brightfield epifluorescent macroscope  (Evident Corporation)

Journal: eLife

Article Title: Synapse-specific opioid modulation of thalamo-cortico-striatal circuits

doi: 10.7554/elife.45146

Figure Lengend Snippet: Figure 1. Mu-opioid agonists suppress thalamic but not cortical inputs to single MSNs in the striatum. (a) Schematic (upper panel) and an acute mouse brain slice example (lower panel) of viral injection design and the axonal projections to the striatum, respectively. Overlaid brightfield and epifluorescent images showing the injection site of Chrimson (left image, red) in the ACC, and CsChR (right image, cyan) in the MThal, and convergent axonal projections from both injections to the DMS (center image). (b) Schematic (upper panel) and representative recordings (lower panel) for optical excitation. (c) Example oEPSCs of individual MSNs evoked by 625 nm (from the ACC, upper traces), and by 470 nm (from the MThal, lower traces) light pulses. The MOR (orange label) agonist DAMGO (1 mM) was perfused followed by the MOR antagonist CTAP (1 mM). Following CTAP, the DOR (teal label) agonist DPDPE (1 mM) was perfused followed by the moderately-selective DOR antagonist naltrindole (0.3 mM). Red bars: 3 ms of 625 nm light stimulation; blue bars: 1 ms of 470 nm light stimulation. (d) Summary data of dual wavelength excitation of the ACC and MThal input oEPSCs recorded from single MSNs. Data are plotted as the percentage of baseline current following exposure to DAMGO or DPDPE for inputs from the ACC and MThal (N = 5, n = 8, Linear mixed model: 3-way interaction, opioid type (mu vs. delta opioid) x input source (ACC vs. MThal) x drug condition (baseline vs. agonist vs. antagonist), F (4,8)=2.938, p=0.091; MThalbaseline x

Article Snippet: Injection sites were imaged on a brightfield epifluorescent macroscope (Olympus MVX10) using identical settings, and uniform thresholds were established using fluorescent values from the top 90% brightest pixels (ImageJ).

Techniques: Slice Preparation, Injection

Journal: eLife

Article Title: Synapse-specific opioid modulation of thalamo-cortico-striatal circuits

doi: 10.7554/elife.45146

Figure Lengend Snippet: Figure 2. Mu-opioid agonists selectively suppress thalamic inputs from vGluT2-positive thalamic neurons. (a) Example overlaid brightfield and epifluorescent images showing Cre-dependent expression of ChR2(H134R)-EYFP (cyan) in the MThal (left panel) and axonal projections into the DMS (right panel) following injection of AAV-DIO-ChR2(H134R)-EYFP in the MThal of Slc17a6-Cre mice, which express Cre-recombinase in vGluT2-positive cells. (b) Experimental schematic showing optical stimulation of glutamate inputs in the DMS (left panel). Representative traces of oEPSCs showing effects of MOR agonist DAMGO (1 mM, middle panel, orange) and antagonist naloxone (NLX, 1 mM, middle panel, black), and the DOR agonist DPDPE (1 mM, right panel, teal) and DOR antagonist naltrindole (0.3 mM, right panel, black). Blue bars: 1 ms of 470 nm light stimulation. (c) Summary data of oEPSCs showing effects of MOR agonist DAMGO and antagonist CTAP or NLX (1 mM), and the DOR agonist DPDPE and DOR antagonist naltrindole. DAMGO/(CTAP/NLX): N = 3, n = 6, SM = 9.33, p<0.01; DPDPE/naltrindole, N = 3, n = 5, SM = 0, p=1.0. Skillings-Mack test followed by paired Wilcoxon signed-ranks test post-hoc analysis. Mean ± standard error of the mean. *p0.05; SM: Skillings-Mack statistic; Str: striatum. DOI: https://doi.org/10.7554/eLife.45146.005

Article Snippet: Injection sites were imaged on a brightfield epifluorescent macroscope (Olympus MVX10) using identical settings, and uniform thresholds were established using fluorescent values from the top 90% brightest pixels (ImageJ).

Techniques: Expressing, Injection

Journal: bioRxiv

Article Title: Synapse-specific Opioid Modulation of Thalamo-cortico-striatal Circuits

doi: 10.1101/525782

Figure Lengend Snippet: (A) Schematic of thalamo-cortico-striatal circuits and putative expression pattern of MORs (orange) and DORs (teal) ( , ). (B) Schematic and example traces of electrically-evoked EPSCs from a MSN in the DMS. ME: non-selective opioid agonist [Met 5 ]-enkephalin (3 μM). (C) Summary data of opioid inhibition of EPSCs, as shown in panel B, plotted as a percent of peak current in the drug condition relative to the baseline. ME: N = 6, n = 11; wash: N = 3, n = 6, SM = 9.712, p < 0.01; baseline vs. ME: W(11) = 1, p < 0.01; baseline vs. wash: W(6) = 1, p = 0.063; NBQX: N = 4, n = 5; baseline vs. NBQX: W(5) = 0, p < 0.05. (D) Independent activation of CsChR and Chrimson using two-wavelength optical excitation. CsChR (blue): virus expressing CsChR injected into the MThal; Chrimson (red): virus expressing Chrimson injected into the ACC. Blue bars: optical excitation with 470 nm light (1 ms, 0.5 mW); red bars: 625 nm light (3 ms, 1.4 mW). (E) Quantification of spectral selectivity of CsChR, ChR2(H134R), and Chrimson plotted as peak optically-evoked EPSC (oEPSC) amplitude following 470 nm (blue) or 625 nm (red) LED optical excitation (CsChR: N = 2, n = 5, I 625nm :I 470nm = −.0002 ± .0081, paired t-test p < 0.01; ChR2 (H134R): N = 4, n = 10, I 625nm :I 470nm = 0.0009 ± .002, paired t-test p < 0.01: Chrimson: N = 5, n = 8, I 470nm :I 625nm = 0.0423 ± 0.014, paired t-test p < 0.05). (F) Overlaid brightfield and epifluorescent images of coronal brain slices showing expression of CsChR in the MThal and axonal projections to the DMS (blue), and expression of Chrimson (red) in the PFC and axonal projections to the DMS (red). (G) Two-wavelength optical excitation elicited oEPSCs in response to 470 and 625 nm light in single MSNs when CsChR was injected into the MThal and Chrimson was injected into the PFC. (H) Summary data of DAMGO (1 μM, orange) and DPDPE (1 μM, teal) effects on oEPSCs elicited from the MThal (blue) and PFC (red) using two-wavelength optical excitation protocol. Linear mixed model: 3-way interaction; opioid type (mu vs. delta opioid) × source (PFC vs. MThal) × condition (baseline vs. agonist vs. antagonist): F(4,7) = 2.171, p = 0.174, N = 5, n = 7; DAMGO: I MThal ; 22.2 ± − 3.6 % of baseline, z = 3.497, p < 0.001, I PFC ; 93.9 ± 5.6 %, z = 0.052, p = 0.958; DPDPE: I MThal ; 96.4 ± 3.9 % of baseline, z = 0.087, p = 0.931; I PFC : 81.6 ±3.9 %, z = 0.672, p = 0.502. MThal baseline × DAMGO vs. ACC baseline × DAMGO ; z = −2.436, p < 0.05. ( I) Example traces for summary data shown in (H). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Mean ± standard error of the mean. N: number of animals; n: number of recorded cells; Str: striatum; PL: prelimbic cortex.

Article Snippet: Injection sites were imaged on a brightfield epifluorescent macroscope (Olympus MVX10) using identical settings, and uniform thresholds were established using fluorescent values from the top 90% brightest pixels (ImageJ).

Techniques: Expressing, Inhibition, Activation Assay, Injection

Journal: bioRxiv

Article Title: Synapse-specific Opioid Modulation of Thalamo-cortico-striatal Circuits

doi: 10.1101/525782

Figure Lengend Snippet: ( a )Schematic (upper panel) and an acute mouse brain slice example (lower panel) of viral injection design and the axonal projections to the striatum. Overlaid brightfield and epifluorescent images showing the injection site of Chrimson (left image, red) in the ACC, and CsChR (right image, cyan) in the MThal, and convergent axonal projections from both injections to the DMS (center image). ( b ) Schematic (upper panel) and representative recordings (lower panel) for optical excitation. ( c ) Example oEPSCs of individual MSNs evoked by 625 nm (from the ACC, upper traces), and by 470 nm (from the MThal, lower traces) light pulses. The MOR (orange label) agonist DAMGO (1 μM) was perfused followed by the MOR antagonist CTAP (1 μM). Following CTAP, the DOR (teal label) agonist DPDPE (1 μM) was perfused followed by the moderately-selective DOR antagonist naltrindole (0.3 μM). Red bars: 3 ms of 625 nm light stimulation; blue bars: 1 ms of 470 nm light stimulation. ( d ) Summary data of dual wavelength excitation of the ACC and MThal input oEPSCs recorded from single MSNs. Data are plotted as the percentage of baseline current following exposure to DAMGO or DPDPE for inputs from the ACC and MThal (N = 5, n = 8, Linear mixed model: 3-way interaction, opioid type (mu vs. delta opioid) × input source (ACC vs. MThal) × drug condition (baseline vs. agonist vs. antagonist), F (4,8) = 2.938, p = 0.091; MThal baseline × DAMGO : z = 4.738, p < 0.001; MThal baseline × DAMGO vs. ACC baseline × DAMGO ; z = −3.026, p < 0.01). Mean ± standard error of the mean. Str: striatum. The following figure supplement is available for : Figure supplement 1 . Mu-opioid agonists suppress thalamic, but not cortical inputs. Figure supplement 2 . Single channelrhodopsin injections reproduced specific effect of mu-opioid-mediated inhibition of thalamic but not cortical inputs.

Article Snippet: Injection sites were imaged on a brightfield epifluorescent macroscope (Olympus MVX10) using identical settings, and uniform thresholds were established using fluorescent values from the top 90% brightest pixels (ImageJ).

Techniques: Slice Preparation, Injection, Inhibition

Journal: bioRxiv

Article Title: Synapse-specific Opioid Modulation of Thalamo-cortico-striatal Circuits

doi: 10.1101/525782

Figure Lengend Snippet: ( a ) Example overlaid brightfield and epifluorescent images showing Cre-dependent expression of ChR2(H134R)-EYFP (cyan) in the MThal (left panel) and axonal projections into the DMS (right panel) following injection of AAV2-DIO-ChR2(H134R)-EYFP in the MThal of vGluT2-Cre mice. ( b ) Experimental schematic showing optical stimulation of glutamate inputs in the DMS (left panel). Representative traces of oEPSCs showing effects of MOR agonist DAMGO (1μM, middle panel, orange) and antagonist naloxone (NLX, 1 μM, middle panel, black), and the DOR agonist DPDPE (1 μM, right panel, teal) and DOR antagonist naltrindole (0.3 μM, right panel, black). ( c ) Summary data of oEPSCs showing effects of MOR agonist DAMGO and antagonist CTAP or NLX (1 μM), and the DOR agonist DPDPE and DOR antagonist naltrindole. DAMGO/(CTAP/NLX): N = 3, n = 6, SM = 9.33, p < 0.01; DPDPE/naltrindole, N = 3, n = 5, SM = 0, p = 1.0. Skillings-Mack test followed by paired Wilcoxon signed-ranks test post-hoc analysis. Mean ± standard error of the mean. * p ≤ 0.05; SM : Skillings-Mack statistic; Str: striatum.

Article Snippet: Injection sites were imaged on a brightfield epifluorescent macroscope (Olympus MVX10) using identical settings, and uniform thresholds were established using fluorescent values from the top 90% brightest pixels (ImageJ).

Techniques: Expressing, Injection