David Blauvelt
Harvard College 2009

One of the first steps in processing a smell occurs at the glomerular layer, which is where axons of olfactory receptor neurons make synapses on the dendrites of mitral cells. While olfactory receptor neurons’ pre-synaptic activity have been extensively researched in anesthetized mice, little is known about odor-evoked synaptic activity in awake, freely moving animals. This project attempts to address this problem by adapting a fiber optic bundle imaging technique to the olfactory bulb. The results show that while behavior causes no consistent changes in olfactory receptor neuron activity across all subjects and odors, it does seem to be linked to differences in the nature of odor responses in individual mice. Importantly, this study is the first demonstration that a fiber optic imaging bundle can be used to image the olfactory bulb of an awake, behaving animal.

Introduction

One of the fundamental concerns of neuroscience is to understand how the brain perceives and processes the outside world. From the first stage of detection by sensory nerves to the forging of a memory of an event, our brains are dynamically interacting with our surroundings. Olfaction is one of the ways we sense the world, yet the study of the olfactory system has many unanswered questions. How do we process different odors? How do we forge and retrieve odor memories? How do our different mental and physical states affect how we respond to odor stimulation?

Sensing an odor begins with the olfactory receptor neurons (ORN) located on the nasal epithelium (Wilson and Mainen, 2006). A single mouse possesses approximately 44 million ORNs (Albeanu, 2008, Mombaerts, 2006, Wachowiak, 2004), with each ORN containing G-protein coupled receptors known as the olfactory receptors (OR). In order to remain functionally distinct, a single ORN only expresses one or perhaps a small number of different types of ORs (Albeanu, 2008, Mombaerts et al., 1996). Every OR can detect multiple odors, and many types of ORs detect each odor. However, each odor is sensed by a unique combination of ORs that allows it to be distinguished from other odors (Malnic et al., 1999). The axons of the stimulated ORN transmit the sensory signal to approximately two locations in a single olfactory bulb known as glomeruli, which are simply the synapses joining the ORN axons with the dendrites of mitral/tufted (M/T) cells, which output the signal to the olfactory cortex (Albeanu, 2008, Mombearts et al. 1996). Since the glomeruli are spatially defined functional clusters, the glomerular layer can be viewed as an odor map for which each odor is encoded by a unique combination of glomerular activation (Mombaerts et al., 1996). Once the signal reaches the glomerular layer, it is passed to the mitral cell layer, which contains the M/T cells (Wilson and Mainen, 2006).

The structure of the olfactory system, however, is not a simple relay. Rather, the system is subject to lateral modulation from regulatory cells. Each glomerulus is surrounded by different types of regulatory juxtaglomerular (JG) cells, including periglomerular (PG) cells and short axon (SA) cells (Albeanu, 2008, Wachowiak and Shipley, 2006). Periglomerular cells, the more populous of the interneurons in the glomerular layer, are inhibitory regulators (Albeanu, 2008, Wilson and Mainen, 2006). PG cells receive stimulatory input from ORNs, M/T cells, and even other PG cells. They then transmit inhibitory signals reciprocally to the ORN axons and the M/T dendrites of the exciting glomerulus. The other type of juxtaglomerular cell is the short-axon cell, which indirectly inhibits M/T cells by exciting PG cells. Contrary to what the name suggests, a short axon cell is a long-range regulatory cell and may act on PG cells several glomeruli away (Albeanu, 2008, Aungst et al., 2003, Wilson and Mainen, 2006).

Mitral/tufted cells also communicate with each other indirectly and directly. M/T cells form dendrodendritic synapses with granule cells in the external plexiform layer, and can inhibit other M/T cells through granule cells (Wilson and Mainen, 2006). M/T cells may also directly influence their neighbors through a process known as spillover. When the M/T cells release glutamate through their dendrites, some of the glutamate excites dendrites of neighboring M/T cells (Isaacson, 1999, Wilson and Mainen, 2006).

Finally, the olfactory system is influenced by centrifugal inputs from the cortex and other areas of the brain (Isaacson, 1999). These inputs may be affected by the behavior of the mouse, genetic predispositions, and other factors. Centrifugal influences can be separated into three major categories. First, odor intake is affected by areas of the brain outside of the olfactory bulb. Passive sniffing rate is determined by central pattern generators in the medulla (Ramirez and Richter, 1996, Wilson and Mainen, 2006) while active respiration is controlled by the forebrain (Wilson and Mainen, 2006). Second, the bulb receives reciprocal feedback from output regions such as the olfactory cortex. Finally, changing levels of neuromodulators such as norepinephrine, acetylcholine, and serotonin can affect odor responses (Wilson and Mainen, 2006).
The complexity of the olfactory system, designed to accomplish what may seem like a simple task, is astounding. This intricacy makes the system susceptible to outside influences such as alterations in behavior. Most in vivo imaging research on the olfactory system examines the bulb while an animal is in an anesthetized state. However, anesthesia greatly limits the scope of the field because it makes it impossible to study the effect of behavior on olfactory response.

One way in which an animal’s behavioral state may affect its response to odors is by influencing the internal bulb activity. Recently, a team headed by Kenasku Mori studied the dendro-dendritic activity in the external plexiform layer (Tsuno et al., 2008). They found that mitral-granule activity was strongest in anesthetized animals and was progressively weaker in lightly sleeping, awake and immobile, and awake and mobile animals.

In addition to direct effects upon the olfactory bulb, behavior may affect centrifugal inputs. For instance, while the sniffing rate of an anesthetized animal is passively controlled by the brainstem, a behaving animal has the ability to actively control this rate. Furthermore, the levels of norepinephrine, acetylcholine, and serotonin can be altered by different behavioral states (Wilson and Mainen, 2006). As an example, Shea et al. (2008) found that norepinephrine release, when coupled with odor presentation, acts in the olfactory bulb to cause suppression of paired odor responses.

To more fully understand the effects of different behaviors on odor responses, the olfactory bulb needs to be monitored while the animal is awake and behaving. While electrical activity in behaving animals has been extensively studied, they are limited to single cell readouts as opposed to imaging studies, which allow for large-scale population readouts. Sub-glomerular resolution imaging studies of the olfactory bulb in awake animals have been performed on head-fixed animals (Carey et al., 2004), but are limited. Furthermore, to date, sub-glomerular resolution imaging of the olfactory bulb in freely behaving animals has not yet been accomplished. The present study attempts to address this issue by offering a way to image the bulb in a freely behaving mouse.

Two important advances have made it possible to explore odor responses in freely behaving mice. The first was the creation of transgenic mice expressing synaptopHluorin in the ORNs, which allowed the visualization of pre-synaptic glomerular activity in the olfactory bulb. pHluorins is a mutant form of GFP that was sensitive to pH and would fluoresce in a neutral environment but not in an acidic environment. SynaptopHluorin (spH) was then made by fusing pHluorin to synaptobrevin (Miesenböck et al., 2000). Synaptobrevin is a vesicle protein required for the release of neurotransmitters into the synapse. The idea is to take advantage of the acidic environment of the neurotransmitter vesicles (pH ~5.7). When inside the vesicles, spH would not fluoresce, but once a vesicle binds with the cellular membrane to release neurotransmitters into the neutral pH of synaptic space (~7.4), spH would fluoresce (Miesenböck et al., 1998). This laid the groundwork for the use of synaptopHluorin as a genetically encoded molecular probe that would allow detection of neural activity using simple fluorescence microscopy.

Though there are many other ways to image ORN synaptic activity such as nasal calcium dye injections, bulk calcium dye loading, and intrinsic signaling (Toga and Mazziotta, 2002, Albeanu, 2008), synaptopHluorin was used because of three significant advantages. First, it is genetically encoded unlike methods involving calcium dyes, which need to be exogenously loaded. Second, the signal is mostly specific to pre-synaptic activity. Finally, because it is localized to the axon termini, spH makes it possible to more easily distinguish individual glomeruli.

The second necessary development for this study was the introduction of an imaging technique using a flexible fiber optic imaging bundle attached to the skull. This method was chosen over other methods such as the head-fixed method (Carey et al., 2004) specifically because it allows the mouse to freely move around while keeping the fluorescence image in the focal plane of the microscope.

The first fundamental question that this study attempts to answer is whether the fiber technique is a viable way to image freely behaving animals. This technology is still in its very early stages, and has never been used to image the olfactory bulb. However, after adapting and optimizing the procedure, this technique will presumably offer a good way to image freely behaving animals.

The second question of this study is whether behavior has an effect on the pre-synaptic activity of glomeruli. As discussed earlier, there are several ways that behavior could influence ORN output. Thus, it seems likely that behavior will cause some change in glomerular activity, although attributing observed changes to specific individual behaviors as well as parsing out the causes is beyond the scope of this study. In order to test this hypothesis, the aforementioned fiber optic technique was used to image transgenic mice expressing synaptopHluorin in the glomeruli. In addition, a customized odor rack was employed to expose the mice to different odors while monitoring the fluorescence response in real-time. The same mice were imaged in both an awake and an anesthetized state, and the responses were compared.

Fundamentally, the goal of this study is simply to open the doors to a burgeoning field of new imaging techniques while answering some questions about how behavior can affect olfactory sensory activity. First, this project attempts to determine whether a fiber optic imaging bundle provides a way to image the olfactory bulb of an awake, freely-moving animal. Second, this study uses the bundle to track olfactory receptor neuron pre-synaptic activity in anesthetized and freely-behaving mice to find similarities and differences based on behavioral state.

Methods

Subjects and surgery

The subjects used were transgenic adult (postnatal days 60-100) synaptopHluorin mice, including both heterozygous and homozygous as well as male and female mice (Bozza et al., 2004). Mice were anesthetized for surgery with a cocktail of ketamine and xylazine (ketamine – 100 mg/kg, IP, Fort Dodge Animal Health #440761; 10 mg/kg xylazine, IP, Phoenix Pharmaceutical, Inc. #4111505). The mice were mounted on a stereotaxic frame and the skin was cut to expose the skull. A small hole, approximately 1.5-2 mm in diameter, was opened over the right olfactory bulb by thinning the bone in a circle and removing the piece. A flexible fiber bundle (Schott Inc., #1137189) with a 1.45 mm outer diameter was lowered onto the brain. The other end of the bundle was placed in the focal plane of a widefield fluorescence microscope fitted with a 10x objective and a high speed imaging camera (SensiCam, Cooke Corp.). The bundle was lowered using an XYZ translator until glomeruli became clear. The fiber bundle provided a continuous two-way light path from the microscope to the glomeruli and back to the microscope, allowing the mouse to move around away from the microscope while still keeping an image of the glomeruli within the focal plane of the microscope. Once the bundle was in the proper place on the brain, it was cemented onto the skull with RelyX Luting Plus (3M ESPE, #3525). The mouse was allowed to recover for several hours before the analgesic, Buprenorphine HCl (0.5 mg/kg, BD, IP, Bedford Labs, #1208141), was given. Anesthetized imaging occurred immediately after surgery while the mouse was still anesthetized. Behavioral imaging was performed after recovery.

Imaging fiber optic bundle

There are two issues regarding the properties of an imaging fiber bundle that need to be addressed. The first is the pixelation of the image. The bundle was composed of thousands of microscopic fibers, each 8 microns in diameter. This led to pixelation that was visible under 10x magnification. However, since the pixels were much smaller than the glomeruli (approximately 90 microns), the pixelation did not significantly interfere with the overall quality of the image. The second issue is the lack of focusing optics in the bundle itself. Ideally, one would use microscopic lenses to focus the light and prevent cross-contamination of signals from nearby glomeruli. Fortunately this was not a problem, since the distance between the bundle and glomerular layer was sufficiently small. As demonstrated by the results, the images obtained were clear enough to distinguish individual glomeruli.

Odor delivery apparatus

In order to systematically present odors, a computer-controlled olfactometer (Figure 1) was designed and constructed. The apparatus contained tygon tubing (McMaster-Carr 5046K11) and two main sets of solenoid valves (ASCO Scientific AL4124). The first set consisted of ten valves, each associated with a single odor. When a valve was opened, air would pass through the valve, going into a test tube containing the odor and out via another path, carrying the odor with it. The odor would then travel to a second set of valves, which included an exhaust valve, an odor valve, and an air valve. The odor could either go through the odor valve or the exhaust valve. If the odor valve was open, the odor would be presented to the animal. Alternatively, if the exhaust valve was open, the odor would leave the system through a ventilation system. The air valve was connected to an air line, allowing clean air to be presented to the animal. Aside from the two main sets of valves, there was a cleaning valve.

Figure 1
Figure 1. Odor rack schematic. During the cleaning phase, air passes though valve 6, cleaning the tubing. Air and contaminants are flushed from the system though valve 3, the exhaust valve. During the air1 phase, either valve 1 or 2 is opened. Odor fills the system but goes out through valve 3. In addition, valve 5 is opened, allowing air to flow to the animal. During the odor phase, valve 1 or 2 remains open, but valves 3 and 5 are closed, replaced by the opening of valve 4, which allows odor to flow to the animal. During air2, valve 4 is closed and valves 3 and 5 are reopened. In addition valve 6 is opened, starting the cleaning process. This process is repeated for each odor.

Each trial involved four different phases that were repeated: cleaning, air presentation 1, odor presentation, and air presentation 2. During the cleaning phase, the cleaning valve was opened to allow fresh air to flow through the system. In addition, the air valve was open, providing fresh air to the animal. During air presentation 1, the air valve remained open and images were taken to obtain an average baseline fluorescence image for comparison to odor images. During the odor phase, the exhaust and air valves were closed, and the odor valve was opened. The odor flowed through the second set of valves to the animal. Finally, during air presentation 2, the odor valve was closed, and the air valve was re-opened, allowing the fluorescence to return to baseline while images were taken to track the return.

Software and experimental design

The odor delivery control and image acquisition was performed by a software program written in LabView (National Instruments) adapted by Tomokazu Sato from a similar program by Edward Soucy. Each experiment could be altered by changing the settings of the program. During the course of the experiment, the mouse was placed in a small chamber to minimize movement during awake imaging and to maximize and homogenize odor exposure. The air in the chamber was constantly evacuated throughout the experiment. However, some delay in clearing an odor from the chamber was expected. While the concentration was not measured, it was assumed that the odor was expunged from the chamber quickly, likely on the order of a few seconds or less.

Analysis

Analysis was done primarily using ImageJ (National Institutes of Health) and Microsoft Excel. Time course image stacks were collected and used to track the responses in real time. Ratio images were used to quickly identify the presence or absence of responses as well as the strength of response. These images are displayed as functions of the inverse of ΔFluorescence/Base Fluorescence (ΔF/F). Hence, dark spots indicate an increase in ΔF/F. Ideally, one would like to subtract out all fluorescence that is not attributable to glomeruli. However, since ratio images compared images taken within minutes of each other, it was assumed that background fluorescence changed minimally.

Results and Discussion

Efficacy of fiber bundle imaging

Using a fiber optic bundle to image the brain of an awake, behaving animal is an underdeveloped technique. Hence, there are several potential complications that may arise in its application to this study. First, the fiber method has never been used to image glomeruli. It is hard to predict whether individual glomeruli will be distinguishable because of their proximity to each other and because of variable optical properties of the imaging fiber. Secondly, due to less constraining structural support, the olfactory bulb may be more subject to movement when the mouse moves its head. If the bulb were constantly moving relative to the fiber, it would be difficult to maintain clear images throughout a chronic experiment. Furthermore, movement of the brain during the course of imaging may introduce motion artifacts, interfering with results. A third potential problem with the fiber technique is that the stressful nature of the surgery may cause the mice to experience stress, pain, or fear upon recovery. This may cause them to alter their behavior in response, which could affect not only odor intake, but also neural response. While these are interesting behaviors that can be studied, too high a level of stress, pain, or fear may limit other behaviors such as natural exploration.
The first goal in the development of the technique was to reproduce detailed images comparable to simple wide-field images. The concerns about pixelation and lack of optics mentioned earlier would affect the quality of the images. However, the fiber technique was successful in this respect, and consistently produced clear images, often showing clear glomeruli (Figure 2A).

 

Figure 2
Figure 2. Odor responses can be visualized with the fiber bundle. A) Image of the olfactory bulb as seen through the fiber bundle. Arrows point to sample glomeruli. B) Ratio image indicating ΔF/F values. Image is inverted with dark spots indicating an increase in fluorescence. Responding glomeruli can be seen as dark circles. Odor used was isopropyl tiglate; subject was the mouse from A. C) Enlargement of the image enclosed by the square in A. The contrast has been increased to more easily visualize fluorescence differences. The circle encloses a glomerulus at resting fluorescence. D) Similar enlargement showing the same glomerulus after odor stimulation. Contrast was increased by the same amount as in C. Comparing the two images reveals a greater amount of fluorescence after odor stimulation. This glomerulus is also the dark spot in the middle of B. E) Graph tracking fluorescence over time. Gray arrow indicates the start of odor stimulation (10 seconds) and the black arrow indicates the stop (30 seconds). This time course was not obtained from the same glomerulus as in C and D.

The next important step was to be able to consistently see glomerular odor responses. (For the rest of this paper, mentions of “glomerular responses” means a change in ORN pre-synaptic activity as a result of odor stimulation.) This was also successful (Figure 2B-E), but not as much as simply achieving clear images. While several animals showed clear responses, others did not. Often individual animals or even litters do not respond very well to odor stimulation for a multitude of possible reasons such as sickness, obstruction of the nasal passages, or other variables. Furthermore, the surgery to implant the fiber bundle is very invasive, and often damage to the dura matter of the brain could cause a failure to respond. This could be due to actual damaging of the glomeruli or, more likely, due to decreased image quality caused by dura matter damage. Finally, the bundle does not cover the entire bulb, so even strong odors may only stimulate glomeruli outside of the field of view. In order to correct for this, a test trial was run before cementing the bundle. If the testing revealed limited responsiveness, the fiber was moved and retested until either responses were seen or it was determined that the mouse would not respond, in which case it was euthanized.

The final step was to determine whether the fiber technique was suitable for imaging awake, freely behaving animals. Strong responses similar to those seen in anesthetized animals were observed. However, there were several shortcomings, as predicted. One major problem is the small size and high curvature of the olfactory bulb, which creates problems with maintaining focus. The olfactory bulb has space to move around, and during the awake trials, images often contain motion artifacts due to brain movement relative to the fiber (Figure 3A,B). There were two types of motion artifacts observed. The most common was caused by horizontal movement of the brain. When the brain shifted horizontally, so too did the glomeruli in the field of view. The movement was manifested in ratio images as a pattern of very dark and bright spots (Figure 3A). Another type of movement artifact was caused by vertical movement of the brain. Vertical movement caused glomeruli to come in and out of focus. If a glomerulus came into focus, the ratio image would show a dark spot at the new position (Figure 3B).

Figure 3
Figure 3. Olfactory bulb’s size and shape make it difficult to get reliable results. A) Movement artifact caused by horizontal movement of the brain. As the glomeruli shift, their original position becomes darker because of the loss of base fluorescence. By contrast, their new position is brighter. This is seen as a characteristic dark and bright pattern in the ratio image. B) Movement artifact caused by vertical movement of the brain. In this case, the glomeruli came more into focus, causing fluorescence increase. Vertical movement is problematic, because it often manifests as an odor response. Note the halo effect. C) Image of the olfactory bulb taken immediately after surgery. Blood vessels and glomeruli are in focus. D) Image of the same olfactory bulb taken one day after surgery. While many glomeruli are still visible, others (arrows) may disappear out of focus.

Another focusing problem that was caused by the size and shape of the bulb was simply maintaining the focus over a long time period. Given the mobility of the bulb, it was hard to keep glomeruli in focus even for a day. By the time a mouse fully recovers, the clarity of an image may be lost (Figure 3C,D). While several glomeruli may still be visible, others will be lost. This is one reason to keep the surgery and recovery as short as possible. Usually, an entire experiment can be finished within one day, preventing problems such as this.

Glomerular Pre-synaptic Activity in Anesthetized and Behaving Mice

As discussed earlier, one of the fundamental concerns of this study is to determine whether behavior has an effect on ORN output. To answer this question, images from three mice were analyzed. The images from these three mice were chosen because they were the only ones that matched a set of three criteria necessary to enhance the probability of getting reliable results. First, the images remained clear in both the behaving and anesthetized trials. Second, clear glomerular responses were observed. Finally, the animals were behaving relatively normally after recovery and did not display signs of excessive stress or pain.

The first characteristic of glomerular response studied was the pattern of the responses. Glomerular activation patterns for individual odors were compared in the same animal to determine if the patterns were similar in anesthetized and behaving animals. It was predicted that the patterns would be the same. While behavior may affect the feedback loops that can modulate the signal, it seemed unlikely that behavior would alter odor-ORN binding affinities or the wiring of the ORN. In all three mice it was found that the patterns were conserved in behaving animals (Figure 4).

Figure 4
Figure 4. Glomerular response patterns are similar in both anesthetized and behaving animals. Shown are the Z-stack projection averages derived from ΔF/F images for each of the three mice over 10 trials. Each row represents images from a different mouse. The left column displays images taken from an anesthetized mouse and the right column displays images from the same mouse while behaving. Some of the image slices were removed from the projections because of large motion artifacts. While some motion artifacts remain, the response map appears to be unaffected by behavior.

The next property studied was whether or not the signal strength was significantly different when the animal was awake as compared to when the animal was anesthetized. It was predicted that there would be a difference, although whether it would be stronger or weaker was unknown. ΔF/F values were obtained for several responding glomeruli in each of the three animals and averaged over 10 trials. Then, the mean values from the anesthetized and behaving conditions were compared. However, since ORN output can vary significantly for different odors and even different glomeruli, comparison was only done between the same glomeruli in each animal for identical odors. Also, in order to correct for motion artifacts, data from trials with large motion artifacts were removed. Somewhat contrary to what was predicted, the results (Figure 5) indicated that only one glomerulus responded to one odor, ethyl valerate, significantly more strongly in the anesthetized condition (p<0.05 2-tailed paired t-test). The other 11 glomerulus-odor combinations examined had statistically insignificant differences.

Figure 5
Figure 5. ORN output is similar in the anesthetized and behaving conditions. Shown is a bar graph pairing the mean ΔF/F values for the anesthetized and behaving conditions for various animals (A), odors (O), and glomeruli (G). These 11 glomeruli were chosen because they showed some response in either the awake or the anesthetized condition. Only A1-O2-G3 showed a statistically significant difference between the anesthetized and behaving conditions (p<0.05 2-tailed paired t-test). Odor 1 (O1): Isopropyl Tiglate; Odor 2 (O2): Ethyl Valerate. Error bars denote standard error.

While the pattern and intensity of the responses appeared to be unaffected by behavior, it was predicted that the nature of the response over time might be affected. In order to evaluate this, time course data was examined for patterns across glomeruli and animals. Unfortunately motion artifacts greatly limited the quantity of usable data, making it difficult to draw global conclusions.
Figure 6A

Figure 6 B,C
Figure 6. Animal 2 has a steeper odor response slope in the anesthetized condition. A) Time courses for the two responding glomeruli in animal 2. Gray arrow indicates stimulus start; black arrow indicates stimulus end. B) Table showing the slopes and R-squared values. The interval for which each slope was calculated varied based on the start and end of the response. The slopes for the anesthetized condition were roughly twice those of the awake condition. Slope is expressed as change in fluorescence over time.

One major difference noted in the time courses was that the slope of the fluorescence change during odor presentation varied. In animal 2, stimulation with ethyl valerate in the anesthetized condition caused a fluorescence change with a steeper slope (Figure 6). By contrast, in animal 3, stimulation with isopropyl tiglate in the anesthetized condition caused a change with a shallower slope (Figure 7). For both animals, this change was seen across all glomeruli but only during the first repeat. Subsequent trials were too variable to draw any conclusions. In addition, data would ideally come from the same odor. Hence, this difference cannot be attributed to animal differences or odor differences. Nevertheless, the differences between the two conditions in both animals are strikingly large. Interestingly, it seems that the slopes all fall around 0.05 for both animals in the anesthetized conditions. By contrast, it seems that the variance comes mostly from the behaving condition, which has a slope of only 0.025 in animal 2 but approximately 0.1 in animal 3. There are several possible explanations for this. One possibility is that animal 3 may have had a higher sniffing rate than animal 2. It is possible that animal 3 found isopropyl tiglate more pleasing than animal 2 found ethyl valerate. Animal 3 could have also been more exploratory or aware than animal 2, which may have not only increased its sniffing rate but also its mental awareness. A further study could test this theory by monitoring sniffing rate and comparing it to the slope of the response.

Figure 7 A

Figure 7 B
Figure 7. Animal 3 has a steeper odor response slope in the behaving condition. A) Representative time courses from glomerulus 1 of animal 3. Gray arrow indicates stimulus start; black arrow indicates stimulus end. B) Table showing the slopes and R-squared values. The interval for which each slope was calculated varied based on the start and end of the response. In this case, the slopes for the behaving condition were about twice those of the anesthetized condition, which is the opposite of the result found in animal 2.

The final characteristic examined was rate of recovery from odor stimulation. Only animal 2 was analyzed because the data from animals 1 and 3 had too much noise or large motion artifacts during recovery. Also, other than a few exceptions that will be discussed later, only the first trial was analyzed for the same reasons. To quantify the rate of recovery, the downward slope was calculated. It is important to note that the data included in this analysis is somewhat arbitrary because there is no time point defined as the peak and each glomerulus peaks at a different time. For both odors and across glomeruli, the odor recovery was slower during the behavior trial (Figure 8). In fact, for isopropyl tiglate, the odor responses did not peak over the course of the entire trial. The small differences in slope and the low R-squared values are less than ideal for drawing solid conclusions. However, when viewed in combination with the time course graphs, there does seem to be a trend for faster recovery under anesthesia. Furthermore, the lowest R-squared values were seen in the behaving condition, and as discussed earlier, behavior seems to cause large fluctuations in the signal as

Figure 8 A

 

Figure 8 BC
Figure 8. Animal 2 recovered from odor stimulus faster in the anesthetized state. A) Representative time courses taken from the first trial of glomerulus 1 and odor 1, isopropyl tiglate. Gray arrow indicates stimulus start; black arrow indicates stimulus end. B) Chart showing the slope and R-squared values for each glomerulus stimulated by odor 1. Note the positive slopes in the behaving condition. C) Chart showing the slope and R-squared values for each glomerulus stimulated by odor 2, ethyl valerate.

the animal recovers from odor stimulation.Furthermore, since the slope is often close to horizontal, these vertical fluctuations can significantly affect the correlation coefficient because the mean variance in the y-direction is determined almost completely by these fluctuations. By contrast, as the slope of the trendline gets steeper, the total variance in the y-direction increases. This means that the proportion of the total variance that is determined by the variance of these fluctuations decreases, thus increasing the R-squared value. Finally, the R-squared value was calculated based on a total sum of squares that only took into account variance of the data points used to create the trendline. It can be argued, however, that total sum of squares ought to be based on all data points, since that is the true variability of fluorescence. In this case, the R-squared values would be higher. All of this would suggest that even low R-squared values do not necessarily signify an erroneous trend, especially in the cases where the slope is very close to zero. The difference in recovery time could be the result of several factors. One possibility is that active exploration after the stimulus presentation may play a role in odor intake or neural response. The expunging of the odor from the stimulus chamber does not happen instantly. Rather, after the odor presentation, a weak vacuum evacuates residual odorants out of the chamber. It is unclear at what concentration or time point the mouse is no longer able to sense the odor, so it is possible that even as the concentration dwindles, the mouse increases its sniffing rate, causing the effect observed. However, as mentioned earlier, while odorant concentration was not measured, it is assumed that it was expunged relatively quickly. Given this, another explanation is that the mouse may actively suppress inhibition in an attempt to try to maintain sensation of the smell even after the odor is cleared.

Conclusions and Future Directions

From the beginning, this study has had two goals. The first goal was to explore the use of a fiber optic imaging bundle as a viable way to image the olfactory bulb in awake, freely-moving mice. The second was to investigate any differences in glomerular response based on whether the mouse was anesthetized or awake and freely-behaving.

This project is the first demonstration that a fiber optic bundle can be used to image the olfactory bulb of awake, freely-behaving mice. The results illustrate that not only can clear images showing distinct glomeruli be attained, but also changes in fluorescence can be observed, captured, and quantified. Furthermore, the unique advantage that the fiber bundle offers over other awake imaging methods such as the head-fixed method is that mice are able to freely explore their environments. Given results that indicate that exploratory behavior may have a significant effect on odor response, the fiber technique offers a way to further investigate this possibility.

However, since this method is still in its early stages, some problems remain. One issue not indicated by the presented results is that the surgery required to install the bundle on the mouse is incredibly difficult. Obtaining and maintaining clear images is a sensitive process requiring a clean and rapid surgery. Also, as shown in the results, motion artifacts could interfere with results. In order to make the fiber technique practical, it is necessary to optimize the technique to address issues such as motion artifacts. However, the success of this technique in its first application to the olfactory bulb is a testament to its potential as an invaluable tool for imaging behaving animals.

The second major finding of this study is that generalized free behavior does not seem to cause any consistent differences in glomerular responses across all animals, odors, and glomeruli. Both the pattern of the response as well as the overall strength of response were unchanged by the behavioral state. As discussed earlier, one of the main reasons for looking at ORN synaptic output was because ORNs are more upstream than the rest of the olfactory system. Knowing that odor responses do not appear to be globally modulated at the level of the ORN, it is now possible to study the mitral cells to see if a global change occurs in that layer. If a difference is observed, one can more reliably attribute the change to mitral cell modulation as opposed to a modification in ORN activity. One way to explore this possibility would be to use transgenic mice expressing the calcium-sensitive dye, GCaMP, in mitral cells.

The final major finding was that behavior causes localized changes individual to different mice, glomeruli, or odors. In two animals, a wider variability was observed after the average response began to stabilize. Differences in the rate of odor response were also seen in two mice. One mouse responded more quickly when it was behaving and the other responded more slowly. Finally, a difference in odor recovery rate was observed. For both odors presented, one of the mice recovered from odor stimulation more slowly when it was behaving. Interestingly, for one of the odors, differences were found even amongst trials for the same glomeruli. In two of the trials, relative to when it was anesthetized, the mouse recovered more slowly when behaving, but in the third, it recovered more quickly. Furthermore, the recovery rate in the anesthetized condition did not vary much. Rather, the discrepancy was caused by a large variation in recovery rate between free-behavior trials. This trend of large variations in odor response in the freely-behaving condition was also seen in the fluctuations of fluorescence, as mentioned earlier. In addition it was seen in the rate of odor response. While the slopes for the anesthetized condition were similar for both animals, the slopes for the freely-behaving condition were dramatically different. Thus it seems that while differences are individualized and seemingly unpredictable on a global scale, the general trend seems to be that behavior causes variation in the nature of the odor response.

The field of imaging awake animals holds an exciting future. Little is known about how behavior and brain activity are linked and countless questions remain unanswered. However, as mentioned earlier, before these questions can be addressed, the fiber technique needs to be optimized. One way the method can be improved is by enhancing the image quality. There are several potential ways to accomplish this. Adding focusing optics such as a gradient-index (GRIN) lens would not only increase the image quality by reducing cross-contamination of light, but it would also allow the fiber to be placed above the bulb as opposed to directly on it. In addition, adding a mechanism by which the fiber can be focused after a surgery is performed would decrease the number of experiments that fail because of bulb movement.

The preliminary results of this study inspire several future studies that can further explore some of the findings presented. Since the results indicate that exploratory behavior, possibly sniffing rate, may have an effect on odor response, a study that concurrently monitors sniffing rate and glomerular response could shed some light on this variation. One potential outcome is that sniffing rate may match closely with the level of fluorescence observed, which would explain why different mice had different odor responses while freely behaving. To further explore this, one could use calcium dyes to more clearly pair sniffing with ORN output. While synaptopHluorin is a good indicator of overall ORN activity, it cannot be used to image individual ORN action potentials. By contrast, calcium dyes react quickly to ORN activity, and one can visualize events on a much shorter time scale, including individual action potentials.

As discussed earlier, the biggest difference between the head-fixed technique and the fiber bundle technique is that with a fiber bundle, mice can freely move and behave. Thus, it would be interesting to examine whether the two techniques produce differences in glomerular response. Hypothetically, any significant differences can be attributed to exploratory behavior, and this may help to resolve whether the differences seen in this study are attributable simply to being awake or rather to being able to move around and freely explore the environment.

Finally, one could explore the effect of training on odor responses. A basic study could compare mice that are previously exposed to the experimentation setup to naïve mice. It is conceivable that mice that trained in this fashion may reduce their exploratory behavior because the environment they are experiencing is not a novel one. Another study could compare mice trained to a certain odor to mice that are just trained to general odor stimulation or even naïve mice. This might also have an effect on how a mouse approaches and explores the different odors presented.

This project is the first step towards understanding the effect of behavior on the olfactory bulb’s response to odor stimulation. While the field is new and there is a large amount of unexplored territory, hopefully this study will lay the groundwork for similar future studies. Given the promising results of this project, the fiber technique has the potential to offer a way to understand the olfactory bulb in the larger context of the whole brain and even the entire body.

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