C. Example Result
XI. Calcium Dye Recording in the Mouse Olfactory Bulb
Voltage-sensitive dyes were effective in revealing spatio- temporal patterns of global activity in the olfactory bulb (Section X). In contrast, selective labeling of olfactory recep- tor neuron terminals with a calcium-sensitive dye was used to visualize the spatial patterns in the input to the bulb. While functional imaging methods such as f MRI and intrinsic imaging reveal patterns of glomerular activity (e.g., Xu et al., 2000; Rubin and Katz, 1999), with these methods it is difficult to relate the signals to a particular class of neurons.
Labeling neurons with voltage- or calcium-sensitive dyes via retrograde or anterograde transport allows selective monitor- ing of activity in defined neuronal populations (e.g., O’Donovan et al., 1993; Tsau et al., 1996; Kreitzer et al., 2000). This approach was first developed for the olfactory system by Friedrich and Korsching (1997, 1998) in the zebrafish and later adapted for use in the turtle (Wachowiak et al., 2002) and mouse (Wachowiak and Cohen, 2001).
Spatially organized patterns of neuronal activity have long been hypothesized to play an important role in odorant recognition (Adrian, 1953; Kauer, 1991). In the mouse, the majority of olfactory receptor neurons express 1 of ~1000 olfactory receptor proteins (Malnic et al., 1999). All of the 10,000–20,000 neurons expressing the same receptor protein converge onto a few (one to three) glomeruli in stereotyped locations of the olfactory bulb (Vassar et al., 1994). Thus, odorant-evoked activity of receptor neurons distributed across the olfactory epithelium is transformed into a spatially organ- ized pattern of input to olfactory bulb glomeruli. We used calcium-sensitive dyes to image receptor neuron input to glomeruli in the dorsal olfactory bulb of the mouse.
A. Initial Dye Screening
We tested one calcium-sensitive dye, Calcium Green-1 (Molecular Probes, Eugene, OR), that was not conjugated to dextran. While it did appear to label receptor neurons in the olfactory epithelium, we did not detect labeling in the recep- tor neuron terminals in the olfactory bulb. Thus, our choice of calcium dyes was limited to those that can be obtained as dextran conjugates. We tried both Calcium Green-1 dextran and Fluo-4 dextran (Kreitzer et al., 2000). In our hands, labeling was more reliable and the fluorescence signals were larger with Calcium Green-1 dextran. We tested both the 3000- and the 10,000-kDa dextran conjugates of Calcium Green-1. No clear difference was observed. We found that a concentration of 4% Calcium Green-1 dextran resulted in sufficiently bright labeling of olfactory bulb glomeruli.
Concentrations of 2% or lower were considerably less bright.
The Calcium Green-1 dextran solution was stored at 4°C for up to 2 weeks before use. Freeze–thawing this solution appeared to result in loading of receptor neurons but little or no transport of dye to the axon terminal. While loading and transport appeared to be successful with Fluo-4 dextran, labeling of glomeruli was less clear, presumably because of the low resting fluorescence of this probe (Kreitzer et al., 2000). Also, odorant-evoked Fluo-4 dextran signals were smaller in amplitude and not as reliably detected across preparations.
B. Methods
Because dextran-conjugated dyes are membrane imperme- ant, loading olfactory receptor neurons with Calcium Green- 1 dextran requires treatment with a permeabilizing agent.
Friedrich and Korsching (1997) found that coapplication of Calcium Green-1 dextran with a dilute solution of Triton X-100 detergent was an effective method for loading zebrafish olfactory receptor neurons. Triton treatment appears to cause a transient loss of the receptor neuron cilia, which contain olfactory receptor proteins as well as the transduction machinery necessary for generating an electrical response to odorant stimulation. The cilia and molecular components necessary for odorant responsiveness appear to regenerate normally after 1–2 days (Friedrich and Korsching, 1997;
Wachowiak and Cohen, 2001). However, the concentration of Triton used in the loading procedure is critical for obtaining adequate labeling with minimal damage to the olfactory epithelium and must be determined empirically for each new species used.
For loading mouse olfactory receptor neurons with Calcium Green-1 dextran, mice were anesthetized with keta- mine (90 mg/kg, ip) and xylazine (10 mg/kg, ip) and 2 µl of 0.25% Triton X-100 in mouse Ringers was injected into the nasal cavity. After 60 s, 8 µl of 4% Calcium Green-1 dextran was injected at a rate of ~0.4 µl/min. Mice recovered from anesthesia and were held for 4–8 days before imaging. We
found that this detergent “shock” procedure labeled more consistently and with less damage to the epithelium than application of Calcium Green-1 dextran dissolved in Triton.
With higher Triton concentrations or more prolonged appli- cation, axon terminals in the olfactory bulb glomeruli were well labeled but the epithelium was severely damaged, as evaluated by the lack of an EOG response or by visual inspection. It was difficult to achieve homogeneous loading of olfactory receptor neurons projecting to glomeruli in all regions of the olfactory bulb. However, if the animal was placed on its back throughout the loading procedure, wide- spread labeling of most, if not all, dorsal glomeruli was achieved with a success rate of >70%.
For imaging, mice were anesthetized with pentobarbital (50 mg/kg, ip). A double trachaeotomy was performed so that an artificial sniff paradigm could be used for precise control of odorant access to the nasal cavity. This helped to ensure a rapid onset of the signal, which was important when multiple trials were averaged. The mice breathed freely through the lower trachaeotomy tube. The bone overlying the olfactory bulbs was thinned and Ringers solution and a coverslip was placed over the exposed area. The heart rate was maintained at 400–500 bpm by periodic injection of pentobarbital and was sometimes stabilized by directing a continuous stream of pure oxygen over the lower tracheotomy tube. The dorsal surface of one olfactory bulb was illuminated with 480 ± 25 nm light using a 150-W xenon arc lamp (Opti-Quip, Highland Mills, NY) and 515-nm long-pass dichroic mirror, and fluorescence emission above 530 nm was collected (Fig. 9, right). Images were acquired and digitized with an 80 × 80-pixel CCD camera (NeuroCCD; RedShirtImaging LLC) at 100–200 Hz and time-binned to a 25-Hz frame rate before being stored to disk. Fluorescence was imaged using a 10.5×, 0.2 NA objec- tive (spatial resolution, 22 µm per pixel assuming no scatter- ing or out-of-focus signals) or a 14×, 0.4 NA objective (16.5 µm per pixel resolution). The olfactometer used was the same as that described above for the turtle experiments.
While odorant-evoked signals were detected in single trials (e.g., Fig. 11C), we typically collected, then averaged, responses of two to four consecutive odorant presentations in order to improve the signal-to-noise ratio and to obtain a measure of trial-to-trial variability. We waited a minimum of 45 s between trials. Repeated presentations of the same odorant at this interstimulus interval evoked signal ampli- tudes that typically varied by less than 10%. The primary source of extrinsic noise was movement associated with respiration and heartbeat. The noise was largest in regions adjacent to major blood vessels, and so pixels overlying these regions were removed from the data set (omitted) prior to analysis. Occasional trials with widespread artifactual signals (primarily due to movement) were discarded. After averaging, data from each pixel were temporally filtered with a 1- to 2-Hz low-pass Gaussian and a 0.017-Hz high- pass digital RC filter (both filters have a low sharpness).
To correct for unequal labeling of glomeruli, the signal from each pixel was divided by its resting fluorescence obtained at the beginning of each trial. Because a significant part of the resting fluorescence arises from dye in axons, which appear not to experience an increase in calcium (Wachowiak and Cohen, 1999), this correction is only par- tially successful. To construct the spatial maps of input to the bulb, response amplitudes for each pixel were measured by subtracting the temporal average of a 400-ms time window just preceding the stimulus from a 400-ms temporal average centered around the peak of the response. For display, maps of response amplitudes were smoothed slightly by increasing the pixel dimensions from 80×80 to 160 ×160 and inter- polating between pixels. Response maps were normalized to the maximum signal amplitude for that trial. No spatial filtering or background subtraction was performed.
Response amplitudes for individual glomeruli were meas- ured from the maps in two ways: by averaging responses from 4 adjacent pixels in the center of the glomerulus and by measuring the amplitude of a one-dimensional Gaussian function fit to a profile (2–4 pixels wide) of the signal through a glomerulus (Meister and Bonhoeffer, 2001). The first method gives a measure of signal amplitude relative to the resting fluorescence, while the second method gives a measure of amplitude relative to the local background signal.
The maps of signal amplitude are a measure of the activation of a population of 10,000 olfactory receptor neurons con- verging onto each glomerulus.
Strongly activated glomeruli could be easily counted by visual inspection. For counting glomeruli with smaller amplitude signals, the signal profile was fit to a Gaussian and evaluated according to criteria for size (half-width within 2 SD of the mean measured for 101 test glomeruli), amplitude (amplitude greater than five times the root mean square spatial noise, measured from adjacent nonactivated areas), and appearance in multiple trials.
C. Example Result
Calcium Green-1 dextran loading resulted in labeling of olfactory receptor axon terminals in the olfactory bulb (Figs. 11A and 11B). We imaged odorant responses from the dorsal olfactory bulb of anesthetized mice 4–8 days after loading. Previous experiments using this imaging method in the zebrafish and turtle olfactory bulbs showed that the Calcium Green-1 fluorescence increases reflect action poten- tial-evoked calcium influx into receptor neuron axon termi- nals (Friedrich and Korsching, 1997; Wachowiak and Cohen, 1999). Odorant presentation evoked rapid (200- to 500-ms rise time) increases in fluorescence up to 6% ∆ F/F (Fig. 11C).
Spatial maps of the response amplitude measured from each pixel showed well-defined foci of fluorescence increases (Fig.
11D), often corresponding to individual glomeruli visible from the resting fluorescence (Fig. 11B). Odorant-evoked
signals showed a range of amplitudes in different glomeruli.
Using an expanded gray scale (Fig. 11D, inset), it is clear that even smaller signals have glomerular localization. The spatial distribution and amplitude of the signals were consistent across repeated odorant presentations and were different for different odorants.
The noise in this measurement of fluorescence from transported dye is also consistent with expectations from a shot-noise-limited measurement (Section V). In these meas- urements made at a frame rate of 100 Hz, the number of photoelectrons per pixel per millisecond was approximately 2 × 104, much lower than in the measurements from the voltage-sensitive dye described above. Because we digitally low-pass filtered the data at 2 Hz, the effective sample period is 500 ms and thus the number of photons/sample period is 107. The shot noise in this measurement should then be approximately 3×10–4 of the resting intensity (Fig. 6). Consistent with this prediction, the noise in the measurements shown in Fig. 11C is less than 2×10–3of the resting intensity. In preliminary measurements, we found
that the shot noise and the noise from respiration and heart beat were similar in magnitude. For some detectors the noise was dominated by shot noise, for others it was dominated by the extraneous movement noise.
The spatial resolution shown in Fig. 11D is on the order of 20 µm, far better than might have been anticipated from the measurements illustrated in Fig. 7. However, both factors that could contribute to blurring are minimized in the meas- urements shown in Fig. 11. First, scattering will be minimal because the Calcium Green-1 dextran is only in the outer layer of the olfactory bulb. Second, out-of-focus signals will be nonexistent because the glomerular layer is only 100 µm thick.
In addition to the rapid, dye-dependent increase in fluores- cence due to presynaptic calcium influx, odorants sometimes evoked a slower and longer lasting decrease in fluorescence, which we attributed to changes in the intrinsic optical proper- ties of the olfactory bulb tissue (absorption or light scattering).
This intrinsic signal was often apparent only at higher odorant concentrations (>1% of saturated vapor) and was strongest onl
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Figure 11 Imaging mouse olfactory receptor neuron activation after in vivo loading with Calcium Green-1 dextran. (A) Confocal image of a section through one olfactory bulb fixed 5 days after loading. Fixable rhodamine dextran (10 kDa) was used instead of Calcium Green-1 dextran to preserve labeling in fixed tissue. Glomeruli are strongly labeled. There is no evidence of transsynaptic labeling. onl, olfactory nerve layer; gl, glomerular layer; epl, external plexiform layer. (B) Resting Calcium Green-1 dextran fluorescence imaged in vivo, 7 days after loading. The image was contrast-enhanced to emphasize individual glomeruli. Blood vessels appear as dark lines. The saturated regions in the upper right are from olfac- tory nerve bundles that obscure underlying glomeruli. Lines originate from two glomeruli whose responses are shown in (C) and (D). (C) 1.9%
hexanal evoked rapid (~200 ms rise time) increases in fluorescence in the two glomeruli indicated in (B) (lines). Each trace shows the optical signal measured from 1 pixel and from a single trial after band-pass filtering from 0.017 to 2 Hz. (D) Gray-scale map of the evoked signal for the trial shown in (C), showing foci of fluorescence increases. A region of the map normalized to 50% of the maximum signal (inset) shows additional smaller amplitude foci. Signal foci in the upper portions of the image are blurred due to out-of-focus glomeruli and light scattering from overlying axons.
along major blood vessels (Fig. 12). Focal signals indicative of glomeruli were not apparent with this intrinsic signal. In freely breathing animals anesthetized with Nembutal, this signal had a slow onset and relatively small amplitude and so did not affect the spatial character or amplitude of the calcium signal. However, artificial respiration with pure oxygen caused an increase in the amplitude and speed of onset of the intrinsic signal such that the calcium signal was partially or completely obscured. A similar effect was seen in animals anesthetized with urethane (3 g/kg, ip). We have not tested other anesthetics.
Intrinsic imaging studies have reported fewer glomeruli activated by comparable concentrations of these same odor- ants (Belluscio and Katz, 2001; Meister and Bonhoeffer,
2001). These intrinsic imaging studies spatially filtered or thresholded the signals before counting glomeruli. To test whether either kind of processing might affect the number of detected glomeruli, Fig. 13B shows the response to 1.9%
hexanal (from Fig. 13A) after smoothing with a Gaussian kernel and subtracting the smoothed data (Meister and Bonhoeffer, 2001), and Fig. 13C shows the same response after thresholding the data at 2 standard deviations above the mean (Belluscio and Katz, 2001). High-pass filtering pre- serves nearly all of the glomeruli visible in the unfiltered image. However, thresholding the data eliminates many of the glomeruli.
Five glomeruli were identifiable across animals based on their location and responses to one or two odorants; all five
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Figure 12Odorant stimulation can evoke decreases in fluorescence that reflect changes in intrinsic optical signals. (A) Resting Calcium Green-1 dextran fluorescence imaged from the dorsal olfactory bulb at 14×magnification. This image was not contrast enhanced. This prepar- ation was anesthetized with Nembutal and artificially ventilated with pure oxygen. (B) Response map of the fluorescence increase evoked by hexanal. The map was constructed as described in the text, with the time window for measuring response amplitude centered at t1(880 ms after odorant onset). In this map, darker shades represent larger increases in fluorescence. There were no decreases in fluorescence apparent at this latency. Hexanal evokes input to many widespread glomeruli. The time course of the response measured from the two glomeruli indi- cated by the lines is shown in (D). (C) Response map of the “fluorescence” decrease evoked by the same presentation of hexanal. The time window for measuring the response amplitude was centered at t2(4000 ms after odorant onset). In this map, lighter shades represent larger decreases in fluorescence, with black indicating no change. There were no increases in fluorescence apparent at this latency. The largest decreases in fluorescence occur along blood vessels. (D) Time course of the optical signal recorded from the two locations indicated by the lines in (B) and (C). Each trace is from a single pixel and is filtered from 0.017 to 1 Hz. The top trace, which is from a location near a major blood vessel, shows a strong decrease in fluorescence (t2) following the initial fluorescence increase (t1).
showed complex response specificities to odorants of differ- ent functional groups and molecular size. Maps of receptor neuron input were chemotopically organized at near-thresh- old concentrations but, at moderate concentrations, they involved many widely distributed glomeruli (Fig. 11D).
These results suggest a high degree of complexity in odorant representations at the level of input to the olfactory bulb.