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1.
Figure 6

Figure 6. Mitral cell responses to VSN stimulation with male and female mouse urine mixtures. From: An ex vivo preparation of the intact mouse vomeronasal organ and accessory olfactory bulb.

(A) Peristimulus time histograms for a cell that was suppressed by stimulating the VNO for 5s (solid black bars) with a mix of preferred (1:200 male urine, blue trace) and non-preferred (1:200 female urine, red trace) stimuli. The 1:200 mixture (purple trace) induced firing rates below those seen for the preferred stimulus alone, indicating intact laterally-connected inhibition within the ex vivo circuit. 6 of 9 cells exposed to these stimuli had suppression indices below zero. (B) An example mitral cell that did not undergo mixture suppression. The mixed stimulus (purple trace) elicited higher firing rates than the preferred stimulus alone (female urine, red trace). (C) Scatter plot of the 9 cells in this study. Suppression index, ranging from -1 (most suppressed) to +1 (enhanced), is plotted against the selectivity index (-1 = male-selective, +1 = female-selective).

Julian P. Meeks, et al. J Neurosci Methods. ;177(2):440-447.
2.
Figure 5

Figure 5. Mitral cell responses to VSN stimulation with dilute male and female mouse urine. From: An ex vivo preparation of the intact mouse vomeronasal organ and accessory olfactory bulb.

(A) Mitral cells in ex vivo preparations respond to dilute conspecific male and female urine. (A1) This mitral cell responded strongly to 5 s VNO stimulation with 1:100 diluted Balb/c strain female urine (red trace, stimulus indicated by solid black bar) but not 1:100 dilute male urine (blue trace) or Ringer's control (black trace). The selectivity index for this response was 0.82916, indicating that this cell was “strongly female-selective”. (A2) Example of a strongly male-selective cell from a separate recording. (B) profiles from 43 mitral cells that responded significantly to either 1:100 Balb/c male or female urine. (C) Histogram of selectivity values for the 43 cells in (B). 24 of the 43 cells met our criteria for classification as “strongly-selective” (selectivity index > |0.5|). 11 of the 43 cells were considered “non-selective” for urine based on sex of origin (selectivity index < |0.25|). Error bars represent standard errors of the mean.

Julian P. Meeks, et al. J Neurosci Methods. ;177(2):440-447.
3.
Figure 2

Figure 2. Ex vivo perfusion chamber schematic. From: An ex vivo preparation of the intact mouse vomeronasal organ and accessory olfactory bulb.

(A) VNO-AOB ex vivo preparations were placed in a custom tissue perfusion chamber designed per this schematic. Warmed, oxygenated aCSF is driven at ∼7.5 mL/min via a peristaltic perfusion pump (not shown) into a small antechamber (right). In this antechamber, the perfusion aCSF was re-oxygenated with 95% O2/5% CO2. This oxygen-rich aCSF entered the main chamber via an opening near the AOB (magenta oval). In some experiments, a small microsuction device was placed at the lateral face (i.e. the surface facing downward into the page) of the main olfactory bulb to encourage deep tissue perfusion. Stimuli were delivered into the VNO (red oval) through a polyimide cannula (orange line) at 0.2-0.4 mL/min. Waste solution was removed from a third chamber physically separated from the main chamber to reduce surface vibrations. (B) Detailed diagram of micro-suction device design. A series of fine nylon mesh screens (10-50 μm in diameter, with smallest pore size at tissue interface) were affixed to a polyimide suction wand. A pressure release cannula was threaded into the lumen of the device and left open to the superfusion solution in order to normalize suction pressure at the tissue interface over time.

Julian P. Meeks, et al. J Neurosci Methods. ;177(2):440-447.
4.
Figure 4

Figure 4. Mitral cell responses to VSN stimulation with High K+ solution. From: An ex vivo preparation of the intact mouse vomeronasal organ and accessory olfactory bulb.

(A) Extracellular mitral cell recording configuration. An extracellular glass electrode was advanced into the AOB (magenta oval) while stimuli were applied to the VNO lumen (red oval) via a polyimide cannula. (B) Recording from a single trial stimulus pair. Top: stimulation of VSNs with 50 mM K+ Ringer's solution (“high K+”, solid black bar) induced spiking in the mitral cell. Inset: sorted spike waveforms from this trial. (C) Peristimulus time histograms in baseline (C1) and following 10 minutes of superfusion with 3 μM SR95531 (gabazine, C2). Exposure to gabazine increased the evoked activity in this cell by an average of 3.1 Hz over the 7-second integration window. Stimulus time: 5 s (solid black bar). (D) Mitral cell spontaneous activity did not significantly increase during exposure to 3 μM gabazine (p = 0.52, n = 7). (E) Mitral cell responses to VNO stimulation was significantly increased during exposure to 3 μM gabazine (p < 0.05, n = 7), indicating inhibitory activity remains present in ex vivo preparations over the 6-hour recording period.

Julian P. Meeks, et al. J Neurosci Methods. ;177(2):440-447.
5.
Figure 1

Figure 1. Ex vivo dissection strategy. From: An ex vivo preparation of the intact mouse vomeronasal organ and accessory olfactory bulb.

The connected VNO and AOB of adult male B6D2F1 mice were isolated by performing the diagrammed steps in ice-cold aCSF. For each step, a digital image is shown at the bottom, and a schematic diagram shown on top. Following decapitation (A), the lower jaw, scalp, and external sensory structures are removed from the skull (B). The skull overlying the cerebral cortex and cerebellum is then removed (C), followed by removal of the majority of the brain posterior to the olfactory bulbs (D1). The soft palate is then detached, revealing the two vomeronasal organs (D2 top, red oval structures). The bone surrounding the VNO capsule is broken on the contralateral hemisphere (not shown) using a scalpel point, followed by a careful hemispherectomy of the contralateral portion of the skull (E1). Upon turning the hemisphere on its lateral edge, the VNO can be seen from its medial face (E2 top, red oval), along with the septal cartilage and bone. The olfactory bulb (E2 top, peach outline) containing the accessory olfactory bulb (E2 top, magenta oval) can be visualized. The lateral face of the hemisphere is then affixed to a plastic plank via tissue adhesive (F) and placed into the perfusion chamber. (G) The final dissection steps are performed in the perfusion chamber at room temperature. Following removal of all contralateral tissue, attached ipsilateral frontal cortex, and the septal bone and cartilage (G1), the AOB becomes visible (G2) and a stimulus cannula is threaded under visual control into the VNO lumen (G3).

Julian P. Meeks, et al. J Neurosci Methods. ;177(2):440-447.
6.
Figure 3

Figure 3. Fluoro-Jade C staining reveals trends in tissue health. From: An ex vivo preparation of the intact mouse vomeronasal organ and accessory olfactory bulb.

Fluoro-Jade C staining labels dead and degenerating neurons regardless of the type of degeneration process (Schmued et al., 2005). (A-F) Montage images of VNO (A-C) and AOB (D-F) sections taken from dissection-only (A, C), physiology chamber plus microsuction (B, E), and unoxygenated, warmed aCSF (C, F, “unperfused”) conditions are shown for reference. (A, D) In control sections, very few bright, punctate areas were present in the vomeronasal epithelium (A) or the AOB glomerular, mitral, or granule cell layers (D). (B, E) Following 6 (VNO, panel B) or 4 (AOB, panel E) hours in the physiology chamber at 33 – 35 °C, relatively few punctate areas were visible in the glomerular and mitral cell layers. (C, F) When VNO-AOB ex vivo preparations were placed in unperfused, unoxygenated aCSF at 33 – 35 °C, we saw substantial global increases in Fluoro-Jade C staining, indicative of widespread neuronal death and degeneration. (G) Quantification of punctate staining in the vomeronasal epithelium after 6 hours showed no increase in cell degeneration in the perfusion chamber compared to dissection-only controls. (H) Quantification of punctate staining in the AOB glomerular and mitral cell layers indicated the perfusion chamber provided significant protection from damage (blue symbols) compared with the unoxygenated tissue (green symbols), especially at the 4 and 6 hour time points (p < 0.001, n = 3). (I) We encountered evidence of degeneration in the granule cell layer under normal superfusion conditions (blue symbols). To encourage perfusion through deeper tissue layers, we drew solution through the tissue using a microsuction device (E). Microsuction for 4 hours in the tissue chamber reduced mean staining in the granule cell layer to a level intermediate between “unperfused” (p = 0.2, n = 5) and “dissection only” (p = 0.5, n = 5; (H, I, red symbols). Abbreviations: VE, vomeronasal epithelium; L, vomeronasal lumen; GL, glomerular layer; MCL, mitral cell layer; GCL, granule cell layer; LOT, lateral olfactory tract.

Julian P. Meeks, et al. J Neurosci Methods. ;177(2):440-447.

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