66 J
reviewed in Refs. [13,30,33]. The APC also appears to specific area of the LH sampled. Microinjection of bal-
have a direct role in the detection of amino acid de- anced amino acid solutions directly into the perifornical
ficiencies. Rats fed IMB have reduced threonine levels in hypothalamus depressed eating [18], suggesting that an
the APC [15], anterior cingulate cortex, locus ceruleus and area adjacent to the medial dorsal LH is sensitive to the
nucleus tractus solitarius NTS, but not in the paraven- effects of amino acids on feeding behavior. Tract tracing
tricular nucleus PVN, lateral hypothalamus LH, or studies from the area of the APC that we have found
ventromedial hypothalamus VMH [17], areas implicated sensitive to threonine injections show projections to the
in the control of food intake [2,21,27]. This suggests that LH, medial and reticular thalamic nuclei [1], suggesting
ingestion of a diet deficient in the DLAA will result in a that repletion of DLAA is recognized by a pathway that
low DLAA concentration in specific regions of the brain includes the APC and the LH.
involved with DLAA chemoreception. Furthermore, in- Sufficient evidence supports the hypothesis that the LH
jection of threonine, but not other essential AA iso- may have a role in the detection of DLAA. If the LH acts
leucine, into the APC stimulates intake of a threonine as a chemosensory area for DLAA, then injections of
IMB but not of a low protein diet [5]. Likewise, threonine threonine into the lateral hypothalamus should increase
injections into the APC stimulate intake of DEV, but not of intake of a threonine devoid diet, while injections into the
a standard diet [34]. Expression of the immediate-early LH should have no effect on COR intake.
gene, c-fos, also provides evidence that neurons in the APC are involved in DLAA chemoreception. Increased
Fos-like immunoreactivity is seen in the APC 1 h after
2. Methods
introduction of an IMB. By 2 h this Fos-like immuno- reactivity is significantly greater than for rats fed an amino
2.1. Subjects acid corrected diet COR [53].
The medial and lateral hypothalamic nuclei, along with Adult, male Sprague Dawley rats from Simonsen Lab-
the APC, are also involved in mediating the anorexic oratories, Gilroy, CA, USA weighing between 196 and 244
response to IMB or DEV diets. Electrolytic lesions of the g were used. Animal care was according to the National
DMH increase 3-h IMB intake [4]. VMH lesioned rats eat Institutes of Health guidelines. Animal protocols were
more of a mild ILE-IMB [29] and a histidine IMB diet approved by the University of California, Davis Animal
[35] but not of a severe ILE IMB [29] or of a threonine Use and Care Committee. The animals were housed
IMB diet [40]. Levels of the DLAA are decreased in the individually in hanging wire-mesh cages in a temperature-
DMH in response to IMB [15]. Onset of c-fos expression controlled room 22628C under a 12 12 h light–dark
in the DMH, infralimbic cortex, claustrum, and dorsal cycle lights off 1200 h with deionized water and food
endopiriform nucleus deep piriform cortex [39] was freely available.
similar to the APC suggesting that these areas may also be involved in the early recognition of AA deficiency [53].
2.2. Implantation of guide cannulas into the LH Evidence suggests that the LH, along with the APC,
may have a role in mediating the anorexic response to IMB Rats were surgically implanted with hypothalamic can-
or DEV diets. Differential activation of neurons was seen nulas according to methods previously described [42].
in the dorsal LH in response to cues associated with lysine Each rat was anesthetized as previously described [8] with
in rats deficient in lysine [17]. Neuronal activation is a ketamine cocktail 1 ml kg body weight, i.p., containing
observed in the LH following injections of threonine into 5 ml ketamine hydrochloride 100 mg ml, Fort Dodge, IA,
the APC of animals fed DEV [34], but little change is seen USA, 1.25 ml xylazine 20 mg ml, Mobay, Shawnee,
in animals fed a standard diet or those receiving saline KA, USA, 0.75 ml acepromazine maleate 10 mg ml,
injections. However, c-fos expression in the LH of rats fed Avco, Fort Dodge, IA, USA. The animals were placed in
IMB was not significantly different from rats eating a a stereotaxic apparatus with the incisor bar positioned 3.3.
threonine basal or COR diets [53]. There is evidence that mm below the ear bar. Bilateral stainless steel guide
suggests a chemosensory function for neurons in the LH. cannulas 1860.05 mm tolerance, 26 gauge, Small Parts,
Electrophoretic application of essential amino acids acti- Miami Lakes, FL, USA were stereotaxically directed to a
vates neurons in the medial forebrain bundle region of the position 1 mm dorsal to the intended area, ventral LH
LH [31]. Detection of brain oxygenation using magnetic [coordinates mm: anteroposterior to interaural line A
resonance imaging revealed that lysine deficiency induces P5A5.9, lateral L 51.9, dorsal D 58.5 9.5] and
changes in the LH and VMH. Microinjection of lysine, but dorsal LH [A5.9, L51.9, D58.4 9.4] using the coordi-
not other amino acids into the LH decreased bar pressing nates of Paxinos and Watson [36]. The cannulas were
for a balanced diet following overnight exposure to a secured to the surface of the skull with dental acrylic
lysine deficient diet [51]. As noted, threonine concen- Hygenic, Akron, OH, USA anchored by stainless steel
trations in the LH do not appear to be reduced with an screws. A 33-gauge obturator was inserted into the guide
IMB diet [17]. Likely, this result may depend on the cannulas in order to maintain patency. An antibiotic
J .E. Blevins et al. Brain Research 879 2000 65 –72
67
[penicillin Aquacillin, Vedco, St. Joseph, MO, USA Systems, West lafayette, IN, USA. Bilateral injections 0.3
300 000 units ml; 60 000 units IM] was administered at ml per side of either saline or
L
-threonine Fluka, Ronkon- the completion of surgery.
komoa, NY, USA were delivered simultaneously at a constant rate of 0.06 ml min within 2 h prior to the start of
2.3. Diet composition the dark cycle. The volume of injectate was verified by
forward movement of an air bubble to a pre-calibrated Experimental diets, which have been described previ-
distance 3 mm, corresponding to the appropriate volume. ously [6] Table 1, and water were available ad libitum.
Injection needles were left in place for approximately 60 s These diets included free L-AAs as the protein source,
after the injection was completed before being removed with cornstarch and sucrose 2:1 as the carbohydrate
slowly. Following each injection, obturators were replaced source, 5 corn oil as the fat source, 1 vitamin mix, and
and each animal was returned to its cage. Food bowls were 5 salt mix. The threonine basal diet was a low protein
returned to the respective cages at the completion of all diet, providing approximately 12 crude protein equiva-
injections. lent, with threonine as the growth-limiting AA, present at
about 40 of its requirement for the growing rat [23]; all 2.4.1. Trial 1: Effects of threonine injections into the
other essential amino acids were added to account for ventral LH in rats fed a DEV diet
approximately 70 of their requirement for the growing Rats n524, 210–244 g fed moistened DEV diet 20
rat. The threonine corrected diet contained a balanced H O v:w received bilateral injections of threonine 0,
2
pattern of amino acids at approximately 100 of the 0.25, 0.5, 1, 2, 4 nmol in saline into the LH. Food intake
requirement. The DEV contained an excess of essential was measured at 3, 6, 9, 12, and 24 h post-injection.
AAs other than threonine and was completely lacking in threonine. For the addition of the AAs, a proportionate
2.4.2. Trial 2: Effects of threonine injections into the amount of the carbohydrate fraction was removed. Ani-
dorsal LH in rats fed a DEV diet mals were placed on the threonine basal diet for 7–16 days
Due to the ineffectiveness following THR injections into post surgery.
cannulas positioned within the ventral LH, in a separate Following implantation of the hypothalamic cannulas,
group of animals n520, 200–217 g threonine was rats were adapted to handling and a 2-h fast prior to the
administered in
an identical
manner via
cannulas start of the dark cycle. Food intake was determined by
positioned 0.1 mm dorsal and intake of dry powdered DEV weighing food bowls to the nearest 0.1 g and correcting for
diet was measured. No differences in meal patterns have spillage.
been observed between moistened and powdered IMB diets unpublished data. Therefore, both diets were ex-
2.4. Injection procedure amined in this study. Although evaporative loss from the
moistened diet was negligible, the powdered diet was used Injections were made by lowering the 33-gauge injector
in the trial 2 and in the COR diet study due to the with its tip extending 1 mm beyond the guide cannula into
possibility of error associated with animals getting the diet the brain site of interest. Each injection needle was
on their fur over time. connected via PE-20 tubing O.D. 0.0430 I.D. 0.0150
Plastics One, Roanoke, VA, USA to a 10-ml syringe fitted 2.5. Effects of threonine injections into the dorsal LH in
into a microinjection pump CMA 100, Bioanalytical rats fed a COR diet
Table 1
Rats n519, 196–212 g fed a COR diet received
Composition of diets of diet by weight
bilateral injections of threonine 0, 0.25, 2.5 nmol in saline into the dorsal LH, the same site where THR
Ingredient BAS
COR IMB
DEV
a
stimulated intake in both moistened and powdered DEV.
Dispensable AA 7.53
7.53 7.53
7.53
b
These doses covered the range of effective doses that
Indispensable AA 4.18
13.42 13.36
13.28
L
-Threonine 0.20
0.45 0.20
–
stimulated intake of DEV 0.25, 2 nmol and tested
c
Vitamins 1.00
1.00 1.00
1.00
whether the stimulatory effect of THR on DEV was
d
Salt mix 5.00
5.00 5.00
5.00
specific to a DEV situation. Food intake was measured as
Corn oil 5.00
5.00 5.00
5.00
described above.
Sucrose 25.66
22.50 22.60
22.70 Cornstarch
51.33 45.00
45.21 45.39
Choline Cl 0.10
0.10 0.10
0.10
2.6. Histological verification of injection sites
Total 100.00
100.00 100.00
100.00
Upon completion of the experiments, animals were
a
Dispensible Amino Acid Mix Ajinomoto, USA.
b
sacrificed by ether inhalation and immediately decapitated.
Basal Indispensible Essential Amino Acid Mix Ajinomoto.
c
This was followed by subsequent removal of the guide
Total Vitamin Supplement United States Biochemical.
d
Rogers–Harper Salt Mixture United States Biochemical.
cannula, cannula crown, and the brain, respectively. Brains
68 J
were stored in 10 formalin for 24 h followed by 10 3. Results
sucrose for at least 48 h prior to freezing and sectioning. Coronal sections 40 mm were taken through the most
3.1. Effects of threonine injections into the ventral LH ventral portion of the cannula track. Sections were
on intake of DEV diet mounted on microscope slides and allowed to dry for 24 h
prior to staining with Cresyl violet. A projection micro- Threonine injections into animals n58 that had can-
scope was used to project the image of the brain section nulas placed within the ventral LH as described in Section
showing the most ventral portion of the injectate Fig. 1, 2 did not affect intake of the moistened DEV diet at any
defined as the injection site, onto the corresponding section time measured data not shown.
from the rat brain atlas of Paxinos and Watson [36]. In trial 1, data were grouped based on cannula placements either
3.1.1. Trial 1. Moistened DEV diet dorsal LH symmetrically within the ventral LH or animals having one
Threonine injections into cannulas placed stimulated injection site within the ventral LH and the other at the
moistened DEV intake n54 at 0.25 nmol through the level of the fornix. Similarly, in trial 2, data were grouped
first 9 P50.056, trend and 12 h P50.046 by 35 and based on symmetrical injection sites within the dorsal LH
30, respectively data not shown. A similar response or had one injection site within the dorsal LH and the other
was seen in an animal whose placement was slightly at the level of the fornix.
ventral to the fornix on one side and dorsal to the fornix, ventral to the internal capsule on the other, 121 and 39
stimulation at 6 h and 29 and 62 stimulation at 9 h at 2.7. Statistical analyses
0.25 and 1 nmol respectively. Food intake over 24 h following saline injections was 12.6461.18 g, which was
Data are presented as means6S.E.M. Results from each not significantly different from intake at this time follow-
study were analyzed separately by one-way ANOVA. ing threonine injections at any dose.
Results were considered significant if P,0.05. Each animal served as its own control in a crossover design with
the exception of the effects of threonine into the dorsal LH 3.1.2. Trial 2. Powdered DEV diet dorsal LH
on intake of corrected diet. All analyses were conducted In a separate group of animals, threonine administered
using version 7.0 of
PC
-
SAS
SAS, Cary, NC, USA. into sites dorsal to the fornix Fig. 2 tended to stimulate
Fig. 1. Representative photomicrograph taken of injection sites within the dorsal LH.
J .E. Blevins et al. Brain Research 879 2000 65 –72
69
Fig. 3. Schematic representation from Paxinos and Watson showing the injection sites corresponding to the dorsal LH in animals exposed to the
DEV diet. The symbol j is used to designate injection sites included in
the data analysis.
4. Discussion
