At the same time, through their mACT axonal projection, iPNs effectively send olfactory signals to the lateral horn (see below). Since both iPNs and vlpr neurons send processes to the lateral horn, IA-elicited Ca2+ signals within the lateral horn (Figure 1I) could be contributed by either or both of these neuronal

types. We next aimed to isolate putative postsynaptic signals of vlpr neurons from presynaptic selleck signals in iPNs within the same lateral horn using a laser transection protocol outlined in Figure 2A. Specifically, we first obtained lateral horn odor responses from control and experimental hemispheres. We then used Mz699-labeled iPN axons as a guide and applied spatially confined laser pulses from the two-photon laser (Ruta et al., 2010) to transect the mACT prior to its entry to the lateral horn on the experimental hemisphere. Following the laser transection, we again imaged lateral horn odor responses in both experimental and control hemispheres. Several lines of evidence suggested that our laser transection of mACT was complete and specific. First, we could observe a small selleckchem cavitation bubble at the mACT from basal GCaMP3 fluorescence with our two-photon microscope immediately following the laser application (Figure S3A), a hallmark of laser transection

(Vogel and Venugopalan, 2003). Second, retrospective immunostaining validated the complete transection of the mACT (Figure S3B, n = 15) with no visible effect on the integrity of the nearby iACT that conveys signals from the ePNs (data not shown). Third, odor-evoked GCaMP3 signals in

mACT near the lateral horn entry site (e.g., Figure 2B2, yellow arrow) were invariably abolished after laser transection of mACT (Figure 2B3, yellow arrow), validating that the responses observed in intact preparations were due to iPN contributions and were lost after mACT transection. Fourth, applying the same energy from the two-photon first laser at locations away from mACT did not cause similar changes in lateral horn Ca2+ signals (data not shown). Fifth, we did not detect changes of iPN responses in the antennal lobe before or after mACT transection (data not shown), suggesting that olfactory input still activates iPNs in the antennal lobe after mACT transection. Thus, we could assume that olfactory response in the lateral horn neuropil after mACT transection is mostly contributed by the vlpr neurons. How does iPN projection contribute to olfactory information processing at the lateral horn, and specifically, how are the responses of putative third-order vlpr neurons modulated by iPN input? To address these questions, we compared Ca2+ signals in response to isoamyl acetate application in the lateral horn (referred to as IA response hereafter) before and after laser transection (Figures 2B and 2C). In all cases, IA responses in the lateral horn were robust (Figure 2C).