1. Volume 21, Issue 2, Pages 335-345 (August 1998)
Point Mutation in trkB Causes Loss of NT4-Dependent Neurons without Major Effects on Diverse BDNF Responses 
Liliana Minichiello, Franca Casagranda, Rosa Soler Tatche, Cheryl L Stucky, Antonio Postigo, Gary R Lewin, Alun M Davies, Rüdiger Klein 
Neuron 
Volume 21, Issue 2, Pages (August 1998)
DOI: /S (00)

2. Figure 1
Generation of the trkBshc Allele by Homologous Recombination in ES Cells
(A) Schematic diagram of the targeted trkBshc allele. Vertical black box represents the juxtamembrane exon of trkB; the mutated shc binding site (NPQF515) is indicated. The PGK-neo cassette (neo) is indicated as an open box and is flanked by FRT sites (indicated as arrow heads). Cleavage sites for restriction endonuclease ScaI are indicated (S). Sizes of ScaI genomic fragments detected with probes 1 or 2 are indicated. The Y to F mutation destroys the ScaI site in the mutant allele.
(B) Southern blot analysis of ScaI digests of genomic DNA isolated from double selected ES cell clones (1–6) and R1 ES cell control DNA. The 2.3 kb HindIII/XbaI DNA probe 1 (see A) identifies a 4.0 kb ScaI DNA fragment in the wild-type allele and a 6.3 kb ScaI fragment in the mutant allele.
(C) Removal of the neo cassette by Flp transgene-mediated excision (see Experimental Procedures). The 2.0 kb HindIII DNA probe 2 (see A) identifies a diagnostic 6.4 kb ScaI fragment of the targeted allele after neo excision, because one ScaI site 3′ of the neo cassette is removed. The neo+ targeted allele and the wild-type allele produce 2.3 kb and 2.4 kb ScaI fragments, respectively, which cannot be resolved in the gel. trkBshc/+;FLP− and +/+;FLP+ mice only show the 2.3/2.4 kb ScaI fragments diagnostic of the unrecombined alleles.
Neuron  , DOI: ( /S (00) )

3. Figure 2 Signaling by Mutant TrkB Receptors in Cortical Neurons
(A) Autophosphorylation and expression of TrkB in cortical neurons. Cortical neurons derived from wild-type (+/+) or mutant mice (trkBshc/shc) were either left untreated (−) or pulse-stimulated with BDNF or NT4 for 10 min. Cell lysates were immunoprecipitated with anti-Trk antibodies followed by immunoblotting with anti-phosphotyrosine (anti-PTyr) antibodies. The blot was reprobed with anti-TrkB antibodies to visualize the levels of TrkB protein.
(B) Lack of Shc binding to mutant TrkB receptors. Wild-type, trkBshc/shc mutant cortical neurons and NIH3T3 cells expressing TrkB (3T3-TrkB) were stimulated with BDNF as above and lysed. Cell lysates were incubated with either GST alone (GST) or with the PTB domain of Shc (GST-ShcB) previously coupled to glutathion beads. After immunoblotting with anti-pan Trk antibodies, in +/+ but not in trkBshc/shc mutant neurons, a specific band appears that migrates at the same position as TrkB expressed in NIH3T3 cells. The same band is detected with anti-PTyr antibodies (bottom).
(C) Tyrosine phosphorylation of Shc proteins. Cortical neurons were treated with BDNF or NT4 as in (A). Cell lysates were immunoprecipitated with anti-Shc antibodies followed by immunoblotting with anti-PTyr antibodies. No tyrosine phosphorylation of Shc proteins was observed in cortical neurons derived from trkBshc/shc mutants. Blot was reprobed with anti-Shc antibodies.
(D) PLCγ1 binding to mutant TrkB receptors. Cortical neurons and 3T3-TrkB cells were stimulated with BDNF, lysed, and incubated with the C-terminal SH2 domain of PLCγ1 (GST- PLCγ1) coupled to glutathion beads. Immunoblotting with anti-TrkB antibodies revealed binding of TrkB receptors in trkBshc/shc mutant neurons.
(E) Tyrosine phosphorylation of PLCγ1. Cortical neurons were treated with BDNF or NT4 as above. Cell lysates were immunoprecipitated with anti-PLCγ1 antibodies followed by immunoblotting with anti-PTyr antibodies. Normal tyrosine phosphorylation of PLCγ1 was observed in trkBshc/shc mutant neurons. Blot was reprobed with anti-PLCγ1 antibodies.
Neuron  , DOI: ( /S (00) )

4. Figure 3
Partial Loss of BDNF-Dependent Vestibular Neurons in trkBshc/shc Mutant Mice
(A–C) Cresyl violet–stained coronal sections through the central portion of a vestibular ganglion of P7 wild-type (control), trkBshc/shc, and trkB−/− mutant mice. Magnification, 200×.
(D) Time course of vestibular neuron loss in trkBshc/shc (5/5) and trkB−/− mutant mice. For each time point and genotype, between three and five mice (six to ten ganglia) were analyzed.
Neuron  , DOI: ( /S (00) )

5. Figure 4
Complete Loss of NT4-Dependent D-Hair Receptors but No Change in Mechanotransduction of BDNF-Dependent SA Fibers
(A) By sampling many single fibers (n = 157) in trkBshc/shc and trkBshc/+ mice (from four to seven mice per group) from the saphenous nerve responding to mechanical stimulation of the skin, it is possible to divide those with myelinated axons into four separate categories: D-hair receptors, A-fiber mechanonociceptors (AM), rapidly-adapting (RA), and slowly-adapting (SA). Among Aδ-fibers (slower conducting), about 35% of recorded neurons are sensitive D-hair receptors, and the remaining 65% are nociceptors (AM). The proportions encountered in trkBshc/+ were found to be identical to that in wild-type mice (left). Among Aβ-fibers, around 55% are RA and 45% SA in wild-type mice (all wild-type data taken from Koltzenburg et al. 1997). The proportion of RA and SA receptor types encountered was the same in all genotypes studied (right). However, in trkBshc/shc mice, almost no D-hairs were encountered in the sample (A). This difference for both genotypes was highly significant (p < Fischers exact test).
(B) Numbers of axons counted in the saphenous nerve. The mean number from 4 to 5 nerves is shown together with their standard deviations (** indicates P < unpaired t-test). A significant 30% loss of axons of was seen in nerves from trkBshc/shc mice.
(C) Cumulative sum distribution of the minimum thresholds for activation of SA fibers. SA fibers from trkBshc/+ mice and a group of nine SA fibers recorded from wild-type mice, in the same series of experiments, had identical median thresholds (2.0 mN), and the data points overlap completely on the cumulative sum plot. For this reason, the data is omitted from the plot for clarity. When SA fibers were analyzed from trkBshc/shc mice, the distribution of thresholds was identical to those from trkBshc/+ mice (open circles and closed squares, respectively).
Neuron  , DOI: ( /S (00) )

6. Figure 5
Reduced MAPK Activation and Phosphorylation in trkBshc/shc Neurons
(A) Reduced ERK2 kinase activity in mutant neurons. Cortical neurons derived from +/+ or trkBshc/shc mutant embryos were stimulated with either BDNF or NT4 (each 5 ng/ml) for the indicated time points and lysed. Cell lysates were immunoprecipitated with anti-ERK2 antibodies and the immunoprecipitates subjected to in vitro kinase activity in the presence of myelin basic protein (MBP) as exogenous substrate. As negative control, only protein A-sepharose beads were used for immunoprecipitation from cell lysates of BDNF-stimulated cells.
(B) Reduced ERK1 and ERK2 phosphorylation in mutant neurons. Cell lysates were immunoblotted with antibodies against activated ERKs. Note that ERK phosphorylation in trkBshc/shc neurons after NT4 stimulation is weaker and more transient than with BDNF, although both ligands are equally potent in wild-type neurons. Blot was reprobed with antibodies against α-tubulin to control for the amount of lysate loaded in the gel.
Neuron  , DOI: ( /S (00) )

7. Figure 6
In Vitro Survival of Sensory Neurons Derived from trkBshc/shc Mutant Embryos
(A) Dose responses of trigeminal neurons from E10.5 trkBshc/+ (S/+) and trkBshc/shc (S/S) embryos to a range of concentrations of BDNF and NT4. (B) Dose responses of nodose neurons from E12.5 trkBshc/+ and trkBshc/shc embryos to a range of concentrations of BDNF and NT4. In these experiments, separate cultures were set up from each embryo of litters resulting from matings between trkBshc/+ and trkBshc/shc parents. Percent survival was assessed after virtually all neurons had died in control cultures (24 hr for trigeminal neurons and 48 hr for nodose neurons). The mean and standard error of data from six trkBshc/shc embryos and from three or four trkBshc/+ embryos are represented by each data point.
Neuron  , DOI: ( /S (00) )

8. Figure 7
BDNF-Dependent Differentiation of CNS Neurons and Foliation of the Cerebellum Are Normal in trkBshc/shc Mutant Mice
(A–D) Parvalbumin immunoreactivity in CA2 and CA3 regions of P17 hippocampus of wild-type (control) (A and C) and trkBshc/shc mice (B and D). Identical expression levels and staining patterns were observed in P17 and adult brains of control and trkBshc/shc mice (n = 3, of all genotypes and time points). Magnifications, (A and B), 200×; (E and F), 1000×.
(E–H) Cresyl violet–stained sagittal sections through the cerebellum of P12 wild-type (wt) (E) and trkB−/− mice (F), and P17 trkBshc/+ (G) and trkBshc/shc mice (H). Note that certain fissures are reduced in length in trkB−/− mice (F), but not in trkBshc/shc mice (H). Abbreviations: itc, intercrural fissure; ppy, prepyramidal fissure; uvu, uvular fissure. Magnification, 25×.
(I–L) Calbindin immunostaining of cerebellar Purkinje cell dendritic trees derived from the indicated wild-type (wt) and mutant mice. Note the reduced size of the dendritic tree of trkB−/− compared to wt mice, while no such reduction is observed in trkBshc/shc mutant mice. Magnification, 400×.
Neuron  , DOI: ( /S (00) )