Biomedical Engineering Reference
In-Depth Information
olfactory epithelium, we did not detect labeling in the 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
(51)
.
In our hands, labeling was more reliable and the fluorescence
signals were larger with Calcium Green-1 dextran. We tested
both the 3,000 kD and the 10,000 kD dextran conjugates of
Calcium Green-1. No clear difference was observed. Yaksi and
Friedrich
(55)
used rhod-dextran but noted that the signal-to-
noise ratio was lower than that obtained with the green calcium
indicators.
Because dextran-conjugated dyes are membrane impermeant,
loading olfactory receptor neurons with Calcium Green-1 dex-
tran requires treatment with a permeabilizing agent. Friedrich
and Korsching
(53)
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.
For imaging, mice were anesthetized with pentobarbital (50
mg/kg, i.p.). A double tracheotomy was performed so that an
artificial sniff paradigm using the upper tracheotomy tube allowed
for precise control of odorant access to the nasal cavity. This
helped to ensure a fixed and rapid onset of the signal which was
important when multiple trials were averaged. The mice breathed
freely through the lower tracheotomy tube. The dorsal surface
of one olfactory bulb was illuminated with 480
2.3.2. Methods for the In
Vivo Mouse Preparation
25 nm light
using a 150 W Xenon arc lamp and 515 nm long-pass dichroic
mirror, and fluorescence emission above 530 nm was collected
(
Fig. 3.8
, right panel). Images were acquired and digitized with
an 80
±
256 pixel CCD camera (NeuroCCD-SM or
NeuroCCD-SM256; RedShirtImaging LLC, Decatur, GA) and
stored on disk at a 25 or 32.25 Hz frame rate. Fluorescence
was imaged using a 10.5
×
80 or 256
×
×
, 0.2 N.A. objective (spatial resolution,
20
m per pixel assuming no scattering or out-of-focus signals)
or a 14
μ
m per pixel resolution). The
olfactometer
(56)
was an improved version of the one used in
Lam et al.
(57)
.
While odorant-evoked signals were detected in single trials
(e.g.
Fig. 3.9C
), we typically collected, then averaged, responses
of two to eight consecutive odorant presentations in order to
improve the signal-to-noise ratio and to obtain a measure of trial-
to-trial variability. To avoid habituating the response, we waited
a minimum of 60 s between trials. The primary source of extrin-
sic 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 sometimes removed
from the data set (omitted) prior to analysis. Occasional trials with
widespread artifactual signals (primarily due to movement) were
×
, 0.4 N.A. objective (15
μ
