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1.
Figure 4

Figure 4. MHC-I Peptides Induce c-Fos Activation in Downstream Neurons in the AOB. From: Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb.

A) Schematic illustrating the exposure of the VNO to either MHC-I peptides or water control to examine the activation of c-Fos in downstream neurons in the AOB. B) Confocal image of a sagittal section containing AOB from a control, water exposed animal (Green=GFP, Red=c-Fos, GL=Glomerular Layer, MCL=Mitral Cell Layer, GCL=Granule Cell Layer). C) Confocal image of a section containing AOB from a peptide exposed animal. D) Normalized c-Fos expression in the mitral cell layer across animals of water exposed control animals (n=5, 191.6 cells +/− 21.6) compared to peptide exposed animals (n=6, 387.8 cells +/− 29.8, p=0.20006).

Kenneth R. Hovis, et al. J Neurosci. 2012 June 6;32(23):7907-7916.
2.
Figure 7

Figure 7. Mitral Cell Dendritic Morphology Undergoes Dramatic Refinement During the First Four Post-Natal Weeks. From: Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb.

A) Neurolucida reconstructions of mitral cells across development (P2–P33). Mitral cells were targeted, patched, and filled with neurobiotin. Slices were fixed post-hoc and stained to be traced and reconstructed using Neurolucida. B–D) Point ending distributions across development. Neurolucida tracings were used and each point-endings within the glomerular layer was identified. The histogram of all the distances between each point ending was calculated and averaged for mitral cells from different age groups. P2–4 (B, n=4), P9–14 (C, n=4), and P30 and older (D, n=4). Scale bars = 50 μm.

Kenneth R. Hovis, et al. J Neurosci. 2012 June 6;32(23):7907-7916.
3.
Figure 8

Figure 8. Electroporation Reveals Highly Precise Adult Connectivity in the AOB. From: Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb.

A) Slice electroporation of an identified glomerulus innervated by V2r1b-GFP expressing sensory neuron axons labels several downstream mitral cells and their subsequent dendrites with a red fluorescent dye. B) Higher magnification view of A showing the targeting of a single GFP-positive glomerulus revealing the morphology of several labeled mitral cells. C) Higher magnification view of C showing multiple dendrites of labeled mitral cells targeting other GFP-positive glomeruli. D & E) Higher magnification view of C showing two individual GFP-positive glomeruli along with multiple red fluorescent dendrites innervating them. Note size of scale bars.

Kenneth R. Hovis, et al. J Neurosci. 2012 June 6;32(23):7907-7916.
4.
Figure 1

Figure 1. The Vomeronasal Duct Is Open At Birth. From: Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb.

A) Schematic of the experimental design. Alexa Fluro 594 Hydrazide dye was painted onto one of the nostrils of a P0 mouse exposing the sensory neurons of one-half of the VNO to the dye for 1 hour. B) Confocal image of a coronal section of the face of an exposed animal illustrating the presence of dye contacting not only the epithelium of the nasal cavity (upper arrow), but also the lumen of the VNO (lower arrow). C) Higher magnification view of B. D) Higher magnification view of the control VNO where only native GFP-expression can be seen in the VSNs. E) Higher magnification view from C showing the exposed VNO which not only contains native GFP-expressing sensory neurons (upper arrow) but some sensory neurons which have been exposed to and taken up some dye (lower arrow). Scale bar = 100 μm.

Kenneth R. Hovis, et al. J Neurosci. 2012 June 6;32(23):7907-7916.
5.
Figure 6

Figure 6. Activity Significantly Affects Axonal Exuberation and Coalescence in the AOB. From: Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb.

A) Example confocal image stacks of glomeruli across three ages in water exposed control animals. B) Example confocal image stacks of glomeruli from peptide exposed animals. C) Histogram distributions using Sholl analysis for the two example glomeruli above each histogram (black=control animal, red=exposed animal). D) Quantification of the half peak width from each histogram across animals at three different ages comparing water controls (Black squares; P1, n=5, 15.2 μm +/− 1.2; P2, n=9, 13.9 μm +/− 1.4; P3, n=9, 12.8 μm +/− 0.7) with exposed animals (Red triangles; P1, n=6, 23.6 μm +/− 1.7, p=0.0029; P2, n=8, 21.7 μm +/− 2.1, p=0.0076; P3, n=9, 23.6 μm +/− 1.1, p=0.001). Scale bar = 20 μm.

Kenneth R. Hovis, et al. J Neurosci. 2012 June 6;32(23):7907-7916.
6.
Figure 2

Figure 2. VSNs form Functional Synapses with Mitral Cells Prior to Birth.. From: Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb.

A) Example recording of mEPSCs from a P0 mitral cell. B) Example recording from a P12 mitral cell. All cells were held at -90mV to amplify events and events were recorded in the presence of 10 μM gabazine. C) Distribution of the average amplitudes of events at three different age groups (P0–1, n=5, 13.7 pA +/− 1.52; P5–6, n=5, 15.8 pA +/− 3.23; P11–12, n=6, 14.2 pA +/− 2.20). Each value was the average amplitude for a given cell from a minimum of one and a half minutes of data. D) The average decay time for all the events of each cell at each of the three different age groups (P0–1, n=5, 6.4 msec +/− 0.92; P5–6, n=4, 8.1 msec +/− 0.75; P11–12, n=6, 6.9 msec +/−0.43).

Kenneth R. Hovis, et al. J Neurosci. 2012 June 6;32(23):7907-7916.
7.
Figure 3

Figure 3. MHC-I Peptide is Present in Urine at Sufficient Concentrations to Activate Sensory Neurons. From: Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb.

A) Example MS spectrum of the native peptide SYFPEITHI using electrospray injection MS/MS at the 553.7 + 2 m/z peak (Red arrow). Collision energy was used to fragment the peptide into smaller pieces for identification (Black arrows). B) Example chromatogram from A (250 nM), which was filtered to peptide fragment mass ranges and then integrated. C) Calibration curve created from the integration of chromatogram peaks across a range of spiked urine concentrations (0 nM, n=3, 76,717 counts +/− 68,574; 50 nM, n=3, 5,318,503 counts +/− 770,200; 125 nM, n=3, 14,155,185 counts +/− 750,388; 250 nM, n=3, 24,637,306 counts +/− 1,716,317). After performing a linear regression, the line equation was then used to calculate the average native concentration of peptide in pure urine samples (red dot).

Kenneth R. Hovis, et al. J Neurosci. 2012 June 6;32(23):7907-7916.
8.
Figure 5

Figure 5. Activation of V2r1b-expressing Sensory Neurons Does Not Significantly Increase Number of V2r1b-expressing VSNs. From: Activity Regulates Functional Connectivity from the Vomeronasal Organ to the Accessory Olfactory Bulb.

A) Point by point reconstruction of a complete VNO traced using Neurolucida and reconstructed using Igor Pro Software. B) Confocal image stack of a 200 μm section of VNO from a transgenic animal whose V2r1b-expressing sensory neurons also express tau-GFP. Scale bar = 20 μM. C) The number of V2r1b-expressing sensory neurons across development in water exposed control animals (black bars; P1, n=3, 86 cells +/− 4.2; P2, n=4, 116.8 cells +/− 8.3; P3, n=3, 134.6 cells +/− 1.6) and peptide exposed animals (red bars; P1, n=3, 94.3 cells +/− 12.2, p=0.55; P2, n=4, 131 cells +/− 7.5, p=0.25; P3, n=5, 131 cells +/− 3.65; P4–5, n=3, 134.5 cells +/− 8.8). D) Comparison of the volume of the VNO’s across ages in control (P1, n=3, 49,375,000 μm3 +/− 1,380,063; P2, n=4, 65,050,000 μm3 +/− 9,058,528; P3, n=4, 59,870,700 μm3 +/− 2,946,470) and peptide exposed animals (P1, n=3, 56,979,833 μm3 +/− 4,453,998; P2, n=4, 60,502,225 μm3 +/− 4,029,919; P3, n=5, 60,960,420 μm3 +/− 2,491,808).

Kenneth R. Hovis, et al. J Neurosci. 2012 June 6;32(23):7907-7916.

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