Prior to testing, all animals were group housed according to sex in standard shoebox cages ... at least 3 licks with ILIs not greater than 1s) (Glatt et. al. 2016), and ...
Look at me, I can draw
A Simple Method for the Rapid Induction of Salt Appetite in Mice Martin A. Raymond, Tasnia Chowdhury, Thomas G. Mast, and Joseph M. Breza Eastern Michigan University, Ypsilanti, MI, United States
Background
Results: Pharmacology
Conclusions
We Look at me, I can draw
In this experiment, two key comparisons were made across a variety of dependent variables. First, comparing Amiloride to the control condition to establish whether the drug is independently
• The ability to manipulate salt appetite in animal subjects possesses considerable utility in behavioral taste
capable of inducing salt appetite in B6 mice. Second, comparing the light treated Amiloride to the
research.
unadulterated solution to establish whether light was capable of reducing the potency of the effect. • Most methods for achieving this end are highly invasive, requiring surgical intervention or subcutaneous
Based on the results of the data analysis, both of these questions are fairly clearly addressed:
injections.
• Amiloride alone is sufficient to induce powerful sodium appetite in C57BL/6J
• Even the least invasive method (dietary sodium restriction) is relatively slow-acting, and involves some
mice.
disruption of the animals’ normal environment.
• Exposure to light has no significant effect on the potency of Amiloride. • Caloiero and Lundy (2004) demonstrated that for rats, this procedure could be accelerated by replacing water with a 100µM solution of Amiloride (a potassium-sparing diuretic).
These findings have several useful implications. First, dietary Amiloride exposure has several advantages over dietary sodium restriction; it works relatively quickly (36hrs), it is relatively
• We sought to replicate and expand those findings, assessing whether self-administration of a 300µM
inexpensive and does not spoil, and since Amiloride is undetectable by mice (Eylam et al. 2003), it
Amiloride solution could induce sodium appetite in C57BL/6J mice without dietary sodium restriction.
involves essentially no disruption of the animals’ normal environment (i.e. diet) prior to testing. Additionally, at least under the parameters of this experiment, no special care needs to be taken to
Methods
protect Amiloride solutions from light in the animals’ environment (though it remains advisable to
Subjects
minimize light exposure during long-term storage).
A total of 18 naive animals were used in this study, including 8 females (26.5 ± 0.96g) and 10 males (37.8 ± 1.93g). All animals were C57BL/6J mice (x̅ = 156 days). Prior to testing, all animals were group housed according to sex in standard shoebox cages with free access (LabDiet #5015) to chow and tap water.
Preference score, burst duration, mean lick total, Mean Primary Interlick Interval, and Lick Efficiency for Sodium solutions all vary significantly between the Amiloride and control conditions, but not between Amiloride conditions. Mean NaCl lick total for Amiloride = 730, Mean NaCl lick total for controls= 94.
Additionally, the consistency of the lick recordings with patterns observed in existing literature suggests that the apparatus was able to reliably capture accurate licking behavior. This consistency is best illustrated by the distribution of interlick intervals in the motivated animals; not
Amiloride
only is the main distribution of licks centered around 120ms, there is also evidence of a “second,
Powdered Amiloride was dissolved in tap water to a concentration of 300µM, and half of this solution was set aside to be used directly. In an attempt to create an inactivated analog for the drug, the remainder of the solution
Amiloride vs H2O
Light Amil. vs Standard Amil.
much smaller symmetrical distribution of ILI frequency centered at twice this value” (Glatt et al.
Preference Score
F(1,33) = 127.90;
p < 0.001, R2 = 0.79
F(1,33) = 0.58;
p=0.45,
R2 = 0.02
2016). That secondary distribution is particularly important here; its routine presence in the
Total Licks (NaCl)
F(1,33) = 28.18;
p < 0.001, R2 = 0.46
F(1,33) = 0.57;
p = 0.46,
R2 = 0.02
recordings captured by this apparatus suggests that the equipment is not merely sensitive enough
72hrs. The two Amiloride solutions were then placed in the home-cage water bottles of the mice in the
Total Licks (H2O)
F(1,33) = 2.33;
p = 0.14,
R2 = 0.07
F(1,33) = 0.32;
p = 0.57,
R2 = 0.01
to capture the typical trends of licking behavior, it is sensitive enough to capture the same obscure
experimental groups.
Licks/Burst (NaCl)
F(1,33) = 34.93;
p < 0.001, R2 = 0.51
F(1,33) = 0.01;
p = 0.92,
R2 = 0.00
phenomena seen in commercially available lickometers.
Lick Efficiency (NaCl)
F(1,33) = 59.51;
p < 0.001, R2 = 0.64
F(1,33) = 0.70;
p = 0.41,
R2 = 0.02
was placed in a clear glass flask and exposed directly to a 40W incandescent lamp (shown in Figure 1). The lamp was turned off periodically to avoid overheating the solution, but total exposure time amounted to approximately
Procedure Approximately 36hrs prior to testing, water bottles were changed on the home cage (water + 300µM amiloride). Access to food (LabDiet #5015) and water were not restricted. At the time of testing, animals were removed from the home cage and placed within a custom-built lickometer apparatus (shown in Figures 2 & 3). Inside the chamber, animals had 30min of free access to two solutions; 0.3M NaCl and tap water. Licking data on both
Future Directions
Results: Technology
spouts were collected by the apparatus, and recorded in the open-source audio program Audacity. Each trial was
A literature review revealed that the wavelength of peak absorption of Amiloride is deep into the ultraviolet,
repeated on the following day with the bottles reversed to account for any inherent side preference.
out of the emission range of incandescent lamps. As such, it remains possible that treating the drug with intense UV light, and/or for a longer period of time, may have the desired effect.
Data Analysis Raw data recordings were exported from Audacity as .CSV files (Spike2), and imported into R (an open-source statistical software package). These data were processed via a custom script written to accompany the apparatus,
Additionally, this research sets the stage for future behavioral study using optogenetic mechanisms.
calculating total lick counts on both spouts, as well as MPI (Mean Primary Interlick Interval; the mean of all ILIs
Knowing that Amiloride alone is sufficient to rapidly induce salt appetite provides the required counterfactual
between 50 and 160ms), lick efficiency (% of ILIs under 160ms), frequency and duration of lick bursts (clusters of
for assessing the impact of light stimulation on the tongues of genetically altered animals. Specifically, some
at least 3 licks with ILIs not greater than 1s) (Glatt et. al. 2016), and frequency and duration of pauses between
pilot data has been collected using animals with photoreceptors (ChR2) selectively expressed in Type 1 cells,
bursts. Once this information had been computed, R was also used to conduct statistical analysis of the data.
showing that while the animals are sodium-deplete, they demonstrate a behavioral preference for water
Statistical tests were comprised primarily of contrast analyses when parametric tests were permissible, and
illuminated with light over plain water. This method will be useful in assessing a potential link between Type 1
Kruskal-Wallis tests with subsequent pairwise Wilcox tests when data were not normally distributed.
cells and an appetitive salt-sensing pathway.
References Caloireo VG, & Lundy RF (2004). A novel method for induction of salt appetite in rats. Brain Research Bulletin 64(1):1-7.
Figure 1: Amiloride exposed to light
Figure 2: Chamber exterior
Figure 3: Chamber interior
Distribution of Interlick Intervals matches expected findings: most intervals fall around 120ms, with a secondary distribution around 240ms, consistent with the literature (Glatt et al. 2016) .
Eylam S, Tracy T, Garcea M, & Spector AC (2003). Amiloride is an ineffective conditioned stimulus in taste aversion learning in C57BL/6J and DBA/2J mice. Chem Senses 28(8):681-689.
Glatt AR, St. John SJ, Lu L, Boughter JD (2016). Temporal and qualitative dynamics of conditioned taste aversions in C57BL/6J and DBA/2J mice self-administering LiCl. Physiology & Behavior 153:97-108.
