 |
INTRODUCTION |
Rats have the ability (within 30 min) to recognize and reject a
diet that is imbalanced with respect to indispensable amino acids
(IAAs; Gietzen et al. 1986a
). Previous work suggests the anterior piriform cortex (APC) is the site of recognition of amino acid
imbalanced diets (IMB) (Leung and Rogers 1971
). Our
results indicate that the anorectic response to IMB is associated with a decrease in the limiting IAA in the APC (Gietzen et al.
1986b
) and that repletion of the limiting IAA by injection into
the APC prevents the anorectic response. This effect was shown to last for 24 h and to depend on protein synthesis (Beverly et al.
1990
, 1991
, and 1993
).
This work examines signals that first indicate repletion of a limiting
IAA in the APC. To achieve repletion, we developed an in vitro model to
examine cellular signaling in the APC after short term deprivation of
an IAA. This study examines the role of calcium signaling in APC slices
on repletion of threonine after short-term culture in threonine-devoid
medium. To examine whether our findings extend to IAAs other than
threonine, we conducted similar experiments with lysine as the limiting IAA.
 |
METHODS |
Dams and litters of Sprague-Dawley rats were housed in
polyethylene cages at 22 ± 2°C (SE) on a 12:12 h
light:dark cycle. Dams were given access ad libitum to rat chow (5012, PMI Feeds) and water. Protocols followed NIH guidelines as approved by
the U. C. Davis Animal Use and Care Committee. Rat pups, aged 7 to 21 days, were killed by decapitation. A portion of the brain containing the APC was prepared by making a transverse cut at the level of the
optic chiasm; then with the use of the cut surface of the anterior
portion of the brain as a base, a horizontal cut was made at the level
of the olfactory bulb. Tissue was glued (cyanoacrylate) to the
microtome stage and submerged in a bath of Earle's Balanced Salt
Solution (EBSS, Sigma) maintained at 0-4° and equilibrated with 95%
O2-5% CO2. Slices 150-200 µm thick were
made using a Campden Instruments 752 M Vibroslice. Slices were 18°
from a true transverse section (i.e., perpendicular to the pial
surface) to preserve the integrity of the apical dendrites of the
pyramidal cells in layer II. Ten to 15 slices, rostral to the closure
of the anterior commissure, were prepared and transferred to oxygenated EBSS. Six to 8 of the best slices were selected from each rat, and
whenever possible, one hemisphere was transferred to complete medium
(control), the other to medium devoid of either threonine (
Thr) or
lysine (
Lys). The medium was prepared according to Brewer et
al. (1993)
with amino acids (injectable medical grade) from
Ajinomoto and all other components (puriss. grade) from Fluka Chemika-Biochemika. Slices were transferred to 30 mm Millicell-CM culture plate inserts (0.4 µm, Millipore), then placed over 1 ml of
the appropriate medium in six-well polypropylene culture plates and
incubated for a period of 4-6 h at 37°C in a 5% CO2 atmosphere. Slices were washed from inserts with EBSS, placed in 2 ml
EBSS containing 5 µM fura-2 acetoxymethyl ester (fura-2 AM, Molecular
Probes) and incubated for an additional 45 min. Slices were rinsed
three times and maintained in EBSS bubbled with 95% O2-5%
CO2. Slices were transferred to a glass coverslip held in a
clamp chamber (Medical Systems, Chicago, IL) and were restrained with a
device made from a ring of polypropylene, covered on one side with a
grid of silk suture thread. Each slice was covered with an initial
volume of 360 µl EBSS. This apparatus was placed on an inverted
microscope (Nikon Diaphot) equipped with a temperature-controlled stage
maintained at 37°C. The APC was located under ×10 magnification and
layer II, the pyramidal cell body layer, was viewed under ×20
magnification. Fura-2 was excited alternately at 340 and 380 nm;
fluorescence emission was recorded at 510 nm. Ratio imaging was carried
out using an ImageMaster Ratio Fluorescence Imaging System consisting
of a Photon Technology International Deltascan 4000 dual wavelength
monochromator, a Hamamatsu intensified CCD camera, and ImageMaster
software. Experiments typically examined three to eight each control
and deficient slices from one rat. For experiments with threonine as
the limiting IAA, a total of 62 control and 70 deficient slices were
imaged from a total of 28 rats. Confirmation of these results with
lysine was carried out by imaging 20 slices from a total of 5 rats,
with approximately equal numbers of control and deficient slices.
Results are expressed as the ratio of fluorescence emission obtained at excitation wavelengths of 340 and 380 nm (R340/380).
R340/380 was chosen for expressing results because regions
of interest (ROIs) imaged within any given slice showed considerable
variation in initial Ca2+ level, but similar magnitude of
change in [Ca2+]i when expressed as the ratio
of fluorescence emission.
 |
RESULTS |
APC slices incubated with either complete,
Thr, or
Lys medium
were imaged during the introduction of a number of amino acids (AAs),
all at a final concentration of either 1 or 10 mM. In
Thr and control
slices the effects of addition of threonine, isoleucine, serine,
alanine, and two other AAs with known neurotransmitter activity,
glycine and glutamic acid, were examined. In
Lys and control slices
the effects of addition of lysine, threonine, and glutamic acid were
examined. The addition of AAs was followed by 50 mM KCl.
In about 20% of the brains examined, there was a clearly
distinguishable rise in [Ca2+]i when the
limiting IAA was added to a deficient slice. This effect was never seen
in control slices. In some brains, more than one deficient slice
responded to the limiting IAA and in others, one or none. The response
was seen in brain slices from rats across the entire age range of 7 to
21 days. Figure 1 shows the response to
threonine in a
Thr slice (Fig. 1A) and its control from
the same rat (Fig. 1B). In Fig.
2, using only one responding deficient
slice and its control per rat, results of threonine addition were
expressed as the change in R340/380 in relation to baseline
before AA addition (Fig. 2A) and in relation to the response
to glutamic acid (Fig. 2B). By either measure, the addition of threonine to
Thr slices gave rise to a significant increase (Student's t-test, n = 5, P = 0.002, and P = 0.01, respectively) in
R340/380, and thus in [Ca2+]i.
That this effect is not unique to threonine as a limiting IAA is shown
by Fig. 3 in which an increase in
[Ca2+]i is seen in response to lysine
addition to a
Lys slice (Fig. 3A) but not its control
(Fig. 3B). This effect was found in slices of two of five
brains examined and was significant (Student's t-test,
n = 2, P = 0.009) when expressed as a
percentage of the response to glutamic acid.

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Fig. 1.
Effect of threonine on [Ca2+]i in anterior
piriform cortex (APC) slices cultured with either threonine-devoid
(A) or complete medium (B). Threonine
(Thr), glutamic acid (Glu), and glycine (Gly) were added at a final
concentration of 10 mM and KCl at 50 mM.
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Fig. 2.
Change in R340/380 in response to threonine addition to
control and Thr slices expressed as percent increase of above
baseline (A; n = 5, P = 0.002) and as percent of response to glutamic
acid (B; n = 5, P = 0.01).
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Fig. 3.
Effect of lysine on [Ca2+]i in APC slices
cultured with either lysine-devoid (A) or complete
medium (B). Lysine (Lys) and glutamic acid (Glu) were
added at a final concentration of 10 mM and KCl at 50 mM.
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|
To determine whether the response in
Thr slices was unique to the
limiting IAA, we conducted experiments in which another IAA,
isoleucine, was added first, followed by threonine. In the responsive
slices (3 of 8 brains examined) the addition of threonine to
Thr but
not control slices resulted in increased
[Ca2+]i, whereas the addition of isoleucine
failed to do so in either control or
Thr slices [analysis of
variance (ANOVA); F3,8=15.766, P = 0.04].
We also examined the effect of other small neutral amino acids on
[Ca2+]i in
Thr slices. These AAs are
presumably transported into brain cells by the same transport protein
as is threonine (Shotwell et al. 1983
). Serine addition
to both
Thr and control slices caused an increase in
[Ca2+]i, with the response being more
pronounced in deficient slices of two of five rats examined. In
addition, serine elicited an increase in
[Ca2+]i in
Thr, but not control slices,
after [Ca2+]i was elevated by prior addition
of both threonine and glutamic acid.
 |
DISCUSSION |
The increase in [Ca2+]i in response to
threonine in
Thr slices and to lysine in
Lys slices suggests that
Ca2+ signaling in APC pyramidal cells may be an early, if
not the first, mechanism by which repletion of a limiting IAA is
recognized in the brain. The fact that this effect was not seen in
every deficient slice, nor in every ROI imaged, may be explained by the
hypothesis that there is a specific region within the APC that
functions as the chemosensor. A precedent for this hypothesis comes
from the study of the APC in seizures, in which it was proposed that a
specific region, the area tempestas, is highly seizurogenic in response
to chemical stimulation (Gale 1989
). Because areas of
the brain other than the APC were not examined, it is not possible to
conclude that the increase in [Ca2+]i in
response to a limiting IAA is unique to the APC.
Neither the addition of the nonlimiting IAA isoleucine to
Thr slices,
nor the addition of the nonlimiting IAA threonine to
Lys slices led
to an increase in [Ca2+]i in either control
or deficient slices. These results suggest that this effect is specific
to the limiting IAA; however, they do not rule out the possibile
involvement of specific AA transporters.
Results of preliminary experiments support the hypothesis that the
mechanism by which threonine exerts its effect on
[Ca2+]i in
Thr slices may involve a
transporter of small neutral AAs. Serine addition led to increased
[Ca2+]i in both
Thr and control slices,
suggesting that it may have neurotransmitter-like activity in our
preparation (Smith 1996
); however, the response to
serine in
Thr slices appeared greater than in control slices and was
observed even after the elevation of [Ca2+]i
by glutamic acid.
These results support the hypothesis that the APC functions in
recognition of a limiting IAA. An increase in
[Ca2+]i may be the first signal by which this
recognition is accomplished, leading to a cascade of events which
ultimately influences the behavior of the animal to accept or reject a
food on the basis of its effect on AA homeostasis.
The University of California, Davis, is a National Institute for
Environmental Health Sciences Center for Environmental Health Sciences
(05707), and support for core facilities used in this work is
gratefully acknowledged. The authors thank Drs. Charles Plopper and
Hilary Benton for scientific consultation and A. Rechs for custom
apparatus preparation. We also thank C. Helton of the School of
Veterinary Medicine Cellular Imaging Facility for technical support and
advice, and E. Kuehnis and R. Moore for imaging assistance.
This work was supported in part by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-50347 and DK-35747 and by
National Research Institute Competitive Grants Program/United States
Department of Agriculture Grant 94-37200-0655.
Address for reprint requests: D. W. Gietzen, Dept. of Anatomy,
Physiology and Cell Biology, School of Veterinary Medicine, University
of California, Davis, One Shields Ave., Davis, CA 95616.