To examine the ultrastucture of the developing dLGN, a total of eight C57/BL6 mice, at ages P7 (n = 2), P14 (n = 2), P21 (n = 2), and adult (9 months, n = 2) were used. Mice were first deeply anesthetized with isoflurane, then given an intraperitoneal injection of avertin (2.5%, 0.5–1 ml), and perfused through the heart with 2% paraformaldehyde and 2% glutaraldehyde in 0.1Mphosphate buffer. The brains were cut into 50–100-µm-thick coronal sections by using a Vibratome (Leica VT100E). Selected sections were postfixed in 2% osmium tetroxide and then dehydrated in an ethyl alcohol series and embedded in Durcupan resin. Because the mouse dLGN is less than 1 mm2 in coronal sections, we mounted the entire dLGN (at its largest extent, approximately 2.3 mm posterior to Bregma in the adult) on resin blocks for ultrastructural analysis. Ultrathin sections (on average 70 nm in thickness) were cut and every fifth section was collected on Formvar-coated nickel slot grids. Every fourth section in the series was stained to reveal the presence of GABA, by using previously published postembedding immunocytochemical techniques (Li et al., 2003 (link); Bickford et al., 2008 (link)) and a polyclonal, affinity-purified rabbit anti-GABA primary antibody (cat. no. A2052, Sigma, St. Louis, MO) diluted 1:2,000, and a goat anti-rabbit IgG antibody conjugated to 15-nm colloidal gold particles diluted 1:25 (British BioCell International, Cardiff, UK).
The immunogen used to produce the GABA antibody was GABA-bound to bovine serum albumin (BSA). The GABA antibody shows positive binding with GABA and GABA-keyhole limpet hemocyanin, but not BSA, in dot blot assays (Sigma product information). In mouse tissue, the GABA antibody stains neurons in the thalamic reticular nucleus and a subset of neurons in the dorsal thalamus. This labeling pattern is consistent with other GABAergic markers used in a variety of species to visualize interneurons. (Houser et al., 1980 (link); Hendrickson et al., 1983 (link); Oertel et al., 1983 (link); Fitzpatrick et al., 1984 (link); Montero and Singer, 1985 (link); Montero and Zempel, 1986 (link); Rinvik et al., 1987 (link); De Biasi et al., 1997 (link); Arcelli et al., 1997 (link); Wang et al., 2001 (link)).
The sections were subsequently stained with uranyl acetate and examined by using a Philips CM10 electron microscope. Images of each synaptic contact (identified by an accumulation of vesicles adjacent to a synaptic cleft) encountered within the examined sections were collected by using a digitizing camera (SIA-7C; SIA, Duluth, GA), or photographic plates that were subsequently scanned and digitized (SprintScan 45i; Polaroid, Waltham, MA). As described in detail in the Results section, each presynaptic and postsynaptic profile was categorized based on a variety of ultrastructural features, as well as the density of gold particles overlying them. Profile areas were measured from digital images of single sections by using Sigma Scan Software (SPSS, Chicago, IL). Images were imported into Adobe Photoshop software (San Jose, CA), where the brightness and contrast could be adjusted.
The immunogen used to produce the GABA antibody was GABA-bound to bovine serum albumin (BSA). The GABA antibody shows positive binding with GABA and GABA-keyhole limpet hemocyanin, but not BSA, in dot blot assays (Sigma product information). In mouse tissue, the GABA antibody stains neurons in the thalamic reticular nucleus and a subset of neurons in the dorsal thalamus. This labeling pattern is consistent with other GABAergic markers used in a variety of species to visualize interneurons. (Houser et al., 1980 (link); Hendrickson et al., 1983 (link); Oertel et al., 1983 (link); Fitzpatrick et al., 1984 (link); Montero and Singer, 1985 (link); Montero and Zempel, 1986 (link); Rinvik et al., 1987 (link); De Biasi et al., 1997 (link); Arcelli et al., 1997 (link); Wang et al., 2001 (link)).
The sections were subsequently stained with uranyl acetate and examined by using a Philips CM10 electron microscope. Images of each synaptic contact (identified by an accumulation of vesicles adjacent to a synaptic cleft) encountered within the examined sections were collected by using a digitizing camera (SIA-7C; SIA, Duluth, GA), or photographic plates that were subsequently scanned and digitized (SprintScan 45i; Polaroid, Waltham, MA). As described in detail in the Results section, each presynaptic and postsynaptic profile was categorized based on a variety of ultrastructural features, as well as the density of gold particles overlying them. Profile areas were measured from digital images of single sections by using Sigma Scan Software (SPSS, Chicago, IL). Images were imported into Adobe Photoshop software (San Jose, CA), where the brightness and contrast could be adjusted.
