Figure 1.
Metabolic pathways of Ang peptides.
Metabolic routes of Ang I and II by neurolysin and other peptidases of the RAS. ACE = angiotensin-converting enzyme, dipeptidyl carboxypeptidase I, Kininase II, EC 3.4.15.1, CD143; ACE-2 = angiotensin-converting enzyme-2, EC 3.4.17.23; APA = aminopeptidase A, glutamyl aminopeptidase, EC 3.4.11.7, CD249; NEP = neprilysin, neutral endopeptidase, EC 3.4.24.11; PRCP = prolyl carboxypeptidase, angiotensinase C, carboxypeptidase P, EC 3.4.16.2; PREP = prolyl endopeptidase, post-prolyl cleaving enzyme, EC 3.4.21.26; TOP = thimet oligopeptidase, EC 3.4.24.15. Adapted from Wright et al. [12].
Table 1.
Summary of autoradiography protocol.
Figure 2.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of a representative neurolysin KO (right panels) and WT (left panels) mouse strain in the presence of PCMB, losartan, and PD123319. Approximate coordinates relative to Bregma: +3.56 mm for WT and +3.32 mm for KO. Top row shows thionin-stained coronal sections adjacent to the sections used to generate the autoradiograms for “total” (middle panels) and “non-specific” (lower panels) of 125I-SI Ang II binding. Binding is represented in pseudocolor. The vertical calibration bar represents the relationship between 125I-SI Ang II binding density and the color spectrum. The blue horizontal calibration bar shown in the upper left panel = 1 mm. This pattern is repeated for Figures 3–12.
Figure 3.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma +2.22 mm (histology) and +2.10 (autoradiograms) for KO, and Bregma +2.16 (histology), +1.92 (total) and +2.04 mm (non-specific) sections for WT.
Figure 4.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma +0.86 (histology) and +0.98 mm (autoradiograms) for KO, and Bregma +0.96 mm (histology and autoradiogram) sections for WT.
Figure 5.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma +0.38 (histology) and +0.5 mm (autoradiograms) for KO, and Bregma +0.36 mm (histology and autoradiogram) sections for WT.
Figure 6.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma +0.08 mm for the KO and WT histological and autoradiogram sections.
Figure 7.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma −0.34 mm for the KO and WT histological and autoradiogram sections.
Figure 8.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma −1.82 mm for KO, and Bregma −1.70 mm for WT histological and autoradiogram sections.
Figure 9.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma −3.40 mm (histology and autoradiograms) for KO, and Bregma −3.32 (histology) and −3.20 mm (autoradiogram) sections for WT.
Figure 10.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma −4.24 mm (histology and autoradiograms) for KO, and Bregma −4.36 (histology) and −4.24 mm (autoradiogram) sections for WT.
Figure 11.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma −5.8 mm for the KO and WT histological and autoradiogram sections.
Figure 12.
125I-SI Ang II binding comparison.
Comparison of 125I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma −7.2 mm for the KO and WT histological and autoradiogram sections.
Figure 13.
Regional distribution: non-AT1, non-AT2 binding.
Regional distribution of non-AT1, non-AT2 Ang II binding in neurolysin KO and WT mouse brains. Brain regions were divided into cerebellum, brainstem and midbrain (Panel A), hypothalamic nuclei (Panel B), thalamoseptalstriatal regions (Panel C), and telencephalic regions (Panel D). In all but two regions, a priori t-tests showed significant reduction in 125I-SI Ang II binding in the brains of the neurolysin KO mice. * p<0.05. AH, Anterior Hypothalamus; AMYG, Amygdala; ARC, Arcuate Nucleus; CCTX, Cingulate Cortex; CP, Choroid Plexus; CPu, Caudate Putamen; CRBLM, Cerebellum; DMH, Dorsomedial Hypothalamus; DTLCMe5, Dorsal Tegmentum, Locus Coeruleus and Mesencephalic Nucleus of the Trigeminal Nerve; ETC, Entorhinal Cortex; HPC, Hippocampus; IPN, Interpeduncular Nucleus; LMC, Limbic Cortex; LS, Lateral Septum; ML BRST, Mediolateral Brain Stem; MnPO, Median Preoptic Nucleus; MPOH, Medial Preoptic Nucleus; NACC, Nucleus Accumbens; NTS, Nucleus Tractus Solitarius; PAG, Periaqueductal Gray; PH, Posterior Hypothalamic Area; PMN, Premamillary Nucleus; PVA-THAL, Paraventricular Thalamic Nucleus, Anterior; PVH, Paraventricular Hypothalamic Nucleus; PVTHAL, Paraventricular Thalamic Nucleus; Red N, Red Nucleus; RSPC, Retrosplenial Cortex; SC, Superior Colliculus; SCN, Suprachiasmatic Nucleus; SN, Substantia Nigra; TSN, Triangular Septal Nucleus; VMH, Ventromedial Hypothalamic Nucleus.
Figure 14.
Regional distribution: neurolysin and non-AT1, non-AT2, non-neurolysin binding.
Regional distribution of 125I-SI Ang II binding to neurolysin (Panel A) and non-AT1, non-AT2, non-neurolysin (Panel B) in the mouse brain. Values represent the difference between non-AT1, non-AT2 Ang II binding in the WT and neurolysin KO mouse brains in 32 regions. AH, Anterior Hypothalamus; AMYG, Amygdala; ARC, Arcuate Nucleus; CCTX, Cingulate Cortex; CP, Choroid Plexus; CPu, Caudate Putamen; CRBLM, Cerebellum; DMH, Dorsomedial Hypothalamus; DTLCMe5, Dorsal Tegmentum, Locus Coeruleus, Mesencephalic Nucleus of the Trigeminal Nerve; ETC, Entorhinal Cortex; HPC, Hippocampus; IPN, Interpeduncular Nucleus; LMC, Limbic Cortex; LS, Lateral Septum; ML BRST, Mediolateral Brain Stem; MnPO, Median Preoptic Nucleus; MPOH, Medial Preoptic Nucleus; NACC, Nucleus Accumbens; NTS, Nucleus Tractus Solitarius; PAG, Periaqueductal Gray; PH, Posterior Hypothalamic Area; PMN, Premamillary Nucleus; PVA-THAL, Paraventricular Thalamic Nucleus, Anterior; PVH, Paraventricular Hypothalamic Nucleus; PVTHAL, Paraventricular Thalamic Nucleus; Red N, Red Nucleus; RSPC, Retrosplenial Cortex; SC, Superior Colliculus; SCN, Suprachiasmatic Nucleus; SN, Substantia Nigra; TSN, Triangular Septal Nucleus; VMH, Ventromedial Hypothalamic Nucleus.
Figure 15.
AT1 and AT2 receptor binding comparison.
Comparison of 125I-SI Ang II binding to the AT1 and AT2 receptors of neurolysin KO (right panels) and WT (left panels) mouse strain brains in the presence of PD123319 or losartan, respectively. Approximate coordinates relative to Bregma: +0.98 mm (histology and autoradiograms) for KO, and Bregma +0.92 (histology), +0.86 (AT1), and +0.98 mm (AT2 and non-specific) for WT. Top row of panels are thionin-stained coronal sections adjacent to the sections used to generate the autoradiograms for “total” 125I-SI Ang II binding to the AT1 receptor (second row), AT2 receptor (third row), and “non-specific” 125I-SI Ang II binding (fourth row), represented in pseudocolor. The vertical calibration bar represents the relationship between binding density of 125I-SI Ang II and the color spectrum. The horizontal calibration bar in the upper left panel = 2 mm. This pattern is repeated for Figures 16 and 17.
Figure 16.
AT1 and AT2 receptor binding comparison.
Comparison of 125I-SI Ang II binding to the AT1 and AT2 receptors of neurolysin KO and WT mouse strain brains in the presence of PD123319 or losartan, respectively. Bregma +0.14 (histology and AT1) and +0.02 mm (AT2 and non-specific) for KO, and +0.14 (histology, AT1, and non-specific) and +0.26 mm (AT2) for WT.
Figure 17.
AT1 and AT2 receptor binding comparison.
Comparison of 125I-SI Ang II binding to the AT1 and AT2 receptors of neurolysin KO and WT mouse strain brains in the presence of PD123319 or losartan, respectively. Bregma −4.48 (histology) and −4.36 mm (autoradiogram sections) for KO, and Bregma +4.36 mm for the WT histological and autoradiogram sections.
Figure 18.
Regional distribution: AT1 and AT2 receptor binding.
Regional distribution of 125I-SI Ang II binding to the AT1 and AT2 receptors in the neurolysin KO and WT mouse brains. Panel A describes binding to the AT1 receptor in 9 brain regions. Panel B describes binding to the AT2 receptor in 10 brain regions. * p<0.05. AC, Anterior Commissure; CCTX, Cingulate Cortex; LC, Locus Coeruleus; LS, Lateral Septum; MnPO, Median Preoptic Nucleus; NACC, Nucleus Accumbens; NTS, Nucleus Tractus Solitarius; OVLT, Organum Vasculosum of the Lamina Terminalis; PVH, Paraventricular Hypothalamic Nucleus; SC, Superior Colliculus.
Figure 19.
Ventricular and total surface area of brain sections.
Comparison of ventricle size and total surface area of neurolysin KO and WT mouse strain brain coronal sections. Panel A describes the average surface area of lateral ventricles from the rostral limit of the hippocampus (∼0.9 mm caudal to Bregma) to the most rostral extent of the corpus callosum crossing (∼1.15 mm rostral to Bregma). Third ventricle surface area was measured from the rostral limit of the hippocampus (∼0.9 mm caudal to Bregma) to the rostral limit of the subfornical organ (∼0.2 mm caudal to Bregma). Fourth ventricle surface area was measured from ∼6.66 mm to ∼5.34 mm caudal to Bregma. Cerebral aqueduct surface area was measured from ∼4.84 mm to ∼4.24 caudal mm to Bregma. Panel B describes the average surface area of coronal brain sections at 120 micron intervals (5 mm caudal to Bregma to 1.48 mm rostral to Bregma). Panel C describes the average surface area corresponding to the sections used to measure the lateral ventricle area in Panel A (left Y-axis) and the ratio (%) of lateral ventricle size to total surface area of coronal brain sections (right Y-axis). * p<0.05.