Figure 1.
The areas of the amygdala sampled and the distributions of ACIII immunopositive cilia in subdivisions of amygdala in WT and Bbs4-/- mice are illustrated.
Figure A shows the subdivisions of amygdala and the coronal plane that was analyzed in this study. Figures B and C are maps documenting distribution of cilia within these regions of WT (B) and Bbs4-/- (C) brains that were acquired at high magnification using Sterioinvestigator image analysis software. Figure D shows the density of cilia ± the SEM within each amygdala subdivision of WT (blue) and Bbs4-/- (gray) animals; asterisks and brackets indicate where statistical analysis demonstrated statistical differences between WT and Bbs4-/- mice. The data for (D) was collected from three WT and five Bbs4-/- mice. High magnification comparisons of the distribution of cilia within comparable regions of each of the amygdala subdivisions are demonstrated in figures E through N. Note the association of cilia with neurons counterstained with neutral red in each region as well as the dramatic reduction in ACIII+ cilia in Bbs4-/- animals. BLA = basolateral nucleus of amygdala; BMa = basomedial amygdala, anterior part; BMp = basomedial amygdala, posterior part; CeA = central nucleus of amygdala with capsular (c), lateral (l) and medial (m) subdivisions; ICM = intercalated cell masses; LA = lateral nucleus of amygdala. Scale bar in figure A = 1 mm, in N = 20 μm; figures E – N are of the same magnification.
Figure 2.
Transmission electron microscopic images of PNC in amygdala of WT (A & B) and Bbs4-/- mice (C & E).
Note the intact basal bodies associated with neurons in WT and Bbs4-/- mice and abnormal cilia starting from basal bodies in C & E compared to healthy PNC with microtubules in A & B. The insert (D) is a higher magnification image of the area incorporated in the rectangle on (C). The yellow highlights the shape of PNC in Bbs4-/- lateral amygdala. The solid arrowheads in D and E point to the ciliary transition zones; open head arrow points to the broken axoneme; red arrow head points to the spherical inclusion bodies. The yellow highlights the shape of PNC in Bbs4-/- lateral amygdala. The data was collected from three WT and five Bbs4-/- mice. BB – basal bodies; c – cilium; N -neuron; Nu – nucleus; arrows point to BB. Scale bars: 0.5 μm for A-E.
Figure 3.
The areas of rostral (dorsal) hippocampus sampled and the distributions of ACIII immunopositive PNC in subdivisions of the hippocampus in WT and Bbs4-/- mice are illustrated.
Figure A shows the subdivisions of hippocampus in coronal plane of chosen in this study for sampling. The boxed areas define the regions in which quantitative data was collected for each area. Figures B and C illustrate the cytoarchitecture of WT (B) and Bbs4-/- (C) hippocampus at this plane of section. Note the reduction in hippocampal mass and the resulting expansion of the lateral ventricle in Bbs4-/- mouse brain. Figures D and E illustrates the density of cilia ± the SEM (D) and length of cilia ± the SEM within each of the analyzed hippocampal subfields of WT (blue) and Bbs4-/- (gray) mice; asterisks and brackets indicate where statistical analysis demonstrated statistical differences between WT and Bbs4-/- mice. The data for (D) and (E) was collected from three WT and five Bbs4-/- mice. High magnification comparisons of the distribution of cilia within these regions of hippocampus are shown in figures F through K. Note the association of cilia with neurons counterstained with neutral red in each region as well as the dramatic reduction in ACIII+ cilia in Bbs4-/- animals. Cornu Ammonis areas: CA1, CA3 and DG (dentate gyrus). Scale bar in figure A = 1 mm, in K = 20 μm and figures F – K are of the same magnification.
Figure 4.
The areas of caudal (ventral) hippocampus sampled and the distributions of ACIII immunopositive PNC in hippocampal subdivisions of WT and Bbs4-/- mice are illustrated.
Figure A illustrates the subdivisions of hippocampus sampled at this caudal coronal plane. The arrows indicate the regions in which quantitative data was collected for each area. Figures B and C illustrate the distribution of cilia within ventral subiculum of WT (B) and Bbs4-/- (C) mice in this plane of section. Note the association of cilia with neurons counterstained with neutral red in each region as well as the dramatic reduction in ACIII+ cilia in Bbs4-/- animals. Figures D and E illustrates the density of cilia ± the SEM (D) and length of cilia ± the SEM (E) within each of the analyzed hippocampal subfields of WT (blue) and Bbs4-/- (gray) mice; asterisks and brackets indicate where statistical analysis demonstrated statistical differences between WT and Bbs4-/- mice. The data for (D) and (E) was collected from three WT and five Bbs4-/- mice. Cornu Ammonis areas: CA1, CA3 and DG (dentate gyrus); vSub = ventral subiculum. Scale bar in figure A = 1 mm, in C = 20 μm and figures B & C are of the same magnification.
Figure 5.
Transmission electron microscopic images of PNC in CA1 hippocampus of WT (A) and Bbs4-/- mice (B & C).
Note the intact basal bodies associated with neurons in WT and Bbs4-/- mice and abnormal balloon-like cilia starting from basal bodies in B & C compared to healthy PNC with microtubules in A. The insert (D) is a higher magnification image of the area incorporated in the rectangle on (C). The yellow highlights the shape of PNC in Bbs4-/- hippocampus. The data was collected from three WT and five Bbs4-/- mice. BB – basal bodies; c – cilium; N - neuron; Nu – nucleus; arrows point to BB; solid arrow heads point to the ciliary transition zones; red arrow heads point to the spherical inclusion bodies. Scale bars: 0.5 μm for A–C.
Figure 6.
Similar distribution of ACIII immunopositive PNC in Bbs4-/- (A–D) compared to WT (E–H) regions of the brain is illustrated.
AcbSh – nucleus accumbence shell; SCN – suprachiasmatic nucleus; Pr5Vl - principal sensory trigeminal nucleus, ventrolateral subdivision; DTg - dorsal tegmental nucleus. The data was collected from three WT and five Bbs4-/- mice. Scale bars = 25 μm for A-H.
Figure 7.
Transmission electron microscopic images of PNC in AcbSh (A), SCN (B), Pr5Vl (C) and DTg (D) of Bbs4-/- mice.
The morphologically intact elongated PNC containing axonemal microtubules and their basal bodies associated with neurons in four areas not known to contribute to the human phenotype or functional deficits in the Bbs4-/- animal model are illustrated. Insets in each figure demonstrate the basal bodies of the cilia at higher magnification. No overt differences in morphology of PNC in these regions were observed compared to those of wild type animals (see Figure 2). The data for was collected from three WT and five Bbs4-/- mice. BB – basal bodies; c – cilium; N -neuron; Nu – nucleus; arrows point to BB. Scale bars: 0.5 μm for A-D. Abbreviations as for Figure 6.
Figure 8.
Statistical analysis of brain regions not affected by the syndrome compared to the corresponding areas in WT mice: cilia length ± the SEM (A) and cilia density ± the SEM (B) within each of the analyzed brain regions of WT (dark gray) and Bbs4-/- (light gray) mice.
Abbreviations as for Figure 6. The data for (A) and (B) was collected from three WT and five Bbs4-/- mice.
Figure 9.
Western blot analysis of ACIII expression in hippocampus, amygdala, AcbSh and SCN of WT and Bbs4-/- mice brains normalized for actin.
Note the lower level of ACIII in hippocampus and amygdala of Bbs4-/- mice compared to WT (A) and no changes in ACIII expression in AcbSh and SCN (B). Shown is a representative western blot from three separate experiments. The data for western blot was collected from three WT and three Bbs4-/- mice. Densitometric analysis (C) ± the SEM of the immunoblots shows the amounts of immunoreactive ACIII in the dissected areas of WT mice brains (dark gray) versus the corresponding dissected areas of Bbs4-/- mice brains (light gray) using densitometric values after correction for the levels of b-actin (*p<0.05). The data for Western blot was collected from three WT and three Bbs4-/- mice.
Figure 10.
Neuronal density ± the SEM within each of the analyzed subfields of amygdala, hippocampus and regions not affected by the syndrome in WT (dark gray) and Bbs4-/- (light gray) mice brains.
Note the absence of a significant difference in the number of neurons in all described regions except for the CA3 of dorsal hippocampus. The data was collected from three WT and four Bbs4-/- mice. Abbreviations as for Figures 1, 4 & 6.
Figure 11.
Variable ciliopathy phenotype in the investigated brain regions.
Dark grey rectangle corresponds to the regions where ether the PNC or the number of neurons are affected in Bbs4-/- mice brains. Light gray rectangle corresponds to the unaffected areas. Abbreviations as for Figures 1, 4 & 7.