Recent studies show that brain-derived neurotrophic factor (BDNF) decreases feeding and body weight after peripheral and ventricular administration. BDNF mRNA and protein, and its receptor tyrosine kinase B (TrkB) are widely distributed in the hypothalamus and other brain regions. However, there are few reports on specific brain sites of actions for BDNF. We evaluated the effect of BDNF in the hypothalamic paraventricular nucleus (PVN) on feeding. BDNF injected unilaterally or bilaterally into the PVN of food-deprived and nondeprived rats significantly decreased feeding and body weight gain within the 0- to 24-h and 24- to 48-h postinjection intervals. Effective doses producing inhibition of feeding behavior did not establish a conditioned taste aversion. PVN BDNF significantly decreased PVN neuropeptide Y (NPY)-induced feeding at 1, 2, and 4 h following injection. BDNF administration in the PVN abolished food-restriction-induced NPY gene expression in the hypothalamic arcuate nucleus. In conclusion, BDNF in the PVN significantly decreases food intake and body weight gain, suggesting that the PVN is an important site of action for BDNF in its effects on energy metabolism. Furthermore, BDNF appears to interact with NPY in its anorectic actions, although a direct effect on NPY remains to be established.
- food intake
- body weight
brain-derived neurotrophic factor (BDNF) is important in differentiation, survival, and plasticity of the central nervous system (CNS). Recent studies also have shown significant roles of BDNF in energy metabolism regulation. Intracerebroventricular administration of BDNF decreases feeding (35, 37, 42) and body weight gain (37, 42). Furthermore, chronic ventricular infusion of BDNF reversed the hyperphagia and obesity of BDNF (+/−) heterozygous animals (16), suggesting that BDNF in the CNS exerts negative effects on feeding. In our recent work (see companion article, Ref. 51a) and other publications (32), BDNF has been found to increase oxygen consumption and heat production by elevating resting metabolic rate, and levels of uncoupling protein 1 (UCP1) mRNA and protein in brown adipose tissue (50), indicating elevated thermogenesis and energy expenditure. Effects of BDNF on energy metabolism have also been observed in human subjects. In a clinical case report, a patient with severe obesity carries a mutation in the BDNF receptor TrkB (61). These data suggest that BDNF is important to energy metabolism regulation.
Most reported studies to date have relied on injections of BDNF in the periphery or into the brain ventricles, and few specific brain sites of BDNF action have been tested. Furthermore, most studies have examined long-term effects of chronically administered BDNF. Short-term effects of acute administration of BDNF in the PVN have not been reported. The PVN is a central structure in the regulation of energy metabolism, and several neuropeptides in the PVN have been reported to affect energy metabolism. Neuropeptide Y (NPY; 3, 8), agouti-related peptide (13, 57), and ghrelin (34, 44) stimulate eating after administration into the PVN. On the other hand, elevated leptin expression in the PVN significantly reduces food intake (1). Melanocyte-stimulating hormone (α-MSH) (17, 58), cocaine- and amphetamine-regulated transcript (CART) (52), urocortin (9, 10, 18), and corticotrophin-releasing hormone (CRH) (24, 27) in the PVN suppress feeding. Based on the distribution of BDNF and its receptor TrkB in the PVN (7, 16, 60), and the major role of the PVN in regulation of energy metabolism, we tested the effect of acute injections of BDNF in the hypothalamic PVN on normal feeding responses, feeding induced by deprivation and NPY. Considering that BDNF effects on food intake are somewhat delayed, we also measured NPY gene expression in the hypothalamic arcuate nucleus (Arc) 24 h after BDNF administration. Single unilateral and bilateral injections of BDNF significantly decreased 48-h feeding and body weight gain at doses not associated with taste aversion, indicating that feeding inhibition is not a result of malaise or other illness. PVN BDNF suppressed short-term NPY-induced feeding, and BDNF in the PVN decreased NPY expression in the Arc compared with the pairfed group. These findings suggest that the PVN is an important site of action of BDNF in its effects on feeding and body weight, and that BDNF may interact with NPY to inhibit feeding behavior.
Male Sprague-Dawley rats (Harlan, Madison, WI) weighing 280–320 g were housed individually in cages with a 12:12-h light-dark photo cycle (lights on at 07:00 AM) in a room at 21–22°C. Teklad Lab Chow and water were allowed ad libitum, except where noted. The protocol was approved by the Veterans Affairs Medical Center Institutional Animal Care and Use Committee.
Cannulation and Verification of Placement
Rats were anesthetized with intramuscular xylazine at 3.5 mg/kg and ketamine at 20 mg/kg and were fitted with a 28-gauge stainless steel guide cannula placed just above the PVN unilaterally or bilaterally in separate experiments. Stereotaxic coordinates were determined from the rat brain atlas of Paxinos and Watson (36) and are as follows: 0.6 mm lateral and 1.9 mm posterior to bregma, and 7.1 mm below the skull surface. The injector extended 1 mm further than the end of the guide cannula. The animals were given at least 1 wk to recover following surgery before experimental trials. After terminal experiments, the rats were decapitated and whole brain tissue was taken for histological examination. The brain tissues were sectioned with cryostat at thickness of 50 μm, mounted on gelatin-coated slides, stained with 0.1% thionin, and treated with ethanol (from 30% to 100%) and Clearing Agent (Electron Microscopy Sciences, Hatfield, PA). After the slides were dried, injection placement was determined microscopically at ×10 magnification, using the brain atlas of Paxinos and Watson (36) as a reference. Injection placements for experiments 1, 2, and 4 are summarized in Fig. 1. A cannula was deemed correct if the histological examination indicated that the injection was within a 0.25-mm diameter from the targeted site. Data from animals with misplaced cannulae were excluded from the data analysis. Histological examination of brain tissue to verify injection site was not possible in some experiments, as rats were undergoing additional studies. In this situation, we used feeding response to injected PVN NPY as a behavioral assay to determine correct cannula placements. A cannula was deemed correct if the animal ate more than 2 g of chow within 2 h after 100 pmol NPY injections. This biological/behavioral assay has been confirmed in our previous work (3). Although this method errs on the side of overestimating incorrect cannula placements, it increases the probability of correct cannula placements in the remaining rats.
BDNF was kindly provided by Regeneron Pharmaceuticals (Tarrytown, NY), and stored at −70°C in 10 mg/ml of 150 mM NaCl, 10 mM NaHPO3 buffer, and 0.004% Tween 20 until use. Just before use, BDNF was diluted with artificial cerebrospinal fluid (aCSF). NPY was purchased from Phoenix Pharmaceuticals, (Mountain View, CA) and was diluted with aCSF just before use.
A volume of 0.5 μl was injected slowly over 30 s, with the injector left in place an additional 15 s to ensure extrusion from the tip and to minimize distribution of drug upward on the cannula tract. In total, animals received about 10 injections. Injection sites were examined by light microscopy for tissue damage in the present studies, and none was found.
To determine potential injection and/or BDNF-induced gliosis, histological measures of glial fibrillary acidic protein (GFAP) were made in rats that had received injections of BDNF or aCSF. This was performed on a set of eight unilaterally cannulated rats that had already received three injections in the course of other experiments: aCSF, BDNF (0.5 μg), and NPY (100 pmol). Each rat received each of these injections once. The rats were then randomly divided into two groups. For the next seven injections, four rats received aCSF and four received 0.5 μg BDNF. After completion of these injections, the animals were perfused and brain tissue was fixed, sectioned, and stained. Immunohistochemical identification of astrocytes was performed by washing sections in blocking solution containing PBS, 3% normal goat serum (NGS), and 0.3% Triton X-100 (Sigma) for 30 min and then incubating 48 h at 4°C in PBS containing 1% NGS, 0.1% Triton X-100, and polyclonal anti-GFAP (1:1,000; DAKO cat. no. Z0334). Sections were washed three times for 15 min each in PBS and then incubated 3 h at 23°C in PBS containing 1% NGS, 0.3% Triton X-100, and cyanine3-conjugated goat anti-rabbit IgG 1:600 (Jackson ImmunoResearch). Sections were washed three times for 10 min each in PBS and then mounted and coverslipped with DPX (Electron Microscopy Sciences).
A consistent area just beneath the injection site, both ipsilateral and contralateral to the PVN, was measured microscopically (×40 magnification, Nikon E 400, Tokyo, Japan), and fluorescence intensity readings were taken by fluorometry (Image-Pro Plus; Media cybernetics, Silver Spring, MD) in six brain sections per animal. Data units for total immunofluorescence calculations are in gray scale pixel values, ranging from 0 (dark) to 255 (saturation level) (56). Mean fluorescence intensity for each side in each animal was calculated.
The GFAP fluorescence intensity for each group was statistically analyzed with ANOVA. There was no difference in GFAP staining intensity between BDNF and aCSF on both injection (P = 0.4898) and noninjection (P = 0.8980) sides, indicating that BDNF does not induce gliosis. These results are consistent with a report in which chronic intracerebroventricular BDNF infusion for 14 days did not increase newly generated astrocytes in the hypothalamus compared with aCSF-treated rats (38), in spite of BDNF-induced neurogenesis.
Experiment 1. Effect of BDNF on normal feeding.
Food was allowed ad libitum before and during the experimental period. Eight rats were injected bilaterally with aCSF or 0.1 μg, 0.3 . μg, 0.5 μg, 1 μg, or 3 μg BDNF on each side at 2:00 PM. Food intake and body weight were measured at 24 and 48 h following injection. Each animal received each treatment once with at least 72–96 h between treatment to allow for clearance of BDNF from the CNS and for normal feeding patterns to be reestablished. Treatments were given in a randomly selected Latin square design to avoid a treatment order confound. All eight rats had correct cannula placements (verified by histology) and therefore were included in the statistical analyses.
Experiment 2. Effect of BDNF on deprivation-induced feeding.
Twenty-nine rats were injected unilaterally with aCSF or BDNF (0.1 μg, 0.3 μg, or 0.5 μg) after 18-h food deprivation, and were given food immediately after injection. Food intake was measured at 1, 2, 4, 24, and 48 h following injection. Body weight change was measured at 24 and 48 h after injection. Each animal received each treatment once with at least 72–96 h between treatment. After excluding six rats that did not respond to NPY, 23 rats were included in the statistical analysis. In another set of 21 rats with double cannulation, they were bilaterally injected with the same dose on each side. After excluding three rats with incorrect cannula placement (verified by histology), 18 rats were included in the statistical analyses.
Experiment 3. Effect of PVN-injected BDNF on preference for saccharin solution.
In two separate group of animals, the two-bottle preference test was used to determine whether BDNF results in aversive consequences after administration into the PVN. The basis for this test is as follows: when rats are exposed to water and saccharin solutions, they show a preference for saccharin. When animals are exposed to 0.1% saccharin solution for the first time and are concurrently injected with a drug that has aversive properties, saccharin is associated with the aversive stimuli. Subsequently, when given water and saccharin at the same time, drinking of saccharin is reduced. Reduced consumption of the drug-paired flavor indicates that the drug administered has aversive properties.
The procedures for this experiment have been previously described (53). In brief, drinking water was scheduled between 10:30 AM and 11:00 AM for bilaterally PVN-cannulated rats each day for 7 days. During water access, two bottles of water were presented. The rats were then randomly divided into five groups (with an even distribution of body weight) and given 15 ml of 0.1% saccharin immediately followed by injection on each side with aCSF or BDNF (0.1, 0.3, 0.5, or 1.0 μg). Due to the delayed effect of BDNF on feeding behavior, we performed a second conditioned taste aversion experiment in which the saccharin exposure was delayed to more accurately match the onset of BDNF-induced feeding inhibition. To more precisely determine onset of BDNF-induced feeding inhibition, a pilot time course feeding experiment was performed in which food intake was measured every 2 h for 24 h in BDNF (1.0 μg) or aCSF-injected animals (n = 5/group). Cumulative food intake became significantly different between groups at 8 h postinjection (P = 0.0152, data not shown), and BDNF significantly inhibited feeding in the 4–8 h interval postinjection (P = 0.034, data not shown). On the basis of these data, a second set of rats were prepared with bilateral PVN cannulae, habituated to the restricted drinking schedule described above, and then exposed to 0.1% saccharin 7 h after administration of BDNF (0.3, 0.5, 1.0, or 3.0 μg) or aCSF to more accurately match the time at which BDNF feeding inhibition (and thus potential aversive sequelae) become significant. This conditioned stimulation was repeated once after 2 days, and the animals were then given the choice of water and saccharin in the absence of drug administration 2 days later. To avoid the possible confound of bottle placement, initial position and order of presentation of the saccharin bottle were counterbalanced across subjects, and the two-bottle test was repeated 2 days later in which the two bottle positions were reversed. Intake of water and saccharin during 24 h was measured, and the mean of the two tests was calculated. In the first experiment, in which pairing of BDNF and saccharin was immediate, 26 rats were included in the statistical analyses. Three rats that did not respond to PVN NPY were excluded. In the second experiment, in which pairing of BDNF and saccharin was delayed, 26 rats were included in the statistical analyses. Six rats that did not respond to PVN NPY were excluded.
Experiment 4. Effect of BDNF on NPY-induced feeding.
Food was allowed ad libitum before and during the experimental period. Nine PVN bilaterally cannulated rats were randomly assigned to one of six treatments: 1) aCSF + aCSF, 2) aCSF + NPY (100 pmol), 3) BDNF (0.1 μg) + NPY (100 pmol), 4) BDNF (0.3 μg) + NPY (100 pmol), 5) BDNF (0.5 μg) + NPY (100 pmol), or 6) BDNF (0.5 μg) + aCSF. The first injection was made bilaterally at 7:00 AM, and the second injection was made unilaterally 4 h later (11:00 AM) on the alternate side on each experimental day. The 4-h delay was used in an effort to match the time course of BDNF effects as the effects of BDNF on feeding behavior in food-deprived animals is delayed. Each animal received each treatment once with at least 72 h between treatment to allow for clearance of BDNF from the CNS and for normal feeding patterns to be reestablished. Food intake was measured at 1, 2, and 4 h following the second injection. After excluding one rat that was incorrectly cannulated (as verified by histology), eight rats were included in the statistical analyses.
Experiment 5. Effect of BDNF on ARC NPY gene expression.
A set of NPY-responsive rats (n = 27), were divided into three groups with body weight distributed evenly: 1) aCSF-injected, 2) BDNF-injected, and 3) aCSF-injected with food intake yoked to that of the BDNF-treated rats (pairfed). The nine rats that were to receive BDNF (0.5 μg) and five rats that were to receive control (aCSF) injections were injected on the first day at 10:30 AM. The nine rats to receive aCSF and be pairfed and four additional controls (aCSF) were injected on the second day at 10:30 AM. Food was given at 4:30 PM ad libitum with the exception of the pairfed animals, which were given the same amount of food the BDNF-treated animals had consumed the previous day. At 8:00 AM the next morning, food was removed, and overnight food intake was measured. These animals were killed at 10:30 AM by rapid decapitation.
Brain punches of the Arc were taken for measurement of NPY mRNA. Total RNA was isolated from the Arc according to the TRI reagent protocol with minor modifications (5). Briefly, tissue was homogenized with Trizol reagent (GIBCO, Paisley, UK) and chloroform. After phase separation, the aqueous phase was removed, and total RNA was precipitated with 70% ethanol and applied to a Qiagen column (Qiagen RNeasy micro kit, Qiagen, Valencia, CA). The concentration and purity of the RNA were determined by the 260 nm and 280 nm readings on a spectrophotometer (Nanodrop ND-1000; Nanodrop Technologies, Wilmington, DE).
The primers for NPY and the housekeeping gene ribosomal protein L32 (Rpl32), were created using MacVector 7.2 (Accerlys, San Diego, CA; Table 1). One-step real-time RT-PCR was performed by using 100 ng of total RNA, reagents provided in the Roche RNA Amplification Kit SYBR Green I, and a Roche LightCycler (Roche Applied Science, Indianapolis, IN). Real-time RT-PCR was performed as follows: reverse transcription for 30 min at 42°C, denaturation for 30 s at 95°C, followed by 35 cycles of cDNA amplification consisting of a 15-s denaturation at 95°C, primer annealing for 20 s at 58°C (NPY), and 59°C (Rpl32), and product elongation for 20 s at 72°C. Data acquisition was taken at the end of each amplification cycle at a temperature slightly lower than the temperature required to melt the PCR product. Each primer set yielded a single product that corresponded to the appropriate nucleotide lengths. Amplification products from the reaction were viewed after a cycle of melting, electrophoresed in a 4% Nuseive gel, and further verified by capillary electrophoresis. The 2−ΔΔCT method was used to calculate relative mRNA levels, and fold changes in mRNA levels (23). Fold change in mRNA is expressed as the ratio of the mean relative expression levels between treatment groups.
Experiment 6. Effect of BDNF in the lateral hypothalamus on deprivation-induced feeding.
To determine whether PVN BDNF effects could be due to BDNF action in neighboring areas, such as the lateral hypothalamus (LH) and/or perifornical area, a set of 15 animals were prepared with bilateral cannulae directed at the rostral LH (coordinates: 1.5 mm lateral, 2.8 mm posterior to bregma, and 7.3 mm below the skull surface). The experimental procedures were the same as that described for experiment 2. After overnight food deprivation, the rats were given aCSF or 0.1, 0.3, 0.5, 1.0, or 3.0 μg of BDNF, and allowed access to regular chow immediately after injection. Food intake was measured at 1, 2, 4, 24, and 48 h after injection, and body weight was measured at 24 and 48 h after injection. Each rat received each treatment once in a counterbalanced order with a 72- to 96-h interval between treatments. After histological placement verification, 13 rats were included in the statistical analyses.
For all feeding experiments, data were analyzed using StatView 5.0 (Cary, NC) and are expressed as means ± SE. Repeated-measures ANOVA was used to analyze food intake and body weight change. Thus each rat served as its own control. When main effects were observed, post hoc analysis was performed using multiple-comparison contrasts. For the conditioned taste aversion (CTA) experiment, data were analyzed by a one-factor ANOVA followed by Fisher's least significant difference t-test to compare means. For the NPY gene expression experiment, statistical comparisons between BDNF and aCSF-treated or pairfed rats were performed using Student's t-test.
Effect of BDNF on Normal Feeding
BDNF dose dependently inhibited normal feeding (Fig. 2A) at different time points than when tested against deprivation-induced feeding. At 24 h after injection, BDNF at 0.5, 1, and 3 μg significantly inhibited normal feeding by 26.2%, 27.2% and 37.2% (P = 0.0007, P = 0.0005, and P < 0.0001, respectively). In the 24- to 48-h interval, BDNF at 0.3, 0.5, 1, and 3 μg significantly inhibited normal feeding by 12.6%, 16.8%, 21.4% and 31.4% (P = 0.0196, P = 0.0025, P = 0.0002, and P < 0.0001, respectively). In the 0- to 48-h postinjection interval, BDNF at 0.1, 0.3, 0.5, 1, and 3 μg inhibited normal feeding by 8.7%, 12.6%, 21.5%, 24.4% and 34.3% (P = 0.0693, P = 0.01, P < 0.0001, P < 0.0001, and P < 0.0001, respectively, data not shown). In the 0- to 24-h postinjection interval, BDNF at 0.5, 1, and 3 μg significantly inhibited body weight gain (P = 0.0003, P = 0.0081, and P < 0.0001, respectively, Fig. 2B). In the 24–48 h interval, BDNF at 1 and 3 μg significantly inhibited body weight gain (P = 0.0256, and P = 0.0024, respectively, Fig. 2B). In the 0–48 h interval, BDNF at 0.3, 0.5, 1, and 3 μg inhibited body weight gain (P = 0.0361, P < 0.0006, P < 0.0001, and P < 0.0001, respectively).
Effect of BDNF on Deprivation-Induced Feeding
In the experiments using unilateral injections, BDNF at 0.3 and 0.5 μg significantly decreased deprivation-induced feeding in 1–2 h by 49.2% (P = 0.0185) and 66% (P = 0.0019), respectively (Fig. 3A); however, BDNF did not significantly inhibit cumulative feeding in the 0–4 h interval (data not shown). BDNF at 0.5 μg significantly decreased feeding in the 4- to 24-h, 0- to 24-h, and 24- to 48-h intervals by 13.6% (P = 0.0054), 11.4% (P = 0.0004), and 8.2% (P = 0.0261), respectively (Fig. 3B). Analysis of cumulative intake indicated that BDNF at 0.3 and 0.5 μg (62.96 ± 1.59 g for aCSF, 59.58 ± 1.93 g for 0.3 μg BDNF, and 56.55 ± 1.90 g for 0.5 μg BDNF) significantly inhibited feeding in the 0- to 48-h interval by 5.7% (P = 0.0461) and 10.2% (P = 0.0005), respectively. BDNF at 0.5 μg also significantly decreased body weight gain at 0- to 24-h and 0- to 48-h by 24.4% (P = 0.0083) and 23.1% (P = 0.0027), respectively (Fig. 4).
We also analyzed the data from six animals with incorrect unilateral cannula placement based on no or low feeding response to PVN NPY. There were no differences in feeding and body weight gain between aCSF and BDNF treatment, except that BDNF at 0.5 μg significantly decreased feeding during 24–48 h (P = 0.0277, data not shown) and 0–48 h (P = 0.0181) after injection.
To test whether bilateral administration of BDNF into the PVN would further block deprivation-induced feeding, another set of rats were given bilateral injections of BDNF at the same dose on each side. Similar to the unilateral scheme, BDNF inhibited feeding for 24 h after injection (Fig. 5B). BDNF at 0.1, 0.3, and 0.5 μg in the 0- to 24-h postinjection interval significantly reduced feeding by 6.8%, 7.0%, and 10.3%, respectively (P = 0.0376, P = 0.0334, and P = 0.0021, respectively). In the 24- to 48-h postinjection interval, BDNF at 0.3 and 0.5 μg significantly decreased feeding by 6.5% (P = 0.050) and 7.3% (P = 0.0294, Fig. 5B), respectively. During the 0- to 48-h postinjection interval, BDNF at 0.3 and 0.5 μg significantly decreased feeding by 7.7% and 8.9%, respectively (P = 0.0087 and P = 0.0026, respectively). In the 0- to 24-h postinjection interval, BDNF at 0.1, 0.3, and 0.5 μg significantly decreased body weight gain by 19.7%, 19.4%, and 26.8%, respectively (P = 0.021, P = 0.0231, and P = 0.0022, respectively, Fig. 6). In the 24- to 48-h postinjection interval, BDNF at 0.3 and 0.5 μg reduced body weight gain by 29.2% and 29.5%, respectively (Fig. 6) although these differences did not reach statistical significance (P = 0.2007 and P = 0.1960, respectively). In the 0- to 48-h postinjection interval, BDNF at 0.3 and 0.5 μg significantly decreased body weight gain by 23.4% and 27.4% (P = 0.006 and P = 0.0015, respectively, Fig. 6). The data from both unilateral and bilateral administration of BDNF are summarized in Table 2.
We also analyzed data from three animals with incorrect unilateral cannula placement based on histology examination. Although BDNF at 0.1 (P = 0.0009) and 0.3 μg (P = 0.0116) significantly decreased feeding during 0–4 h (data not shown), BDNF at 0.1, 0.3, and 0.5 μg significantly increased feeding during 4–24 h (P = 0.0023, P = 0.0258, and P = 0.0196, respectively); and there are no differences during 0- to 24-h, 24- to 48-h, and 0- to 48-h periods. There are no differences in body weight gain between the treatments during any time frames (data not shown).
Effect of PVN-Injected BDNF on Preference for Saccharin Solution
In the first taste aversion test in which animals were given injection immediately after consumption of saccharin, there was no main effect of BDNF on the percentage of fluid intake attributed to saccharin solution (P = 0.690 for main effect, Fig. 7A). In the second test in which the animals were exposed to saccharin 7 h after administration of BDNF, there was no significant difference in consumption of saccharin percentage among the treatments (P = 0.2835 for main effect, Fig. 7B).
Effect of BDNF on NPY-Induced Feeding
In this experiment, the second injection (NPY) was made 4 h after the first injection (BDNF). Injection of NPY into PVN significantly increased food intake at 1 h after injection (P = 0.0006, Fig. 8), and BDNF at 0.3 and 0.5 μg decreased NPY-induced feeding by 56.3% (P = 0.0252) and 81.7% (P = 0.0017), respectively (Fig. 8). At 1–2 h and 2–4 h, there are no main effects on the feeding (Fig. 8). However, in the 0- to 2-h post-NPY injection interval, NPY-induced feeding (P = 0.0015) was decreased by 0.3 and 0.5 μg BDNF by 44.6% and 85.9% (P = 0.0565, P = 0.0006, respectively; data not shown). In the 0- to 4-h interval, NPY-induced feeding (P = 0.0322) was inhibited by 0.3 and 0.5 μg BDNF by 27.3% (P = 0.1931, data not shown) and 37.4% (P = 0.0769, data not shown), respectively.
Effect of BDNF on Arc NPY Gene Expression
At 24 h after injection, BDNF significantly decreased food intake compared with controls (17.53 ± 1.73 g for BDNF vs. 22.91 ± 1.60 g for aCSF, P = 0.03846, Fig. 9A). The pairfeeding protocol was successful as there was no difference in 24-h food intake between the BDNF and pairfed group (16.19 ± 1.15 g for pairfed, P = 0.5214, Fig. 9A). The relatively mild food restriction in the pairfed group significantly elevated Arc NPY mRNA (P = 0.04433, Fig. 9B), and BDNF treatment with similar food intake resulted in normalization of Arc NPY mRNA levels (0.546 ± 0.106 for BDNF vs. 0.616 ± 0.098 for aCSF, P = 0.6415, Fig. 9B).
Effect of BDNF in the LH on Deprivation-Induced Feeding
There were no significant differences in food intake and body weight gain at any time point. The main effect on P values for feeding at 0–24, 24–48, and 0–48 h were 0.5919, 0.5403, and 0.4637, respectively. The main effect on P values for body weight gain at these periods were P = 0.3828, P = 0.2731, and P = 0.2345, respectively.
BDNF has been demonstrated to be important to the regulation of energy metabolism. Hyperphagia and obesity occur in animal models with BDNF deficiency (16, 25, 39). Chronic ventricular or peripheral administration of BDNF decreases food intake and body weight gain (35, 37, 42), and reverses the phenotype of obese and hyperphagic BDNF +/− mice (16). However, few specific sites of action for BDNF have been identified in the brain with direct administration of BDNF. The PVN is an important site for central control of energy metabolism, and a good candidate site for BDNF action because BDNF peptide (26, 33) and its receptor are present in the hypothalamus; and BDNF immunoreactivity in nerve fibers has been demonstrated in the PVN (7, 28). The present study demonstrates for the first time that BDNF acts within the PVN to inhibit feeding behavior. Our studies show that BDNF in the PVN inhibits deprivation-induced feeding, normal dark-phase feeding, and NPY-induced feeding without producing a CTA even at a relatively high dose. These findings are consistent with the neurochemical and neuroanatomical basis for BDNF action within the PVN.
Single injection of BDNF significantly inhibited normal feeding (Fig. 2A) with reduction 26.2–37.2% at 0–24 h, 12.6–31.4% at 24–48 h, and 8.7–34.3% at 0–48 h, respectively. Body weight gain was also significantly reduced at same periods (Fig. 2B). Similar to a study targeting the dorsal-vagal complex of brain stem (2) in which BDNF at 0.1–1.0 μg dramatically decreased feeding and body weight gain, BDNF administered to the PVN has more potent effects on feeding behavior than that observed after ventricular or peripheral administration. In the present study, a single and comparatively low dose of BDNF (0.5 μg) in the PVN significantly reduced feeding for up to 48 h to levels seen in studies using peripheral doses of 20 mg·kg−1·day−1 for 3 wk (32), 10 mg·kg−1·day−1 for 6 days (31), 6 mg/day for 14 days (37), 50 mg·kg−1·wk−1 (49), and the ventricular dose of 50 ng/h for 14 days (59). This relative sensitivity of the PVN to BDNF anorectic effects suggests that the PVN may be an important mediator of BDNF effects on energy metabolism.
Unilateral administration of BDNF in the PVN also decreased feeding induced by food deprivation (Fig. 3), which was associated with low body weight gain (Fig. 4). This inhibition occurred 4–24 h after injection and lasted for 48 h, which is different from urocortin in the PVN, which inhibits feeding in the first hour and is effective for up to 24 h after injection (54). The late-onset and long-lasting effect of BDNF feeding inhibition suggests that BDNF may engage other pathways in mediating this effect, an idea that needs further testing.
We tested unilateral and bilateral administration of BDNF to see whether bilateral application of BDNF would result in a more robust effect on feeding and body weight gain. Bilateral administration of BDNF resulted in more robust and potent anorectic effects than that seen after unilateral BDNF administration: bilateral BDNF reduced food intake in the 0- to 24-h interval at the dose of 0.1 μg and in the 24- to 48-h interval at the dose of 0.3 μg (Table 2), while no such inhibition was observed at these doses using unilateral administration (Fig. 3 and Table 2). However, at the 0.5-μg dose of BDNF, bilateral administration did not further increase feeding inhibition beyond that observed after unilateral BDNF injection (Table 2). A similar pattern is seen for inhibition of body weight gain: compared with unilateral injection, bilateral BDNF at 0.5 μg did not significantly change inhibition potency during the 0- to 24-h and 0- to 48-h periods (Fig. 6 and Table 2). However, inhibition potency was dramatically increased with bilateral injection at the 0.1-μg dose of BDNF in the 0- to 24-h interval, and at the 0.3 μg dose of BDNF in the 0- to 24-h and 0- to 48-h intervals (Table 2).
To explore specificity of effects to PVN, data from animals deemed to have incorrectly placed injection sites as determined by histology were statistically analyzed. In these cases, no significant effects were observed for any end point. In some cases, cannula placement was deemed incorrect based on feeding response to NPY, and in one of these studies, i.e., that of deprivation-induced feeding, BDNF at 0.5 μg decreased feeding in the 24- to 48-h and 0- to 48-h intervals. However, significant body weight change was not seen in these rats, but was observed in the rats with correctly placed cannulae (Fig. 4). Thus it is possible that there are other sites of action near the PVN, but it should be noted that cannula placement as indicated by behavioral assay, which is the only available option for reducing data from animals with misplaced cannule when histological analysis is not possible, is not an absolute determination. It is possible that some of these rats had correctly placed cannulae, and thus the biological effects observed could have been due data from these rats, but this is impossible to determine. Additional evidence that effects were localized to BDNF action within the PVN comes from the finding that BDNF in the rostral lateral hypothalamic area, which is anatomically near the PVN, had no effect on feeding behavior or body weight.
To verify that the feeding inhibition observed after PVN-injected BDNF is not due to potential aversive effects of BDNF, two CTA studies were performed with bilateral injection of 0, 0.1, 0.3, 0.5, 1, or 3 μg BDNF, in which animals were exposed to saccharin immediately before or 7 h after injection of BDNF, respectively. BDNF at doses ≤ 3.0 μg, for which significant feeding inhibition was observed (Figs. 2, 3, and 5), did not induce taste aversion (Fig. 7). These results indicate that the observed feeding inhibition by these doses of BDNF was not due to malaise or other aversive sequelae. In our present study, BDNF at 1 μg reduced food intake from 26.8 to 19.5 g (21.4% inhibition). Using a chronic infusion paradigm, Bariohay et al. (2) reported that BDNF at 0.1 and 1 μg/day in the dorsal vagal complex decreased feeding from more than 20 g to ∼10 g by 24 h after administration, which is more than a 50% reduction in food intake. However, no CTA was performed, and thus it is uncertain whether the anorectic effect of BDNF in that study was due to interference with feeding regulatory pathways or to potential aversive properties of chronic BDNF infusions into this region.
Inhibition potency in normal feeding (Fig. 2) is stronger than that observed for deprivation-induced feeding (Fig. 5), suggesting a role for BDNF in normal feeding behavior. In a situation of food deprivation, orexigenic peptides, such as NPY (6, 41), AGRP (30), orexin A (4), and ghrelin (47) are upregulated, while anorectic peptides, such as leptin (48), POMC (α-MSH) (29), and CART (21, 40) are downregulated. This results in a stronger appetitive drive in food-deprived compared with nondeprived animals. Although in normally feeding animals the above neuroendocrine changes also contribute to the normal appetitive drive, they are likely to be much more robust in food-deprived animals. Thus the diminished ability of BDNF to inhibit feeding in food-deprived animals (vs. in normally feeding animals) is likely due to these food-deprivation-induced neuroendocrine changes.
In the food-deprivation experiment, we found that BDNF was most effective in suppressing food intake in the 4- to 24-h postinjection interval, and that this feeding inhibition was not compensated for by excess food intake in the 24- to 48-h interval, suggesting that BDNF feeding inhibition is relatively long-lasting and potent. The timing of effects in this experiment is different from parallel studies on energy expenditure after PVN BDNF. In those studies, PVN BDNF increased metabolic rate immediately after injection and lasted for up to 7 h (see a companion article, Ref. 51a). The difference in timing for BDNF effects on feeding and metabolic rate suggests different mechanisms for BDNF feeding inhibition and BDNF-induced thermogenesis. In both the deprivation and normal feeding studies, BDNF reduced body weight gain, which was likely influenced both by the reduction of food intake observed and increases in energy expenditure as indicated above. Thus BDNF in the PVN in the current studies likely decreased body weight by impacting pathways involved in feeding and energy expenditure.
NPY, which is important to both feeding and energy expenditure, is synthesized in the Arc and is axonally transported to the PVN where it is released. Injection of NPY in the PVN induces a significant increase in feeding (43, 46) and a reduction in energy expenditure (3, 11, 20). It is possible that BDNF inhibits feeding and affects energy expenditure by interfering with NPYergic pathways. Our previous studies showing blockade of NPY-induced feeding by CART (55) and urocortin (54) suggest interactions between different signaling neuropeptides in this region. NPY immunoreactive axons make synaptic contact with PVN parvocellular neurons expressing CRH-immunoreactivity (22), and BDNF mRNA has been identified in CRH-containing neurons in the parvocellular PVN (45). Thus it is possible that NPY also influences BDNF cells within the PVN. The current data show that PVN injection of BDNF at 0.3 and 0.5 μg significantly decreased feeding induced by PVN-injected NPY at 1 h after administration, and BDNF at 0.5 μg decreased NPY-induced feeding at 2 and 4 h after injection (Fig. 8), suggesting that PVN BDNF stimulation inhibits PVN NPY feeding effects. The potential mechanism of this interaction remains to be studied, but could include BDNF neurotrophic effects as the 4-h preinjection time for BDNF in the present study would have allowed sufficient time for neurotrophism (51). The inhibition of PVN NPY-induced feeding by BDNF (Fig. 9) suggests that BDNF may be important to NPY regulation of feeding. As an orexigen, fasting or food restriction appropriately elevates NPY gene expression in Arc (6, 41), presumably as a counterregulatory response to energy deficit. We tested whether BDNF interferes with this process by administering BDNF and then measuring Arc NPY gene expression 24 h later. As shown in Fig. 9B, BNDF normalized NPY gene expression to control levels (BDNF vs. pairfed controls) suggesting that the normal rise in Arc NPY gene expression during food restriction is dependant upon reductions in BNDF actions. Together, these data suggest NPY-BDNF interactions in the regulation of feeding behavior and response to energy deficits, although a direct effect of BDNF on NPY remains to be established.
It has been proposed that BDNF is an important downstream effector of melanocortin-4 receptor (MC4R) signaling (59). However, the timing of melanocortin-induced feeding inhibition is inconsistent with that for BDNF found in the present study. After ventricular injection of Ac-Nle4-c[Asp5, d-Phe7,Lys10]α-MSH-(4-10)-NH2 (MTII; agonist for MC4R), food intake was suppressed immediately at 1, 2, 3, 4, 6, 8, and 12 h after injection (12). PVN MTII decreased feeding at 2 h after administration (13). PVN α-MSH also reduced food intake at 2 h and 4 h after injection and the inhibitory effect was no longer seen at 24 h (58). In contrast to the above observations, feeding inhibition by BDNF was not observed within the first 4 h after injection, and the effect lasted up to 48 h after injection. This discrepancy in the timing of feeding suppression suggests that different mechanisms support feeding inhibition after BDNF and melanocortins.
Unlike the immediate effects of BDNF on energy expenditure (see companion article, Ref. 51a) BDNF exhibits delayed and prolonged feeding inhibition, suggesting that different mechanisms mediate these two responses. A potential mechanism for BDNF-induced feeding inhibition is via Arc NPY/AgRP neurons, an idea supported by the finding that BDNF reverses food-restriction-induced increase in Arc NPY gene expression (Fig. 9B). Behavioral effects of reduced gene expression might be expected to be late-onset and long-term, like BDNF effects on feeding behavior. CRH receptor signaling may also be involved as studies have shown functional and anatomical connections between BDNF and CRH (14, 45). Importantly, a single intracereboventricular injection of BDNF gradually increases CRH mRNA levels in the PVN for up to 48 h postinjection (14). It is possible that administration of BDNF increases expression and translation of CRH, which in turn inhibits feeding. Finally, studies also show anatomical and functional connections between BDNF and thyrotropin releasing hormone (TRH) (45). Central TRH reduces food intake (19), and BDNF increases the early expression of TRH mRNA in fetal hypothalamic neurons in vitro and may directly affect TRH biosynthesis (15). Together these data support the possibility that BDNF may induce expression of TRH and via central TRH actions, reduce feeding.
In conclusion, unilateral and bilateral injection of BDNF in the PVN decreased normal feeding, deprivation- and NPY-induced feeding, and body weight gain, with the inhibition lasting for up 48 h and without producing conditioned taste aversion. These data, combined with other evidence, suggest that BDNF decreases body weight by reducing food intake and elevating energy expenditure. These data provide the first evidence that the PVN is a specific central site of BDNF action, and together, these data indicate that BDNF in the PVN may be an important mediator of feeding and energy regulation.
This work was supported by the Department of Veterans Affairs and by the Minnesota Obesity Center (Pilot and Feasibility Program No. 14).
We thank Regeneron Pharmaceuticals (Tarrytown, NY) for providing BDNF and Mark Margosian, Mary Mullet, Jennifer Teske, and Martha Grace for their technical support in the present study.
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.
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