The goal of this research was to find the multiple mechanisms by which hypocretin 1 increases food intake when injected into the lateral hypothalamus (LH) of the brain. Three possibilities were investigated: 1, it causes the release of opioids or 2, the release of glutamate, and 3, the LH communicates with the paraventricular nucleus, telling it to release neuropeptide-Y. I found that hypocretin 1 stimulates feeding via the release of opioids, but not glutamate. The third test was inconclusive.

Obesity is a problem that plagues a large number of people in the developed world. Unfortunately, the definite cause of this condition is not known. One possibility that many scientists are studying is that the brain plays a large role in determining eating behavior. The approach is to determine how specific sites of the brain interact and what neuro-chemicals stimulate activity that may result in an increase in food intake. Numerous experiments must be conducted to learn about specific functions so that the collected information can eventually be put together to formulate a theory of how the brain plays a role in obesity. Once this is done, scientists may begin to formulate ways to solve the problem. However, specific brain regions must be examined to understand how they function to regulate food intake.

The lateral hypothalamus (LH) and the paraventricular nucleus (PVN) are regions in the brain that are known to control eating behavior¹. Injection into the LH of hypocretin 1, a 33-amino acid peptide naturally secreted in the LH, causes an increase in food intake^2,3. When a rat eats food, chemicals called opioids are released by the brain to signal the brain that the food tastes good⁵. Injecting additional opioids in the brain causes an increase in food intake because the rat believes that any food tastes delicious³. Naltrexone is an opioid receptor antagonist; it blocks the receptors that receive and process the opioids. If hypocretin 1 stimulates feeding by releasing opioids, then injecting naltrexone to block the opioid receptors will inhibit the stimulation of feeding induced by hypocretin 1. The administration of hypocretin 1 into the LH has also been proven to stimulate the release of glutamate¹. It is not known why this happens. However, injecting glutamate in the LH has shown to stimulate feeding¹. There is the possibility that hypocretin 1 stimulates feeding by releasing glutamate. A chemical called CNQX is a glutamate receptor antagonist. If hypocretin 1 stimulates feeding via the release of glutamate, then injecting CNQX to block the glutamate receptors will inhibit the stimulation of feeding.

The injection in the PVN of Neuropeptide-Y (NPY), a brain chemical naturally secreted in the PVN, strongly stimulates and increases food intake⁴. It has been detected that after hypocretin 1 is injected in the LH, neurons in the PVN become activated⁵. There is the possiblity that when hypocretin 1 is injected into the LH, the LH signals the PVN to release the chemical NPY which consequentially, increases food intake.

The goal of this research is to find the multiple mechanisms by which hypocretin 1 increases food intake when injected into the LH. Three possibilities will be investigated: it causes the release of opioids, the release of glutamate, and third, the LH communicates with the PVN, telling it to release Neuropeptide-Y. After performing these experiments, one can see if hypocretin 1 uses any or all of the three possible techniques to increase feeding.

1. Preparation for experimentation and care for rats.

Eighteen male Sprague-Dawley rats(about 225-250 g) each had a cannula surgically placed in the LH region of the brain. The procedure was perfomed by the lab technician for the purpose of other experimentation. All 18 rats were later made available and used for all of these experiments over the course of two months. After the procedure, the rats were given a week to recover. The rats were housed at room temperature, about 21-22° C, in individual hanging wire cages and were given tap water and chow pellets (Teklad 8604) from a wire hopper. The room received 12 hours of light each day starting at 7:00 A.M.

Prior to first use, Hypocretin 1 was dissolved in 0.9% saline immediately, and when not in use, was stored at 4° C for a maximum of three weeks. Before each experiment, new paper was put under the cage to collect any food spillage. The first and second chemicals of a treatment were injected with a 15 minute interval in between to allow the first chemical to diffuse. All injections and handing of the rats were done by the supervising scientist. Food and hoppers were then weighed at baseline. Food intake was measured as the weight difference from baseline minus the weight of spillage. The food intake results for experiments 1 and 2 were analyzed using a repeated measures ANOVA. For this type of anaysis, every rat received a control treatment, so each rat was its own control. The control chemical is artifical cerebral spinal fluid (Acsf) which resembles the natural fluid in a rat’s brain. By giving each rat a control injection, one can see how much the rat ate when given the other treatments, and compare it to how much it ate when given the control injection. Then it would be known that the rat ate more or less strictly due to the chemical. The data collected for the third experiment was analyzed using a single factor ANOVA.

Each rat received all three treatments over the course of a week with a day rest between treatments so that the rats could resume their normal eating habits. The three treatments are as follows:

The purpose of the control treatment is to find the amount the rat eats when given a simulated treatment, that is, a treatment without any drugs. The second treatment is to prove that hypocretin 1 stimulates feeding. The final treatment is to see whether the opioid blocker inhibits the effects of hypocretin 1. The dose of Naltrexone(NTX) to be given to each rat was calculated according to the equation: 5 mg NTX/kg of the rat’s body weight. This chemical had to be injected in the belly(intraperitoneally) so it would circulate throughout the brain because it is not known where it works in the brain. One and two hours after the second injection, food intake was measured.

The four treatments that every rat received are as follows:

Food intake was measured 1, 2, and 4 hours after the injections.

5. Experiment 3: Does Hypocretin 1 stimulate feeding by initiating communication between the LH and PVN?

Half of the rats received 2000 pmol Hypocretin 1 in the LH, and the other half received artificial csf in the LH. Two hours after the injections, the supervising scientist euthanized the rats and removed several body samples for other experimental purposes. He then provided this experiment with the PVN of each rat. Later, the protein from each PVN sample was extracted. The specific amount was then calculated. The protein samples were prepared and the NPY levels were found by the lab technician. To account for different protein levels in varying PVNs, the amount of NPY in a sample was divided by its respective amount of protein. The different concentrations of NPY were then compared to see if there was a higher level of NPY in the PVN of the rats injected with hypocretin 1 compared to those of the rats injected with artificial csf.

1. Experiment 1: Does Hypocretin 1 stimulate feeding via the release of opioids?

The data is displayed below. Figure 1 shows the effects from 0-1 hour, and figure 2 shows the effects from 0-2 hours. As shown in figure 1, the rats injected with Acsf+Hrct-1 ate significantly more than the control rats so I can presume that the Hcrt-1 chemical was effective. The rats injected with the opioid blocker ate significantly less than the Hrct-1 rats. The p-value for this analysis is .0024. There is significance if the p-value is below .05. In figure 2, which was from 0-2 hours, the trend was the same. The p-value for figure 2 was .0029. Figure 3 is an analysis I produced that will help show the effect of Naltrexone. From all the data, I assigned the number 1, which meant that the rat attempted to eat food, if the rat ate more than 0.1g of food. If it ate 0.1g or less, I assigned a number 0 which meant that the rat did not attempt to eat food. From figure 3, the rats injected with the opioid blocker had a significantly greater amount of attempts than the control groups. The p-value for figure 3 is .0001.

 

Figure 1.



 

 

Figure 2.

 

Figure 3.



 

2. Experiment 2: Does Hypocretin 1 stimulate feeding via the release of glutamate?

Figure 4.

Figure 5.

Figure 6. 

Figure 7.

The data is displayed in figure 8 below. The P-value for it is .2692. The rats injected with Hcrt-1 seem like they had a higher concentration of NPY in their PVNs, but the results were not significant.

Figure 8.

1. Experiment 1: Does Hypocretin 1 stimulate feeding via the release of opioids?

Because the Naltrexone treatment inhibited the stimulation of feeding induced by Hypocretin 1, I can presume that Hypocretin 1 stimulates feeding via the release of opioids. The rats did not eat as normal because eating provided no satisfaction. The transfer of opioids was blocked, so the food did not taste good. The effect of the drug was the same for the first and second hour. From figure 1, we can see that the rats injected with the opioid blocker ate a nearly equal average amount of food as the rats injected with the control treatment did. According to figure 3, the amount of rats that attempted to eat when injected with the opioid blocker was significantly greater than the amount of rats that attempted to eat when injected with the control treatment. I can then conclude that the hypocretin 1 stimulated the rat to eat, but the rat did not feel any pleasure in eating so it stopped eating. When injected with the control treatment, some rats ate a decent amount, and some did not eat at all. When injected with the naltrexone treatment, most of the rats ate, but they did not eat very much. This is because the transfer and processing of opioids in their brains was halted.

2. Experiment 2: Does Hypocretin 1 stimulate feeding via the release of glutamate?

The glutamate blocker, CNQX, did not have show any significant effect over the entire four hours after injected. Rat injected with CNQX and Hypocretin 1 did not eat significantly more or less than the rats injected with Acsf+Hcrt-1. I can conclude that Hypocretin 1 does not stimulate feeding via the release of glutamate. This result was contrary to what I had expected. From experience, the supervising scientist knew that CNQX inhibited feeding of rats deprived of food for 24 hours. Therefore, I decided to perform the same test to see if I got the same result. Then I would know whether the CNQX that I used was still functional. I performed the experiment and found that the CNQX did inhibit the food intake of rats deprived of food for 24 hours. The batch of chemical I had was functional so my first result was valid.

3. Experiment 3: Does Hypocretin 1 stimulate feeding by initiating communication between the LH and PVN?

According to Figure 8, it appears that there was a higher average concentration of neuropeptide-Y (NPY) in the paraventricular nucleus (PVN) of rats injected with Hcrt-1 than in those injected with artificial csf. However, the p-value for the analysis is .2692, which is not significant. The results were inconclusive and the hypothesis must retested.

I was able to reach several conclusions from my experimentation. First, hypocretin 1 stimulates feeding via opioid release in the lateral hypothalamus in the brain. Second, the brain does not stimulate feeding via glutamate release in the lateral hypothalamus. Finally, it seems as though hypocretin 1 stimulates feeding by activating the LH to instruct the paraventricular nucleus (PVN) to release Neuropeptide-Y. However, this final statement is still only a hypothesis because the data was not significant so it must be retested.

I would like to thank the entire lab of Dr. Cathy Kotz at the VA Medical Center for the wonderful experience. I would especially like to thank Dr. Kotz, Don Sweet, Chuanfeng Wang, and Dr. Jacob Miller for their support and help to give me insight into the world of science.

1. Kotz, Dr. Cathy. Personal interview, a series of interviews beginning 6 June 1999 and ending 25 August 1999.
2. Sakurai, T. et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior, Cell 92 475-481(1998).
3. Sweet, Don. Personal interview, a series of interviews beginning 6 June 1999 and ending 25 August 1999.
4. Stanley, B.T., Magdalin, W., Seiraft, A. et al. Peptides 13, 581-587(1992).
5. Horvath, T.L., Diano, S., can den Pol, A.N. J. Neuroscience. 19, 1072-1087 (1999).