Fig 1.
Visualization of bushy cell axons in Math5Cre x Brainbow mice early postnatally.
A, Ventral view of the brainstem of a P8 Math5Cre x Brainbow mouse. The YFP fluorescence of the Brainbow reporter was excited by 470 nm excitation light in a stereomicroscope equipped for fluorescence. The extent of the VCN and of the superior olivary complex (SOC) are outlined; the midline is indicated by the straight dashed line. Note the strongly YFP-positive ventral acoustic stria exiting the VCN, and the lateral lemniscus (star symbols). B, Schematic overview of a coronal section of the mouse auditory brainstem. Bushy cells in the VCN projecting to the ipsilateral LSO and to the contralateral MNTB are indicated. C-E, Confocal images of coronal sections of the auditory brainstem of Math5Cre x Brainbow mice at three ages (C, P0; D, P3; E, P7). The sections were stained with an anti-GFP antibody (green channel; to detect the Cre-dependent YFP and CFP expression driven by the Brainbow reporter mouse; labelled "Math5"), and with an anti-Calbindin antibody as a marker for MNTB neurons (red channel). Note the presence of Math5—positive axons with few collaterals and no large nerve terminals at P0 (C, right); with more numerous collaterals at P3 (D), and with large calyceal nerve terminals at P7 (E, right).
Fig 2.
Modified angle during preparation of coronal hindbrain slices to preserve the VCN—MNTB connectivity.
A, Ventral view of the brainstem. Based on several observations with organotypic slice culturing of hindbrain tissue from newborn mice, we infer that the MNTB nuclei are located more anteriorly than VCN (see also Fig 1A). The dashed line indicates the desired slicing configuration using the 15° rotation. B, Dorsal view of the slicing configuration. Grey line indicates the orientation of the tissue chopper blade. C, D, scheme of type 1 and type 2 organotypic slice cultures that we observed.
Fig 3.
Math5- and Syt2-positive large nerve terminals are found in MNTB but not LSO neurons in organotypic slices.
A, Overview image of a slice culture prepared from a Math5Cre x Brainbow mouse at P0, using a slice angle of 15° during preparation of the coronal slices (see Fig 2). After 6 days in-vitro the slice culture was fixed and stained with an anti GFP-antibody (green channel), an anti-PV antibody (blue channel), and an anti-Syt2 antibody (red channel). B, VCN at a higher magnification. C, Maximal intensity projection image (stack of 60 images; z step 0.5 μm) from within the MNTB area (boxed region in A); the right image shows the anti-Syt2 immunohistochemistry (red channel) in isolation. Large calyx-like terminals, captured on the level of their largest cross-section, are outlined in white. White arrow, example of a nerve terminal positive for Math5, Syt2 and PV; white arrowhead; nerve terminal positive for Syt2 alone; yellow arrow, nerve terminal positive for Syt2 and PV; yellow arrowhead, nerve terminal positive for Math5 alone. D, Chart of the co-localization of the three markers Math5, Syt2, and PV in large calyx-type nerve terminals in the MNTB area resulting from the analysis of the slice culture shown in A—C. The presence, or absence of each fluorescent channel in each nerve terminal (abscissa) is indicated. Note that most Math5—positive nerve terminals express Syt2, and more than half also co-express PV. E, Histogram of nerve terminal size found in the MNTB for this organotypic slice culture (n = 61 large nerve terminals), measured as shown in C. F, Confocal image of the LSO subarea indicated in A with a box. Note the abundant axons which are either Math5—positive, or PV-positive, or stained by both antibodies, as well as numerous small bouton-like nerve terminals, most of which are Syt2-negative. The image on the right shows an inset at higher resolution. The masks drawn over Math5—positive nerve terminals are shown in white. G, Histogram of nerve terminal size found in the LSO of this organotypic slice (n = 906 terminals analyzed in two image stacks as the one shown in F). Note the significantly smaller nerve terminal size in LSO (G) as compared to MNTB (E; p < 0.001).
Fig 4.
Example of a type 2 organotypic culture with preserved ipsi- and contralateral VCN-MNTB circuit.
A, Overview of a type 2 slice prepared with 15° slicing angle from a Math5Cre x Brainbow mouse at P0. The organotypic slice was fixed after 9 days in-vitro and stained with an anti-GFP-antibody (green channel), anti-PV antibody (blue channel), and an anti-Syt2-antibody (red channel). The arrow indicates a midline-crossing axon. B, Maximal intensity projection image (35 images; z step 0.5 μm) of the ipsilateral MNTB, midline region and contralateral MNTB for the GFP channel. Note the presence of Math5—positive axons which elaborate large nerve terminals. C, Reconstruction of n = 5 ipsilateral axons (shown in red, blue, light blue, yellow, and pink), and of a large- diameter axon crossing the midline (green). This latter axon established large calyx—like synapses with n = 15 PV—positive neurons in the contralateral MNTB (see panel F for a more detailed view). Another two Math5—positive crossing axons (blue) did not establish large nerve terminals. D, E, Maximal intensity projection images of all three fluorescent channels (31 images; z step 0.5 μm) of a region of the ipsi- and contralateral MNTB (D and E, respectively; see boxes in panel B). F, The reconstructed contralateral axon seen in C (green) at a higher resolution, and with the approximate positions of the postsynaptic neurons indicated. Note that this axon makes contact with n = 15 PV—positive MNTB principal neurons (filled blue circles). One PV—positive neuron (non-filled blue outline) received a large non Math5—positive nerve terminal from another source (see also panel E, white arrow).
Fig 5.
The BMP-SMAD inhibitor LDN-193189 reduces the size of calyces of Held developing in organotypic culture.
A, B, examples of organotypic cultures prepared at P0 from two Math5Cre x Brainbow littermate mouse pups. The slices were cultured in the presence of DMSO alone (A) or 5 μM LDN-193189 (B), fixed after 8 days in-vitro, and stained with anti-GFP antibody, and anti-Syt2 antibody. A’, B’, high-magnification confocal images on the level of the VCN. Note Math5- positive bushy cells visible under both conditions. A”, B”, confocal images on the level of the MNTB, in the areas indicated by rectangles in A and B. Note the presence of large, Syt2-positive nerve terminals under control conditions (A''), but the conspicuous absence of large nerve terminals under LDN-193189 at the same magnification (B''). The inset in B'' is a higher magnification image of B''. The blue lines in A'' and B'' are the nerve terminals outlines at their widest extension. C, histograms of Syt2- and Math5- positive nerve terminal size of the entire MNTB region of the cultures shown in A and B (n = 68 and 38 nerve terminals for control, and for LDN-193189). D, histogram of nerve terminal size from n = 3 organotypic cultures under each condition. Note the presence of large nerve terminals in the range of ~ 5–50 μm2, which are largely missing in slices cultured in the presence of LDN-193189. E, Same data as in D, shown as cumulative histograms for each culture. Short- versus long dashed lines show results from type 1—and type 2 cultures, respectively; the fat black and red lines represent the average cumulative histograms across all cultures (n = 3 each). Note the clear shift towards smaller nerve terminal size for cultures made in the presence of LDN-193189. F, Summary plot of the median nerve terminal size analyzed in individual organotypic cultures under each condition. The median nerve terminal size was significantly smaller in the LDN-193189 group as compared to the control group (p = 0.0019). Results from type 1 and type 2 cultures are shown by different symbols as indicated.