Article Figures & Data
Figures
- Figure 1.
BDNFLacZ expression in the substantia nigra. BDNFLacZ/+ embryos, neonates, and postnatal mice demonstrate the presence of BDNF within the substantia nigra pars compacta. The black box indicates the location of the SNC. Coronal sections (40 μm) of different ages, starting at E16.5 and ending at 1 year (1yr) (indicated in the top right portion of each panel), were stained with X-gal, counterstained with Neutral Red, and then photographed at 20× magnification. Scale bar, 1 mm.
- Figure 2.
Expression of Cre in midbrain BDNF-expressing neurons. A, X-gal stain of 40 μm coronal sections (counterstained with Neutral Red) from the midbrain-hindbrain of P21 Wnt1-Cre;R26R mice. The dark blue stain indicates areas of Cre activity. Outside the MHB, there are only scattered CNS cells that undergo recombination. B, Immunofluorescent double labeling using antibodies to tyrosine hydroxylase and β-gal of 10 μm cryostat sections from the anterior portion of the SNC of a BDNFLacZ/+ mouse documents expression in multiple SNC cells. Images were photographed at 400× magnification. Lateral is to the left of the image, and the midline is to the right of the image. C, Effectiveness of Wnt1-Cre at excising BDNF from DA neurons. Images of sections are processed as in B of the SNC of a Wnt1-Cre; BDNFlox/+ mouse.
- Figure 3.
Reduced BDNF protein in MHB leads to motor deficits and reduced striatal TH in Wnt-BDNFKO mice. A, BDNF protein was quantified by ELISA for MHB and expressed as nanograms of BDNF protein per gram of wet tissue. These extracts were obtained from P7 and P9 mice and pooled to increase the n (for P7/P9, n = 5 for Wnt-BDNFKO and wild-type; n = 4 for heterozygous; **p < 0.01; ***p < 0.001; one-way ANOVA with a Newman-Keuls post hoc test). B, Performance of 4- to 5-week-old mice on an accelerating rotarod. Each mouse had three trials each day, which were averaged for the day (Wnt-BDNFKO, n = 7; heterozygous, n = 16; wild type, n = 6; **p < 0.01 heterozygous vs wild type for day 1 only; ***p < 0.001 Wnt-BDNFKO vs heterozygous and wild type all 3 d). C, Wnt-BDNFKO, heterozygous, and wild-type mice at 1, 2, and 4 months of age were suspended by their tails for 1 min. Clasping was defined as the balling up of one or both of the hindlimb paws, accompanied by pulling them into the body and movement of the limbs toward the midline. Four-month-old wild-type, heterozygous, and Wnt-BDNFKO mice undergoing tail suspension are shown. D, Western blot analysis of TH levels in total striatal protein from P35 Wnt-BDNFKO and control mice (n = 4 for Wnt-BDNFKO and wild type; n = 3 for heterozygous; *p < 0.05; one-way ANOVA with a Newman-Keuls post hoc test). White bar, Wnt-BDNFKO; gray bar, heterozygous; black bar, wild type.
- Figure 4.
Wnt-BDNFKO mice have reduced TH expression in the SNC but not in the VTA. A, The extent of the SNC appears reduced in P21Wnt-BDNFKO mice compared with controls, and the number of TH fibers also seems to be reduced. The most anterior section is at the top, and the most posterior section is at the bottom. Coronal cryostat sections (40 μm) taken at 240 μm intervals stained for TH and counterstained with cresyl violet are shown. B, Optical fractionator estimates of the number of DA neurons in both hemispheres of the SNC of Wnt-BDNFKO mice and controls at P0, P21, and P120 (P0, n = 3/genotype; P21, n = 9, 8, and 9 for Wnt-BDNFKO, heterozygous, and wild type, respectively; P120, n = 3/genotype; *p < 0.05; **p < 0.01; one-way ANOVA with a Newman-Keuls posthoc test). C, There is no significant reduction in the total number of TH-positive cells of both hemispheres of the VTA of Wnt-BDNFKO mice compared with controls (P21, n = 11, 7, and 8 for Wnt-BDNFKO, heterozygous, and wild type, respectively, p = 0.6454; P120, n = 3/genotype, p = 0.8170). White bars, Wnt-BDNFKO; gray bars, heterozygous; black bars, wild type.
- Figure 5.
NeuN-positive neuron loss in the SNC of Wnt-BDNFKO mice was not detected. A, NeuN-positive cells were counted in the SNC, defined by outlining the TH-stained population of neurons on adjacent sections, and estimates were obtained for total numbers of neurons in the SNC at P21 (n = 4 for Wnt-BDNFKO and heterozygous; n = 3 for wild type; p = 0.5093). B, Stereological estimates of calbindin-positive cell population in the SNC. C, Estimates of calretinin-positive cell population in the SNC. For both experiments, n = 3/genotype. No significant difference was found between genotypes by one-way ANOVA. White bars, Wnt-BDNFKO; gray bars, heterozygous; black bars, wild type.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supplemental Figure 1 Neuronal processes in the dorsal lateral striatum Wnt-BDNFKO and heterozygous mice appear to less dense fiber innervation in the dorsal lateral striatum than wildtype. Images were taken from sagittal sections of P21 mice using a 63X objective oil immersion lens and polarizing filter. Scale bar equals 50�m.
- supplemental material - Supplemental Figure 2 Calbindin expression in wild-type and mutant mice Comparison of images of the SNC from mutant and control genotypes shows the size of the substantia nigra pars reticulata appears reduced and the intensity of calbindin expression lessened in mutants compared to controls. The most anterior section is on the left and the most posterior on the right. Midline of a section is on the right of the image. Sections in upper panels stained for TH are adjacent and posterior to lower panel sections stained for calbindin.
- supplemental material - Supplemental Figure 3 Calretinin expression in wild-type and mutant mice Although there was no significant reduction in calretinin(CR)-expressing cells in the SNC, comparison of CR expression between genotypes as shown in the last two columns of images suggests a possible reduction in the number of CR-positive cells within the substantia nigra pars reticulata. The most anterior section is on the left and the most posterior on the right. Midline of a section is on the right of the image. Sections in upper panels stained for TH are adjacent and posterior to lower panel sections stained for CR.
