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1 Neuroscience Program, Surgical Metabolism and Nutrition Laboratory, Department of Surgery, SUNY Upstate Medical University, University Hospital, Syracuse 13210; and 2 Department of Quantitative Methods, Syracuse University, Syracuse, New York 13244-2130
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Our past microdialysis studies in ventromedial hypothalamic nucleus (VMN) and lateral hypothalamic area (LHA) of changes in dopamine concentrations in response to changes in food intake [characterized as feeding pattern (changes in meal number and size)] in anorexia of cancer show abnormal presynaptic dopaminergic neurotransmission. To determine postsynaptic receptor status, studies were done in tumor-bearing (TB) and non-tumor-bearing (NTB) free-feeding control rats while continuously measuring their food intake via a rat eater meter. When TB rats developed anorexia, TB and control rats were killed, and postsynaptic D1- and D2-receptor mRNA expression in LHA and VMN were measured via RT-PCR. At anorexia, food intake decreased initially by a decrease in meal number, whereas a concurrent increase in meal size occurred for 24 h in an attempt to maintain food intake constant. Then meal size also decreased. At this time, D1- and D2-receptor mRNA expressions in LHA and VMN of TB vs. controls were significantly upregulated. Verification of D1- or D2-receptor changes to changes in meal number and size at anorexia was made by injection of intra-VMN or -LHA dopaminergic receptor antagonists. Intra-VMN D1-receptor antagonist (SCH-23390) in TB rats decreased food intake mainly via a decrease in meal size. Intra-VMN D2-receptor antagonist (sulpiride) in TB rats increased food intake via an increase in meal number and in NTB free-feeding rats by an increase in meal size. Intra-LHA D1-receptor antagonist in TB rats had no effect on food intake or feeding pattern. Intra-LHA D2-receptor antagonist in TB and in NTB free-feeding rats increased food intake via an increase in meal number. Our data provide evidence that postsynaptic dopaminergic receptor subtypes in the hypothalamus are involved in the regulation of meal size, meal number, and thus food intake in anorectic TB rats.
messenger RNA; dopaminergic receptor antagonist; sulpiride; SCH-23390; meal size; meal number
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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TUMOR GROWTH IS FREQUENTLY associated with anorexia and reduced food intake (FI), which significantly contributes to the development of malnutrition and eventually cachexia (17), worsening cancer patients' prognoses. The mechanism(s) of anorexia in cancer is multifactorial and includes both peripheral and central factors (35), and it awaits further definition so as to develop rational treatment to prevent its detrimental outcome.
Daily FI is a function of meal size (MZ) and meal number (MN) (FI f MZ × MN), which constitute the feeding pattern. Under normal conditions, an increase in MZ is offset by a decrease in MN, and vice versa, to maintain constancy of daily FI. This suggests that MZ and MN are regulated independently via closely coordinated systems (20, 24). The Automated Computerized Rat Eater Meter (ACREM) (23) measures individual MZ and MN in each rat over prolonged periods. It provides us with the ability to characterize the behavioral expression associated with the biochemical findings in the hypothalamus of normal rat FI behavior and to compare it to that at the onset of the anorexia of cancer.
In the methylcholanthrene (MCA)-induced sarcoma-bearing male Fischer rat, anorexia develops with progression of tumor growth so that a characteristic feeding pattern is observed. At the onset of anorexia, a decrease in FI occurs initially via a decrease in MN. Concurrently, an increase in MZ occurs that lasts for ~24 h to partially compensate for the decrease in FI. Then MZ also decreases and anorexia becomes profound (25, 26) and persistent, leading ultimately to the rat's demise. The study of simultaneous changes in MZ and MN, as opposed to studying only FI, provides a more insightful dynamic picture reflecting integrated behavior.
In the tyrosine hydroxylase knockout mouse model, which lacks dopamine (DA), aphagia occurs. This is normalized with tyrosine hydroxylase gene delivery into the striatum (42). In comparison, in the neuropeptide Y (NPY) knockout mouse model, normal FI and body weight are observed (12). Thus relatively speaking, DA forms a critical link and plays a significant role in the mechanism of feeding behavior. However, the target structure(s) for DA in the hypothalamus as it relates to FI regulation has yet to be fully defined. Anatomic, electrophysiological, neurochemical, and functional studies (31, 40) link the lateral and medial hypothalamus in their reciprocal regulation of FI and energy metabolism. We have thus focused our studies on measuring the changes in DA concentrations in the lateral hypothalamic area (LHA) and in the ventromedial hypothalamic nucleus (VMN) in relation to changes in MZ, MN, and thus FI (22).
Our previous findings in normal and in obese rats show increases in DA concentrations in the LHA, as measured via in vivo microdialysis, which correlate to increases in MZ (21, 27, 48, 49). In addition, decreases in DA concentrations in the VMN are related to prolongation of intermeal intervals, which defines the number of meals eaten; i.e., decreases in DA concentrations in the VMN that occur after a meal are associated with a longer intermeal interval that characterizes MN (22, 28, 50). In a series of neural graft studies in obese Zucker rats, we demonstrated a stimulatory (14) or an inhibitory (21) effect of dopaminergic cell grafts on MZ and FI when injected into the medial or lateral hypothalamus, respectively. These data suggest that long-term changes of hypothalamic DA concentrations in the obese Zucker rat modulate feeding pattern. Furthermore, in the obese Zucker rat, FI is accompanied by a hypothalamic release of DA that is quantitatively greater than that in the lean controls (49). More recently, we showed that a rise in DA and a fall in serotonin (5-HT) concentrations in the VMN and the LHA, as measured simultaneously via in vivo microdialysis, were related to increases in FI (13). Finally, in a series of parallel studies using anorectic tumor-bearing (TB) rats, we also reported a high turnover of DA together with a decrease in DA concentration in the VMN, which occurred concurrently with a decrease in FI at the onset of anorexia (4). The sum of our data correlating changes in feeding pattern to hypothalamic DA changes in normal rats, in hyperphagic obese rats, and in anorectic TB rats indicates abnormal presynaptic dopaminergic neurotransmission. However, the status and the function of the postsynaptic dopaminergic receptors in the specific hypothalamic FI-related areas during the anorexia of cancer have not been investigated.
We hypothesize that with the onset of cancer anorexia, changes in postsynaptic dopaminergic receptor expression occur in the LHA and the VMN that stimulate postsynaptic neurons to change the feeding pattern. Hence, we measured dopaminergic D1- and D2-receptor mRNA expression in the LHA and the VMN of anorectic TB rats and their controls. Furthermore, to validate the function of the postsynaptic dopaminergic receptors, we injected D1 and D2 dopaminergic receptor antagonists into the LHA or the VMN of anorectic TB and non-tumor-bearing (NTB) free-feeding rats, while measuring the resultant changes in FI, MZ, and MN.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The SUNY Upstate Medical University, Committee for the Humane
Use of Animals, approved the experiments. Animal care was in accordance
with the guidelines established by the National Institutes of Health.
The experimental groups studied are summarized in Table 1.
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Animals, Cancer Model, Controls, and Definition of Anorexia
Male Fischer 344 rats (240-250 g; Taconic, Georgetown, NY) were housed in holding cages for 7 days to acclimate them to the constant study surroundings: 12:12-h light-dark cycle (lights on 0500-1700), 26 ± 1°C room temperature, and 45% relative humidity. Rats had free access to fresh coarsely ground chow (Diet #5008; Ralston Purina, St. Louis, MO) and tap water. Body weight was measured daily.All TB groups had 106 trypan blue-viable MCA sarcoma cells in 1-ml volume inoculated subcutaneously into the right flank. NTB free-feeding control rats received an equal volume of a saline injection. By definition, anorexia occurred when daily FI, per 100 g body wt, was at least 1 g less than the average before tumor inoculation in each TB rat for 3 consecutive days as defined by Chance et al. (9), indicated as days 2-4 in Figs. 2 and 3.
ACREM
FI, MZ, and MN (constituting the feeding pattern) were measured via an ACREM (23). The ACREM continuously measures MZ, MN, and FI for a prolonged period, without preconditioning or pretraining the rats. Access to chow occurred via a feeding tunnel and was continuously monitored via photocells. Food consumption was measured via an electronic scale. A meal was defined as a bite or a series of bites preceded and followed by at least 5 min of feeding inactivity (23).Experiment 1: Measuring Feeding Pattern and D1- and D2-Receptor mRNA Expression with Onset of Anorexia
Rats were randomly divided into two groups: TB (n = 8) and NTB free-feeding control (n = 7). The TB and the NTB free-feeding rats were placed in the ACREM cages to measure FI, MZ, and MN throughout the experiment. Daily data collection was always done at 0930. In TB rats, anorexia developed 18.0 ± 1.2 days after tumor inoculation at which time rats in both groups (TB and NTB free feeding) were decapitated. Bilateral LHA and VMN were harvested and sonicated. Total RNA was purified using RNA pure reagent (GenHunter, Nashville, TN). Relative quantitative RT-PCR was done according to the method described by Schnell et al. (38), with slight modifications. Total RNA (2 µg) was reverse transcribed using oligo d(T)12-18 as a primer in a first-strand cDNA synthesis kit (GIBCO-BRL, Grand Island, NY). Each 50 µl of PCR contained 0.2 µg of first-strand cDNA as a template, 10 µM of each primer, 0.2 mM of 2-deoxynucleotide 5'-triphosphate, 1.5 mM of MgCl2, and 1 U of Taq DNA polymerase (GIBCO-BRL) in 1× PCR buffer (44). As shown in Table 2, specific oligonucleotide pairs were used. Preliminary experiments were done to determine the optimal conditions for PCR amplification. We obtained a significant positive linear correlation between the amount of PCR products and amplifications from 28 to 34 cycles for D1 and D2 receptors (r2 = 0.98 and 0.99, respectively) and from 23 to 29 for cycles of
-actin
(r2 = 0.92). On the basis of our
preliminary studies, we selected 30 cycles for D1 and
D2 and 25 cycles for -actin for PCR amplification (Fig.
1). We confirmed the specificity of all
PCR products with nested PCR using a specific primer, and the
nucleotide positions of the primers are shown in Table 2. We used a
non-RT template as a negative control and cDNA obtained from homogenate
of whole brain in normal Fischer 344 rats as a positive control.
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After electrophoresis, the gel was stained with ethidium bromide, and
the amount of PCR products was measured using a gel analysis software
(SigmaGel, SPSS, Chicago, IL) and was compared as percentage changes
relative to -actin (see Fig. 4). D2-receptor mRNA
expression was analyzed with total intensity of its two isoforms (long
and short).
Experiment 2: Injection of D1- and D2-Receptor Antagonist into the VMN or LHA
Rats were placed in the ACREM cages. FI, MZ, and MN were measured. After a baseline period, a rodent anesthesia mixture [(in mg/ml) 100 ketamine HCl, 30 xylazine HCl, 10 acepromazine] was administered at a dosage of 0.5 ml/kg body wt intramuscularly. Then a 22-gauge guide cannula (Plastics One, Roanoke, VA) was stereotaxically implanted into the VMN (AP =
3.5 mm, L = 0.7 mm, depth = 7.5 mm) or into the LHA [AP = 2.8 mm, L = 1.8 mm, depth = 7.5 mm (Ref. 33)]. After a 1-wk recovery
period, 106 trypan blue-viable MCA sarcoma cells in 1-ml
volume were inoculated into the right flank of the TB rats. NTB
free-feeding control rats received an equal volume of saline. On the
day of anorexia (indicated as day 0 in Figs. 6-9), TB
rats and their NTB free-feeding controls were further randomized into
subgroups as summarized in Table 1. The intra-VMN or the intra-LHA
antagonists or control injections were given at 1630 (during light
phase), just before the onset of the dark period, so as to enhance the
effect of the antagonist on dark phase FI, MZ, and MN. Thus FI and the
feeding pattern on the day of anorexia were not under the influence of the antagonists or control injection for 7 h of the light phase. The effect of each antagonist persisted for 7 days, but the maximum effect was seen on the first 2 days after the injection. For practical reasons, only anorexia day 0 and day 1 data are
plotted, as shown in Figs. 6-9.
1) Injection of D1-receptor antagonist SCH-23390 into the VMN. In the TB-SCH-23390 (SCH) and NTB-SCH groups (Table 1), the D1-receptor antagonist R-(+)-SCH-23390, at a dose of 2.5 µg/0.5 µl saline (Sigma Chemical, St. Louis, MO), was injected into the VMN using a 28-gauge stainless steel needle (Plastics One) that protruded 1 mm beyond the tip of the guide cannula. The controls, TB-C and NTB-C groups, received the same volume of saline. The dose of the D1-receptor antagonist used was less than that used by Carruba et al. (8). However, this dose induced the reinforcing action of cocaine when injected into the amygdala (6). SCH was freshly dissolved in saline before each use.
2) Injection of D2-receptor antagonist sulpiride into the VMN. In the TB-sulpiride (Sul) and NTB-Sul groups (Table 1), the D2-receptor antagonist Sul, at a dose of 4 µg/0.5 µl saline (Sigma Chemical), was injected into the VMN. The dose of the D2-receptor antagonist selected was based on Parada et al. (32). Sul was dissolved in saline containing 2 N acetic acid with a final pH 5.0. The control groups, TB-SC and NTB-SC (Table 1), received the same amount of saline adjusted to a pH 5.0.
3) Injection of D1-receptor antagonist SCH into the LHA. In this experiment, we only studied TB groups. NTB free-feeding groups were not studied because data in the literature suggest the absence of D1 receptors in the LHA (8, 46). Consequently, we anticipated a minimal to no response (8).
On the day of anorexia, rats were randomized into two groups as shown in Table 1. In the TB-SCH-LHA group, R-(+)-SCH-23390 (2.5 µg/0.5 µl saline) was injected into the LHA using a 28-gauge stainless steel needle (Plastics One) that protruded 0.5 mm beyond the tip of the guide cannula. The control group TB-C-LHA (Table 1) received the same amount of saline.4) Injection of D2-receptor antagonist Sul into the LHA. In the TB-Sul-LHA and NTB-Sul-LHA groups (Table 1), Sul, at a dose of 4 µg/0.5 µl saline with pH 5.0 (Sigma Chemical), was injected into the LHA. Their controls, TB-SC-LHA and NTB-SC-LHA (Table 1), received the same volume of saline at an adjusted pH 5.0.
Histological Examination of Receptor Antagonist Microinjection Site
At the end of each experiment, rats were anesthetized. Then, via cardiac puncture, the brains were perfused with normal saline and 10% Formalin. The brains were removed and further fixed in 10% Formalin. Serial coronal sections (100 µm) were cut on a Vibratome (Technical Products International, St. Louis, MO) through the hypothalamus, mounted on slides, stained with hematoxylin, and examined by light microscopy to confirm the microinjection site.Statistical Analysis
In experiment 1, FI, MZ, and MN were analyzed via regression model MINITAB 10 (MINITAB, State College, PA) to characterize the trend effects associated with anorexia. The day before FI decreased was defined as day 1. Because FI started to decrease from day 2 onward, the regression for FI, MZ, and MN was applied from day 1 to day 5 (see Fig. 2). The slopes of the regression lines estimate the decreasing rates of FI, MZ, and MN over time. The effect of anorexia was estimated by comparing the slopes. MZ was previously documented to increase with the onset of anorexia (25, 26). If the decrease in MZ were similar to the decrease in MN and FI, the measured MZ on day 2 would not be expected to be different from the calculated MZ. The calculated MZ was estimated from a fitted line of MZ from day 1 to day 5, excluding day 2. Hence, we estimated the calculated MZ on day 2 from the fitted line based on data from day 1 to day 5 in each rat without including the observed MZ on day 2 (see Fig. 3). In addition, we compared the observed-to-calculated MZ on day 2 via paired Student's t-test. In experiment 1, the expression of D1 and D2 receptor mRNA was analyzed via unpaired Student's t-test. The data were expressed as percent relative to-actin expression (100%; see Fig.
4).
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In experiment 2, FI, MZ, and MN after a specific antagonist
were calculated relative to baseline data defined as the day before injection (day 1) and equal to 100%. The day of injection
was defined as day 0 (see Figs. 6 to 9). Data were analyzed
via paired Student's t-test in the comparison between
baseline and day 0 and day 1 in the same group
and via unpaired Student's t-test in the comparison between
the study and the control groups on each day. To estimate the effect of
antagonist on FI between the TB and NTB free-feeding control groups,
average FI in both control groups on each day was subtracted
from measured FI of each rat in the TB and NTB free-feeding
control study groups. Their maximum values were compared via unpaired
Student's t-test. Data are shown on day 0 and
day 1. A P value equal to or less than 0.05 was
accepted as significant. Data indicate means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiment 1: Feeding Pattern with Onset of Cancer Anorexia
As shown in Fig. 2, in the TB rats, FI, MZ, and MN significantly decreased at the onset and with the development of anorexia. All the slopes of the regression lines were significantly negative (t =7.53, 2.46, and 3.91,
respectively), indicating that FI, MZ, and MN statistically decreased
with anorexia. In contrast, in the NTB free-feeding control rats, FI,
MZ, and MN remained unchanged and did not statistically decrease. The
intercepts in FI, MZ, and MN between the TB and NTB free-feeding rats
were not significantly different (t = 0.29, 0.24, and
0.55, respectively), indicating that the different groups of rats
were similar and behaved similarly before the onset of anorexia.
As shown in Fig. 3, a fitted line for MZ from day 1 to
day 5, excluding the measured MZ on day 2, was
estimated as: MZ = 1.21 0.08 × Days,
r2 = 0.96. The measured MZ on day
2 (1.24 ± 0.16 g/meal) was significantly higher than the
calculated MZ on day 2 (1.05 ± 0.09 g/meal;
P = 0.054). Thus, when FI decreased via an initial
decrease in MN, a concurrent increase in MZ occurred in an attempt to
compensate for the decrease in FI. Thereafter, measured MZ also
decreased. This pattern whereby FI decreases is characteristic of what
we have repeatedly observed and reported to occur in early cancer anorexia (25, 26).
Experiment 1: D1- and D2-Receptor mRNA Expression with Onset of Cancer Anorexia
As shown in Fig. 4, in the NTB free-feeding rats, D1-receptor mRNA was not expressed in either the LHA or VMN, but D2-receptor mRNA was expressed in the LHA and VMN of NTB free-feeding rats. In the anorectic TB rats, D1-receptor mRNA expression in the LHA and VMN was significantly upregulated. Similarly, D2-receptor mRNA was also significantly upregulated in the VMN. However, the upregulation of D2 in the LHA was not significantly different from NTB free-feeding controls (P = 0.07), a result that could be related to a lack of power (not enough animals per group).Experiment 2
Histological sections of the brains confirmed the intended injection site in all rats. An example of a histological section through the VMN is shown in Fig. 5.
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Experiment 2: D1-Receptor Antagonist SCH Injection into the VMN
As shown in Fig. 6, the intra-VMN injection of the D1-receptor antagonist in TB rats (TB-SCH) led to a significant and persistent decrease in FI, initially via a significant decrease in MZ and then via a significant decrease in MN. Blocking the D1 receptor via intra-VMN SCH in the NTB free-feeding rats (NTB-SCH) had no significant effect on FI, MZ, or MN, although a decrease in FI occurred on day 1, relative to baseline.
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D2-Receptor Antagonist Sul Injection into the VMN
As shown in Fig. 7, the injection of intra-VMN D2-receptor antagonist significantly stimulated FI in the TB rats (TB-Sul) relative to their control (TB-SC). This occurred via a significant increase in MN. In the NTB free-feeding rats (NTB-Sul), intra-VMN Sul also increased FI significantly vs. baseline, occurring only via an increase in MZ, although it was statistically not significant. The increase in MZ is, however, biologically important because in all other groups, MZ decreased. The maximum effect of Sul injection into the VMN on FI was observed on day 0 in both groups (TB: 51.8 ± 15.5% vs. NTB: 8.7 ± 3.3%). The difference in the effect of Sul was significantly greater in the TB vs. NTB group.
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D1-Receptor Antagonist SCH Injection into the LHA
As shown in Fig. 8, when the D1-receptor antagonist was injected into the LHA of the TB rats (TB-SCH-LHA), FI significantly declined vs. baseline. However, these changes in FI, MZ, and MN were similar to those that occurred in the TB-C-LHA group, in this tumor anorexia model (as shown in Fig. 2). Thus an injection of SCH at this dosage into the LHA of anorectic TB rats did not influence FI and feeding pattern.
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D2-Receptor Antagonist Sul Injection into the LHA
Figure 9 shows that when Sul was injected into the LHA of the TB rats (TB-Sul-LHA), a significant increase in FI vs. their controls (TB-SC-LHA) occurred via a significant increase in MN. Intra-LHA Sul in the NTB free-feeding rats (NTB-Sul-LHA) also increased FI via a significant increase in MN. The maximum effect of the intra-LHA Sul was observed on day 1 (68.3 ± 30.9%) in the TB group and on day 0 (10.8 ± 2.8%) in the NTB free-feeding group. This change in the TB group was significantly greater than that in the NTB group.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Our data provide evidence that postsynaptic dopaminergic receptor subtypes in the hypothalamus are involved in the regulation of MZ, MN, and thus FI in anorectic TB rats. Until recently, only the effects of presynaptic dopaminergic system were characterized in FI regulation via in vivo microdialysis in TB rats and in normal rats (27, 28, 48, 50).
As mentioned above, under normal conditions, MZ and MN are regulated reciprocally to maintain constancy of daily FI. However, in anorectic TB rats, this close relationship between MZ and MN is dissociated. The functional relationship between MN and MZ in the normal rat requires a neurochemical mechanism that senses and measures MZ. It links MZ to the time elapsed until the next meal occurs and then compensates for changes between the two to maintain a constant daily FI.
The deviation of appetite (anorexia) in TB rats as expressed by more intense satiety and less intense hunger is manifested by fewer MNs and thus longer intermeal intervals and an attempt to correct it by initiating larger MZs. This suggests that the underlying neurochemical mechanism may use both stimulatory and inhibitory neurotransmitter pathways to inhibit or stimulate FI. DA can stimulate or inhibit its target neurons via its receptor expression.
Currently, five subtypes of dopaminergic receptors are recognized. They are divided into two major classes: the D1-like (i.e., D1 and D5) and the D2-like receptors (i.e., D2, D3, D4). Apart from having different biochemical and pharmacological properties, they mediate different physiological functions. Among them, the D1 and D2 receptors per se represent the major subtypes that are widely distributed in the brain (46). Because D1 and D2 receptors are colocalized on the same neuron in the striatum (1, 2, 30), we postulated that the D1 and D2 receptors are also colocalized on the same hypothalamic neurons. When the D1 and D2 receptors are stimulated, they have opposing effects. As an example, the D1 receptors stimulate adenylyl cyclase activity (16), which in the VMN facilitates prolactin release from the pituitary (10). In addition, prolactin is known to stimulate FI (29). In contrast, the D2 receptors suppress adenylyl cyclase activity inhibiting prolactin release (11, 16). It is likely that the D1 receptors in the VMN may be involved in stimulating FI via prolactin release. Thus the difference in the effect of intra-VMN or intra-LHA SCH in anorectic TB rats could be explained by the effect of prolactin. In contrast, the D2 receptors in the hypothalamus, especially in the perifornical LHA, are involved in the process of satiation (19, 32). Although the D2 receptors are abundantly expressed in the LHA and VMN of normal rats, the D1 receptors are not abundantly expressed in either the LHA or VMN (8, 46).
On the basis of a well-established rat tumor model in our laboratory (18, 22, 25, 26), with the onset of anorexia, a decrease in MN and a concurrent increase in MZ occur (25, 26). These behavioral manifestations of anorexia correspond to biochemical changes in the brain, particularly in the hypothalamic FI-related nuclei. Because the D2 receptors in the hypothalamus play a role in the process of satiation (19, 32), we postulated that D2 receptors in the hypothalamus are highly expressed and contribute to a reduction of FI associated with the characteristic feeding pattern in anorectic TB rats. We interpret our biochemical findings in the hypothalamus as showing that a significant increase in D1-receptor mRNA expression occurs in the LHA and VMN. These are associated with the increase in MZ, and the increase in D2-receptor mRNA expression in the LHA and VMN is associated with the significant decrease in MN. We verified this interpretation by giving D1- or D2-receptor mRNA blockers. As summarized in RESULTS, blocking the effects of DA on D1 receptors led to a decrease in FI and MZ and that of D2 receptors led to an increase in FI via an increase in MN. However, because expression of receptor mRNA is not always associated with the expression of its protein (46), determination of D1- and D2-receptor protein expression in the hypothalamus, via the use of immunohistochemical technique, remains to be determined in anorectic TB rats; studies are currently being conducted.
Sul is a D2-receptor antagonist associated with an affinity to D3 and D4 receptors (7, 45), similar to the other D2-receptor antagonists. These receptors are expressed in the hypothalamus (5, 37). Thus, in this present study, Sul could, in theory, also inhibit D3 and D4 receptors. However, the effects of these additional receptors on the FI regulation have yet to be characterized, and their receptor expressions in the hypothalamus of anorectic TB rats have yet to be measured. However, because D2-receptor expression in the hypothalamus of anorectic TB rats is significantly higher than that in non-TB normal rats, the observed difference in the feeding pattern after Sul injection into the VMN is likely to be due to the effect on the D2 receptors.
In normal rats, the injection of a D1-receptor antagonist into the VMN did not change FI. This finding is in agreement with a published study wherein a higher dose of SCH was injected into the perifornical LHA or into the PVN (8) where D1-receptor mRNA is not abundantly expressed (8, 46). Thus it seems that because of low D1-receptor expression in the VMN in normal rats, the D1-receptor antagonist SCH does not appear to influence FI.
The D1-receptor antagonists, including SCH, have a significant affinity to serotonergic (5-HT) 2A and 2C receptors (43, 47). These serotonergic receptors inhibit FI (41). Both receptors exist in the VMN (18, 34) and are related to the regulation of MN (18, 41). Thus, in our present data, the decrease in MN after an injection of SCH into the VMN may also be due to the effect of this antagonist on 5-HT 2A and 2C receptors. The levels of expression of these serotonergic receptors in the hypothalamus of anorectic TB rats are currently under study, but our data, wherein intra-VMN SCH decreased MN in anorectic TB rats, suggest that 5-HT 2A and 2C receptors are likely to be also highly expressed in the VMN of anorectic TB rats. Furthermore, SCH might affect yet other unknown receptors so as to decrease MZ. However, because hypothalamic D1-receptor expression in anorectic TB rats appeared to be significantly higher than that in the normal rats, it is more likely that SCH injected into the VMN affected D1 receptors, rather than other receptors, to decrease MZ.
D1 and D2 receptors are localized on the same
neuron (1, 2, 30) and can also be localized pre- and
postsynaptically (15, 36). In addition, in vivo exogenous
DA has a biphasic action on the neurons of the caudate nucleus. Low
concentrations excite the neuron via D2 receptors, whereas
high concentrations inhibit it via D1 receptors
(2). Similarly, Shen et al. (39) observed
that both D1 and D2 receptors mediate
excitation as well as inhibition of caudate neurons. Hence, we deduce
that hypothalamic DA acts on a postsynaptic FI-responsive neuron, which
may contain FI-stimulatory neuropeptides such as NPY,
melanin-concentrating hormone, orexin, or inhibitory neuropeptides such
as corticotropin-releasing factor and 
-melanocyte-stimulating
hormone. These act positively or negatively on FI, according to
D1- and D2-receptor expression. An integrated
balance of the expression of these receptors would then be expected to
regulate the constancy of daily FI. In contrast, an imbalance of the
expression of these receptors, as occurs and as observed in anorectic
TB rats, would alter FI and feeding pattern to reflect the
characteristic pattern described in TB rats with anorexia.
Our data show that dopaminergic D1- and D2-receptor mRNA in the hypothalamus were highly expressed in anorectic TB rats compared with NTB free-feeding normal rats. These changes were associated with anorexia and the characteristic decrease in MN and concurrent increase in MZ at the onset of anorexia. Specifically, the highly expressed D1 receptors in the VMN relate to the increase in MZ, whereas the highly expressed D2 receptors in the LHA and VMN contributed to the anorexia via a decrease in MN. In our previous studies, we reported a correlation between DA in the LHA and MZ (13, 22, 27, 48, 49) and an association between DA in the VMN and intermeal interval, characterized as MN (22, 28, 50). On the basis of these observations, we hypothesized that MZ and MN are regulated independently via different systems (20, 24). According to the results of this study, in which we examined the function of D1- and D2-receptor mRNA status in the LHA and VMN, we are now able to conclude that these dopaminergic receptors also play a contributory role in the regulation of MZ and MN in an effort to maintain constancy of FI.
Perspectives
Further delineation of the hypothalamic dopaminergic system as it relates to MZ and MN regulation is needed. The relationship between DA and 5-HT in these hypothalamic areas is currently under investigation, as is the relationship to other neuromodulators such as NPY and leptin, known factors involved in the regulation of FI under normal conditions. Currently, the immunohistochemical connections between subtypes of dopaminergic receptors as well as serotonergic receptors on peptidergic food-related neurons such as NPY in various hypothalamic areas are under investigation. It is conceivable the aminergic neurotransmitters influence the peptidergic neuromodulators via G protein signaling.Our current data provide further insight into the mechanism of hypothalamic dopaminergic regulation of FI in cancer anorexia. They suggest that the hypothalamic dopaminergic system is a potential opportune target for pharmacological manipulation to ameliorate the anorexia of cancer, to prevent weight loss, and to enhance survival and optimize the outcome of primary cancer treatment.
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ACKNOWLEDGEMENTS |
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We thank G. Miyata and E. Samoilova for assistance. We thank K. Rossi and D. Nobile for editorial assistance.
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FOOTNOTES |
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This work was supported in part by National Cancer Institute Grant CA-70239.
Address for reprint requests and other correspondence: M. M. Meguid, Dept. of Surgery, SUNY Upstate Medical Univ., Univ. Hospital, 750 East Adams St., Syracuse, NY 13210 (E-mail: meguidm{at}upstate.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 August 2000; accepted in final form 22 August 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). The day before food intake decreased was defined as
day 1. Day 2 is the day that food intake started
to decrease. The observed meal size significantly increased on
day 2 to 1.24 ± 0.16 g/meal. From the fitted line, the
calculated meal size on day 2 was 1.05 ± 0.09 g/meal.
The observed meal size was significantly different. *P = 0.054 vs. calculated meal size.
End of guide cannula. Arrowhead indicates the internal cannula
track. 3V, third ventricle.
