oMCT2 KD received AAV-PhP.eB-CAG2-DIO-mSlc16a7-P2A-GFP virus (AAV-272101, Vector Biolabs) to re-express MCT2 in these mice.

Techniques: Virus, Control, Immunofluorescence, Expressing, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Astrocytic metabolic control of orexinergic activity in the lateral hypothalamus regulates sleep and wake architecture

doi: 10.1038/s41467-024-50166-7

Figure Lengend Snippet: a Experimental design. Representative traces ( b ) and average firing rate ( c , f ) showing reduced firing in orexin neurons of aMCT4 KD (Controls and aMCT4 KD; n = 7 cells/4 mice; two-tailed Mann–Whitney test, *** p = 0.0006) and oMCT2 KD mice (Controls, n = 7 cells/4 mice; oMCT2 KD, n = 9 cells/4 mice; two-tailed Student t test, * p = 0.0131). d , g Membrane voltage (Vm) in aMCT4 KD (Controls, n = 7 cells/4 mice; aMCT4 KD, n = 6 cells/3 mice; two-tailed Student t test, * p = 0.0252) and oMCT2 KD mice (Control, n = 7 cells/4 mice and oMCT2 KD n = 9 cells/4mice; two-tailed Student t test ** p = 0.0011). e , h Average input resistance (Rin) in aMCT4 KD (Control, n = 6 cells/3 mice; aMCT4 KD, n = 5 cells/3 mice; two-tailed Student t test, ** p = 0.0082), and oMCT2 KD (Control, n = 7 cells/3 mice and oMCT2 KD n = 9 cells/4 mice; two-tailed Student t test, p = 0.28). i − k Lactate depolarization of an aMCT4 KD neuron was suppressed by 4-CIN. Representative trace ( i ) and average firing rate ( j ) ([Basal], n = 6 cells/4 mice; [Lactate], n = 6 cells/4 mice; [Lactate + 4-CIN], n = 5 cells/4 mice; Mixed-effects analysis followed by Tukey’s multiple comparison test, * p = 0.0107, * p = 0.0267). Same experiment as in ( j ) but in Control mice ( k ), ([Basal], n = 6 cells/3 mice; [Lactate], n = 6 cells/3 mice; [Lactate + 4-CIN], n = 4 cells/3 mice; Mixed-effects analysis followed by Tukey’s multiple comparison test, ** p = 0.008, * p = 0.0176). No data points were excluded from the study. l , m Extracellular lactate had no effect on orexinergic activity from oMCT2 KD mice, while tolbutamide increased the activity. The bottom traces show an expanded timescale of the recording ( l ). Average firing ( m ) ( n = 7 cells/5 mice; Friedman Test followed by Dunn’s multiple comparisons test, * p = 0.022, ** p = 0.009). n Tolbutamide increased orexinergic activity from aMCT4 KD ( n = 5 cells/3 mice; two-Tailed paired Student’s t test, ** p = 0.0037) but had no effect in Control mice ( n = 4 cells/3 mice; two-Tailed paired Student’s t test; p = 0.33). Pooled data are expressed as Mean values ± SEM. Source data are provided as a file.

Article Snippet: MCT4 f/f or MCT1 f/f mice received either AAV-PhP.eB-GFAP (0.7)-EGFP-T2A-iCre virus (aMCT4 KD or aMCT1 KD mice) or AAV-PhP.eB-GFAP (0.7)-EGFP virus (control mice) (#VB1131 Vector Biolabs) or vehicle only (control mice) (ACSF, #3525 Tocris). oMCT2 KD received AAV-PhP.eB-CAG2-DIO-mSlc16a7-P2A-GFP virus (AAV-272101, Vector Biolabs) to re-express MCT2 in these mice.

Techniques: Two Tailed Test, MANN-WHITNEY, Membrane, Control, Comparison, Activity Assay

Journal: Nature Communications

Article Title: Astrocytic metabolic control of orexinergic activity in the lateral hypothalamus regulates sleep and wake architecture

doi: 10.1038/s41467-024-50166-7

Figure Lengend Snippet: a Schematic representation of lactate biosensor recording/placement coupled with EEG/EMG, and immunofluorescence micrograph showing positioning of lactate biosensor (highlighted by orange line) in the LH near Orexin-A positive cells (magenta) and eGFP positive astrocytes in green. Scale bar = 100 µm. b Representative trace of extracellular lactate recording during the dark phase color coded according to EEG analysis. Mean stereotypical traces for control and aMCT4 KD mice ( c ), and oMCT2 KD mice ( e ) upon wakefulness (blue line “Wake”). Pooled data are shown as mean ± SEM. EEG=electroencephalogram; EMG=electromyography ( d , f ) cumulative frequency plots (Two-sample Kolmogorov−Smirnov test) of lactate concentrations during all wakefulness events in the first hours of the dark phase (ZT12-18). aMCT4 KD mice (red, n = 4) compared to controls mice (black, n = 5) (Kolmogorov−Smirnov test, p = 3.4135 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 −57 ); ( f ) oMCT2 KD (violet, n = 6) mice compared to controls mice (black, n = 5) (Kolmogorov−Smirnov test, p = 1.887 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 −17 ). Source data are provided as a file.

Article Snippet: MCT4 f/f or MCT1 f/f mice received either AAV-PhP.eB-GFAP (0.7)-EGFP-T2A-iCre virus (aMCT4 KD or aMCT1 KD mice) or AAV-PhP.eB-GFAP (0.7)-EGFP virus (control mice) (#VB1131 Vector Biolabs) or vehicle only (control mice) (ACSF, #3525 Tocris). oMCT2 KD received AAV-PhP.eB-CAG2-DIO-mSlc16a7-P2A-GFP virus (AAV-272101, Vector Biolabs) to re-express MCT2 in these mice.

Techniques: Immunofluorescence, Control

Journal: PLOS Biology

Article Title: KCTD proteins regulate morphine dependence via heterologous sensitization of adenylyl cyclase 1 in mice

doi: 10.1371/journal.pbio.3002716

Figure Lengend Snippet: ( A, B ) HEK-μR/AC1 cells were transfected with Myc-Gβ1, Myc-Gβ2, or HA-KCTD5 as indicated before AC sensitization protocol was applied to the cell. The expression of transfection was probed with anti-Myc and anti-HA antibody. HEK-μR cells were transfected with HA-KCTD5 ( C, E ) or Flag-AC1 ( D, F ) for 48 h. After transfection, the cells were pretreated with either 1 μm MLN4924 or vehicle for 30 min before being treated with 10 μm morphine or vehicle for another 2 h. Immunoprecipitation was performed to examine the interactions of Gβ with KCTD5 ( C, E ) and with AC1 ( D, F ) using anti-HA, anti-flag, or anti-Gβ antibodies. ( G ) HEK-μR cells were transfected with Scr, CUL3 siRNA, or KCTD5 siRNA. After 48 h transfection, whole cell lysate was probed with anti-CUL3, anti-KCTD2/5/17, or anti-Gβ antibodies. ( H ) HEK-μR cells were transfected with HA-KCTD5 or pcDNA before being treated for 2 h with vehicle or 10 μm morphine. Whole cell lysate was probed with anti-Gβ, anti-Gβ1, anti-Gβ2, or anti-HA antibodies. HEK-μR cells ( I ) or HEK-μR/AC1 ( J ) cells were pretreated with 1 μm MLN4924 or 1 μm bortezomib for 30 min before sensitization protocol was applied. Data are representative of 3 independent experiments. The mean intensity of bands was quantified using Image J and normalized to their corresponding input loadings ( C 1 to F 1 ), or to their corresponding loading controls ( G 1 to H 3 ), n = 3. Comparisons between 2 groups were done using Student’s t test. Comparisons among multiple groups under 1 condition were done using one-way ANOVA and comparisons among multiple groups under 2 different conditions were performed using two-way ANOVA followed by Tukey’s test. Mean ± SEM. The data underlying the graphs shown in the figure can be found in . AC, adenylyl cyclase; KCTD potassium channel tetramerization domain.

Article Snippet: The AAV-KCTD5-P2A-mCherry, Lenti-shRNA (KCTD5)-mCherry, and AAV-shRNA (CUL3)-EGFP viruses were purchased from Obio Technology.

Techniques: Transfection, Expressing, Immunoprecipitation

Journal: PLOS Biology

Article Title: KCTD proteins regulate morphine dependence via heterologous sensitization of adenylyl cyclase 1 in mice

doi: 10.1371/journal.pbio.3002716

Figure Lengend Snippet: HEK-μR/AC1 ( A, B ) or HEK-μR cells ( C, D ) were transfected with scrambled (Scr), siRNA targeting KCTD5 (KCTD5-siRNA), pcDNA vector (pcDNA), or HA-KCTD5 plasmid (KCTD5) as indicated before sensitization protocol was applied to the cell. ( E, F ) The overexpression or siRNA knockdown was probed with anti-myc or anti-CAND1 antibodies, and anti-CUL3 or anti-Gβ antibodies were used to probe CUL3/CUL3 N8 or Gβ protein expression. HEK-μR cells were transfected with ( G ) myc-CUL3 or ( H ) myc-CAND1. After transfection, the cells were treated with 10 μm morphine or vehicle for 2 h before the cell lysis was prepared for co-IP to examine the interaction between CUL3 and CAND1. ( I ) HEK-μR cells were treated with vehicle or 10 μm morphine for 2 h. Whole cell lysis was used to probe CUL3/CUL3 N8 protein with anti-CUL3 antibody. ( J ) HEK-μR cells were transfected with HA-KCTD5 before being pretreated with either 1 μm MLN4924 or vehicle for 30 min and treated with 10 μm morphine or vehicle for another 2 h. The cell lysis was prepared for co-IP to examine the interaction between KCTD5 with CUL3 or Gβ. HEK-μR cells were transfected with HA-KCTD5 in conjugation with pcDNA or myc-CAND1 ( K ), or with Scr or CAND1 siRNA ( L ). The cells were treated with 10 μm morphine for 2 h, and the interaction between KCTD5 and CUL3 was detected by co-IP. Data are representative of 3 independent experiments. The mean intensity of bands was quantified using Image J and normalized to their corresponding loading controls ( E 1 to F 3 , I 1 , L 1 ), or to their corresponding input loadings ( G 1 , H 1 , J 1 to K 1 , L 2 ), n = 3. Comparisons between 2 groups were done using Student’s t test. Comparisons among multiple groups under 1 condition were done using one-way ANOVA and comparisons among multiple groups under 2 different conditions were performed using two-way ANOVA followed by Tukey’s test. Mean ± SEM. The data underlying the graphs shown in the figure can be found in . AC, adenylyl cyclase; CAND1, cullin-associated and neddylation-dissociated 1; KCTD potassium channel tetramerization domain.

Article Snippet: The AAV-KCTD5-P2A-mCherry, Lenti-shRNA (KCTD5)-mCherry, and AAV-shRNA (CUL3)-EGFP viruses were purchased from Obio Technology.

Techniques: Transfection, Plasmid Preparation, Over Expression, Knockdown, Expressing, Lysis, Co-Immunoprecipitation Assay, Conjugation Assay

Journal: PLOS Biology

Article Title: KCTD proteins regulate morphine dependence via heterologous sensitization of adenylyl cyclase 1 in mice

doi: 10.1371/journal.pbio.3002716

Figure Lengend Snippet: ( A ) Schematics of the AAV-CUL3 shRNA. ( B ) Experimental timeline for panels C to G. ( C, D ) Mice withdrawal signs were videotaped and analyzed. The body weight loss, withdrawal score, and CPA score were calculated as described in the methods. ( E ) The PVT tissue of the morphine-dependent mice and controls were dissected, and the AC activity was measured as described in the methods. ( F ) The expression of CUL3 in PVT was examined using anti-CUL3 antibody to check the efficiency of RNAi knockdown. The mean intensity of bands was quantified using Image J and normalized to corresponding loading controls, then one-way ANOVA followed by Tukey’s test was applied, n = 3. ( G ) Representative image for showing the site of injection. Two-way ANOVA followed by Bonferroni’s test was applied to behavioral results, n = 7–11. Mean ± SEM. Scale bar, 1 mm. The data underlying the graphs shown in the figure can be found in . AC, adenylyl cyclase; PVT, paraventricular thalamic nucleus.

Article Snippet: The AAV-KCTD5-P2A-mCherry, Lenti-shRNA (KCTD5)-mCherry, and AAV-shRNA (CUL3)-EGFP viruses were purchased from Obio Technology.

Techniques: shRNA, Activity Assay, Expressing, Knockdown, Injection