Journal: bioRxiv

Article Title: Synaptic properties of layer 6 auditory corticothalamic inputs in normal hearing and noise-induced hearing loss

doi: 10.1101/2025.07.08.663770

Figure Lengend Snippet: A, Left: Schematic illustration of stereotaxic injections of retrobeads to label thalamocortical neurons (MGB neurons, red) and viral vectors (AAVs) for expression of ChR2 in corticothalamic (L6 CT) inputs in the MGB (green). All in Ntsr1-Cre mice. Right: Schematic illustration of whole-cell recordings, including CT ChR2-expressing terminals projecting to MGB principal neurons (thalamocortical neurons), which were labelled retrogradely with red retrobeads injection in AC. Photo-evoked EPSCs were recorded from MGB neurons in response to blue light stimulation. B, Representative MGB image of coronal brain slice from Ntsr1-Cre mice showing distinct MGB subdivisions based on calbindin immunostaining (green). The dorsal (MGd) and medial (MGm) MGB are strongly immunoreactive to calbindin staining. The ventral (MGv) MGB shows little immunoreactivity to calbindin staining. D: dorsal; L: lateral C, Representative image of an acute coronal brain slice that contains MGd, MGv, and MGm in bright-field (4x). Scale bar is the same as in B. D, Representative MGB image (4x) of coronal section illustrating ChR2-expressing CT projections in the MGB. Scale bar is the same as in B. E, Representative image (4x) red labelled MGB neurons, by retrobeads injected in the AC. Scale bar is the same as in B.

Article Snippet: Slices were then incubated in blocking buffer containing primary antibodies against calbindin (1:1000, Mouse IgG1 anti-calbindin, Swant CB300) for 72h at 4°C.

Techniques: Expressing, Injection, Slice Preparation, Immunostaining, Staining

Journal: bioRxiv

Article Title: Synaptic properties of layer 6 auditory corticothalamic inputs in normal hearing and noise-induced hearing loss

doi: 10.1101/2025.07.08.663770

Figure Lengend Snippet: CT→MGv synapses are stronger than to CT→MGd synapses, due to higher n in CT→MGv synapses. A, Average light-evoked EPSC amplitude in CT→MGd and CT→MGv synapses (left) and representative traces (right). P = 0.0002 (Mann-Whitney test, CT→MGd: 20 cells; CT→MGv: 30 cells). Data for CT→MGd and CT→MGv are pooled from the 1d SE and 10d SE of CT→MGd and CT→MGv ( A,F) recordings, respectively. B, CT→MGB EPSC traces from the same slice. Recorded neurons are numbered from 1 to 5, in order from MGd to MGv. C, Average light-evoked quantal EPSCs (Sr 2+ -mEPSCs) in CT→MGd and CT→MGv synapses (left bottom), and representative average traces (left top). Right: The top right trace (black) is a control trace from Ca 2+ -containing ACSF; the middle and bottom traces are from Sr 2+ -containing ACSF. The arrowhead indicates the onset of light stimulus. The solid line indicates the 400 ms time window beginning 100ms after light stimulus that was used to analyze the amplitude of Sr 2+ -mEPSC. P = 0.1296 (unpaired t test, CT→MGd: 17 cells; CT→MGv: 16 cells). Data for CT→MGd and CT→MGv are pooled from the 1d SE and 10d SE of CT→MGd and CT→MGv recordings, respectively. D. Average paired pulse ratio (PPR) in CT→MGd and CT→MGv synapses (left) and representative traces (right). P = 0.4006 (Mann-Whitney test, CT→MGd: 18 cells; CT→MGv: 15 cells). Data for CT→MGd and CT→MGv are pooled from the 1d SE and 10d SE of CT→MGd and CT→MGv recordings , respectively E, Average 1/CV 2 in CT→MGd and CT→MGv synapses. P = 0.0324 (Mann-Whitney test, CT→MGd: 20 cells; CT→MGv: 30 cells). Data for CT→MGd and CT→MGv are pooled from the 1d SE and 10d SE of CT→MGd and CT→MGv recordings , respectively. F, Average minimal EPSC amplitude (left) and representative averaged minimal EPSC traces from one cell (right) from CT→MGd (grey) and CT→MGv (black) synapses. P = 0.728 (unpaired t test, CT→MGd: 6 cells; CT→MGv: 8 cells). G, Representative image of coronal brain slice showing calbindin immunostaining (magenta) and ChR2 fluorescence (green). H, Quantification of ChR2 immunofluorescence intensity in the different MGB subdivisions. P = 0.0027 (One-way ANOVA, MGd: 10 slices; MGv: 10 slices; MGm: 10 slices). Data for MGd and MGv are pooled from the 1d SE and 10d SE of MGd and MGv ( ,H,I) respectively. Detailed statistical values are listed in .

Article Snippet: Slices were then incubated in blocking buffer containing primary antibodies against calbindin (1:1000, Mouse IgG1 anti-calbindin, Swant CB300) for 72h at 4°C.

Techniques: MANN-WHITNEY, Control, Slice Preparation, Immunostaining, Fluorescence, Immunofluorescence

Journal: bioRxiv

Article Title: Synaptic properties of layer 6 auditory corticothalamic inputs in normal hearing and noise-induced hearing loss

doi: 10.1101/2025.07.08.663770

Figure Lengend Snippet: Unchanged ChR2 fluorescence intensity of CT terminals in the different MGB subdivisions after NIHL. A-B, Representative images of coronal brain slices showing calbindin immunostaining (magenta) and ChR2 fluorescence (green) from 1d SE (A) and 1 NE (B). C-E, Average ChR2 immunofluorescence intensity in the different MGB subdivisions in 1d SE and 1d NE (C, MGd; D, MGv; E: MGm). C, P = 0.3441 (unpaired t test, 1d SE: 5 slices; 1d NE: 5 slices); D, P = 0.7193 (unpaired t test, 1d SE: 5 slices; 1d NE: 5 slices); E, P = 0.3843 (unpaired t test, 1d SE: 5 slices; 1d NE: 5 slices). F-G, Representative images of coronal brain slices showing calbindin immunostaining (magenta) and ChR2 fluorescence (green) from 10d SE (F) and 10d NE (G). H-J, Average ChR2 immunofluorescence intensity in the different MGB subdivisions at 10d SE and 10d NE (H, MGd; I, MGv; J: MGm). H, P = 0.5064 (unpaired t test, 10d SE: 5 slices; 10d NE: 5 slices); I, P = 0.7387 (unpaired t test, 10d SE: 5 slices; 10d NE: 5 slices); J, P = 0.1008 (unpaired t test, 10d SE: 5 slices; 10d NE: 5 slices). Detailed statistical values are listed in .

Article Snippet: Slices were then incubated in blocking buffer containing primary antibodies against calbindin (1:1000, Mouse IgG1 anti-calbindin, Swant CB300) for 72h at 4°C.

Techniques: Fluorescence, Immunostaining, Immunofluorescence