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Effect of Nucleus Accumbens Shell Inhibition on Salt Intake Frehiwot Gebrehiwot RET Fellow 2010 RET Mentor, Dr. David Wirtshafter Department of Behavioral Sciences University of Illinois at Chicago NSF RET Grant – EEC 0743068 (Andreas Linninger, PI) Introduction The nucleus accumbens is a small region in the brain that controls motivation and reward to natural stimuli such as food or any activity that elicits pleasure. It is also thought to play a role in laughter, fear and aggression. It is a collection of neurons that is part of the limbic system and located near the midline in the frontal region, beneath the frontal lobe1. Each half of the brain has one nucleus accumbens. The nucleus accumbens is divided into two major subdivisions the shell and the core each with different morphology and function. The primary output neurons of both of these areas are medium spiny neurons. The neurotransmitter produced by these neurons is gamma-aminobutyric acid (GABA), one of the main inhibitory neurotransmitters of the central nervous system. One of the major inputs to the nucleus accumbens include dopaminergic neurons located in the ventral tegmental area (VTA) that provide a pathways for the neurotransmitter dopamine2. These terminals are also the site of action of highly-addictive drugs such as cocaine and amphetamine, which act by increasing the level of extracellular dopamine in the nucleus accumbens shell. Figure 1: Nucleus Accumbens, key structure in the brain responsible for reward and motivation Two of the greatest threats to public health in the developed world are drug addiction and obesity. In the US, annual deaths attributable to nicotine and alcohol dependence or abuse are estimated at 450,000 3. Obesity is another silent killer. According to a study published in January of 2010, 33.8% of the population in the United States is now obese (BMI>30) and are at greatly elevated risk for many disorders, such as heart disease, hypertension, and diabetes. The nucleus accumbens and its associated circuitry constitute a system that subserves motivated behaviors, such as feeding, drinking, sexual behavior, and incentive learning, and it is this system that is also most clearly implicated in drug addiction7. Thus understanding the specific role of the nucleus accumbens and it neuro-circuitry has much significance. Further study of the nucleus accumbens may lead to possible treatments for addiction, obesity and other eating disorders. Previous studies of the AcbSh have established its role in the motivation for food intake. This relationship was first discovered by accident when researchers were injecting the AMPA/kainite receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione) into the area of the nucleus accumbens in an attempt to examine spatial learning. It was noted that animals that had received intra-AcbSh DNQX had highly increased feeding when returned to their home cages after behavioral testing. (4) The AcbSh/feeding response to various perturbations has since been subject to exploration. Various receptor agonists, antagonists, neurotransmitter blockers and lesions (8) have been tested in relation to feeding, but of particular interest to us is the GABAA agonist muscimol. It was noted that inhibition of the AcbSh induced an increased feeding response similar to electrical stimulation of the lateral hypothalamus (LH), (4) and because it was known that many of the AcbSh neurons that synapse upon the LH were GABAergic in nature, it was speculated that disabling the AcbSh with a GABA agonist like muscimol would generate similar increases in feeding. We now know this to be the case, though it was later determined that the GABAergic neurons of the AcbSh do not synapse directly upon the LH but instead work through a mediator area like the ventral palladium, (9) but the nature of the feeding response is still not completely clear. For instance, inhibition of the AcbSh with muscimol induces a feeding selectivity not seen with electrical stimulation of the LH. (4) Nutritive foods, like sucrose, are highly preferred by rats that receive muscimol in the AcbSh(4)(12)(13), while non-nutritive foods, even palatable substances like saccharine (a low calorie sugar substitute) are either consumed at the same rate compared to control, or even less. The goal of the present study is to further define the relationship between the accumbens shell (AcbSh) and feeding motivation. We will examine the effect of inhibition of the AcbSh with the GABAA agonist muscimol on salt feeding in salt deprived rats. Salt is a strong natural motivator and plays an important physiological role in the body. Given the opportunity, rats and humans overconsume salt (NaCl) (5). This observation has led researchers to use the rat as one model of human salt consumption. In the past, most studies of behavioral sodium regulation in rats have used salt water intake as a measure of salt appetite(2).Given a choice between hypotonic or isotonic saline and water, many strains of rats will ingest more saline than water when sodium replete(8). This preference for salt water has been studied extensively, partly to understand better the regulation of sodium intake. It is known that inhibition of the AcbSh has a statistically negligible effect on regular salt feeding in rats; however several studies have noted that repeated salt deprivation leads to an increase in dendritic branch and spine formation in the AcbSh similar to that seen in long term amphetamine exposure. Even more interesting is the tendency for repeatedly salt deprived rats to gain a permanent increase to their salt consumption habits, similar to the mechanisms of tolerance and addiction seen in drug users. (10) Furthermore, salt deprivation has been shown to increase presentation of c-Fos, an indirect marker of neural activity, in the AcbSh in rats. (11) The creates an interesting conflict; on one hand, the AcbSh seems to be uninvolved in normal salt feeding motivation, however salt-deprivation has a pronounced effect on the AcbSh not only in its activity (11) but on its plasticity as well. Thus in our investigation we hope to address this inconsistency by salt depriving the rat while simultaneously deactivating the AcbSh. Our goal is to examine if feeding response under these conditions will differ from that elucidated under the effects of AcbSh inhibition alone. Methods and Materials 1) Subjects: a total of 24 male, albino Sprague Dawley strain lab rats were used 2) Surgery and Recovery a. Rats are anesthetized using sodium phenobarbitol (with ketamine available as an anesthetic booster). b. After being sedated guide cannulas are implanted using standard, flat-skull stereotaxic techniques using predetermined coordinates (anteroposterior, 1.7; lateromedial, ±0.9; dorsoventral, -5.9, in millimeters from bregma). (12) Approximate localization of cannula tips can be seen in Fig. 1. c. The cannulas are then anchored in place with bone screws and dental cement and the rats are allowed to recover for a period of several days. 3) Lickometer Acclimation and Baseline Data a. After recovery rats are water deprived nightly and given one hour each day in the lickometer apparatus to drink. The lickometer is a specialized cage with a voltage difference between the feeding bottle spout (+5V) and the cage bottom (0V/Ground). When the rat licks the spout to drink, a small current passes through its body and each instance is recorded by an attached computer running the MedPC software. All lickometer sessions are one hour in duration. b. The rats are given distilled water for the first two days of acclimation, and then a 3% salt solution for the next three days. c. After this follows four more days of 3% NaCl but without water deprivation. This constitutes the baseline data. 4) Experimental Data Collection a. On the day immediately following the conclusion of the non-deprivation 3%NaCl sessions the rats are given an intracerebral injection of sterile saline solution (0.5μL/side). The purpose of this procedure is twofold. First, the injection of fluids into the area of the AcbSh may prove discomforting to the rat and to minimize this as a variable during the actual experimental injections this injection is given to acclimate them to the sensation. Second, the ends of Figure 2: Illustration A shows the approximate location of the cannula after surgery, the cannula are actually situated about 2mm above represented by the thicker portion of the long the AcbSh so as not to disturb the AcbSh or the black line. The thin black line represents the surrounding tissue. When the injectors are inserted 2mm protrusion of the injector which is they project 2mm beyond the end of the cannula so inserted into the cannula. The darkened that materials are injected directly in the vicinity of globular region represents the approximate area of the AcbSh. Illustration B involves a the AcbSh similar procedure targeting the lateral hypothalamus, but is not relevant to this proposal. Reproduced with permission from Ref. 12. (see Fig. 2). When these injectors are inserted they cause fresh tissue damage which must be given time to heal before actual experimental injections can be performed. b. After this sham injection, the rats are given 48 hours to recuperate. c. After recuperating the rats are given an injection of 10% furosemide. Furosemide is a loop diuretic which prevents reuptake of Na+ and Cl- ions in the kidneys. This causes the rats to pass a large amount of their bodily NaCl in their urine. After three hours the rats are moved to new cages and given a preweighed amount of salt-free food pellets for 24 hours. d. The rats are then split evenly into two groups based on similarities in their baseline data. The first group is given an injection of muscimol (0.5μL/side of a 100ng/μL solution, equivalent to 50ng/side) and the second group is given an injection of sterile saline (0.5μL/side). The rats are then placed in the lickometers with 3%NaCl solution to drink and their feeding behavior monitored. e. After this the rats are placed in new cages and the amount of salt free food and distilled water that they had consumed from the previous cages is measured. f. After 72 hours the rats are placed in the lickometers with 3% NaCl for two consecutive days. 48 hours after the second day steps c through e are repeated but with the groups reversed (rats that previously received muscimol injections receive sterile saline instead and vice versa). 5) Placement Checks, Euthanasia and Dissection a. 72 hours later the rats are run on two consecutive days of lickometer sessions with a 10% Sucrose solution. Because it is well established that AcbSh inhibition drastically increases sucrose feeding (4) (12) (13) we use this as a test for determining whether the injections were accurately targeting the AcbSh. b. After four consecutive days of sucrose feeding, the rats are again given muscimol and sline injections as on the first day of 3% NaCl experimentation. c. 24 hours later the rats are run on a 10%Sucrose session. d. 24 hours later the rats are again given muscimol and sterile saline injections but with the groups reversed. e. 48 hours later the rats were anesthetized using a fatal dose of sodium pentabarbitol and then perfused using a 10% formalin solution. The brain is removed, flash frozen and sliced to reveal the cannula tracks in the tissue. These slices are then examined to determine exact placement of injections and to discover any infection. Data Analysis Information about the rats’ consumptatory behavior is collected in a multitude of ways. The MedPC program to which the lickometers are attached calculates 15 different variables based on the rats’ licking patterns. Not all of these will be used for analysis in this protocol, but a brief list and descriptions of the relevant data follows; 1) Total Licks This is the most obvious indicator of rats’ preference for the solution they are eating and correlates directly with their motivation for consuming the given substance. Each lick averages an equivalent volume of 1/150mL. 2) Number of Bursts This data, along with number of clusters and the timing arrays for both are more subtle indicators of rats’ preference. There is a correlation between the type of motivation and the number of bursts and clusters that appear in the consumptatory behavior. For example, a high lick count with a comparatively high number of bursts and clusters may indicate a rat that craved the substance but disliked the taste or sensation of consuming it. This would theoretically lead to frequent pauses and shorter drinking clusters. 3) Number of Clusters See Number of Bursts. Clusters represent longer pauses in feeding behavior than bursts, but are much more variable in nature. Because inter-cluster intervals (ICIs) are defined as pauses greater than 0.5 seconds, they can represent anything from a very brief cessation of normal drinking patterns to extended periods over which the rat engages in other, nonconsumptatory behaviors (resting, foraging, etc.). For example, a low number of clusters combined with short length ICIs might suggest a grouped drinking pattern where the rat consumes as much as it wants in a small window of time and spends the rest of the time engaged in other behaviors. 4) Mean Burst Size/Mean Cluster Size These two variables, together with previous variables, can be used to further qualify a given rat’s feeding behavior. As mentioned in the discussion on Number of Bursts, a possible scenario is a rat with a higher number of Bursts/Clusters but a relatively short Burst/Cluster size. This might indicate a strong dislike for consuming the substance, but a substantial motivation to do so anyway. While this data is useful for making certain assumptions about rat behavior, it is difficult to make any qualitative conclusions based on bursts and clusters. Therefore such data may be used to “color” the quantitative results obtained by statistical analysis of the lick counts, but the true analysis rests solely on the volumes consumed by the rats. The theoretical volume, calculated based on a theoretical consumption of 1mL per 150 licks, and the theoretical licks, based on working backwards from the recorded change in volume of the feeding tubes, will both be analyzed using a repeated measures ANOVA for statistical testing of the null hypothesis that muscimol has no different effect than sterile saline on salt consumption. Before this statistical analysis can be performed we must first omit data from animals with massive infections or misplaced cannulae. Infections can result from the initial operation or possibly even from entry of foreign materials through the cannulae post-surgery. Animals in our lab have been test for infection and we have found that there is a possibility that the cannulae with the obturators in them are acting as a “vertical petri dish” being filled as they are with cerebro-spinal fluid (CSF) and residual tissue debris from surgery. They are also isolated from the body’s immune system, making this an ideal place for bacteria and other pathogens to thrive. It is feasible that when the obturators are removed on the day of the first injection and the injectors subsequently inserted, any pathogens residing in the cannulae are pushed down into the tissue. The resulting tissue damage, combined with the potentially large amount of pathogens introduced into a small area may be enough to overwhelm the rat’s immune system. There is a strong possibility that we can address this issue by regularly changing the screw caps that cover the obturator/cannula assembly. Our investigations have shown that animals which regularly lost and had their screw caps replaced had much smaller, or even no infections. Misplaced cannulas are another concern which we must address. While our stereotaxic method for inserting the cannulas is almost always accurate, there is an inherent error due simply to the differences between animals and human error. We perform placement checks, as mentioned in the methods above, to ensure that the injectors are reaching the AcbSh, and also check the brains physically after perfusion to determine the exact locations. If we do find that a cannula assembly was misplaced, that rat would naturally be dropped from the study. Results and Discussion Consumption of 3% Salt Solution After Injections into the Accumbens Shell 4000 Number of Licks 3000 2000 1000 0 Saline Muscimol Drug Treatment Figure 3: Mean of number of licks recorded during 60 minutes testing. Difference between the two groups is 6.750. t= 0.0201 (P= 0.984) Average Number of Licks Per 5 Minute Bin After Injections Into the Accumbens Shell 1000 Average Licks per Bin 800 600 400 200 0 0 1 2 Muscimol Saline 3 4 5 6 7 8 9 10 11 12 13 Bin Figure 4: Data shows that there is no significant difference in pattern of consumption of salt solution during testing between the two groups. Rats were tested in groups of six with four experiments run separately and the data was then aggregated. Figure 3 shows the mean consumption of 3% solution during the 60 minutes of testing. The mean in the number of licks for the saline group (control) is 1640.083 while for the muscimol group it is 1633.333 with standard deviation of 777.244 and 863.573 respectively. The difference in the mean values (6.750) of the two groups is not great enough to reject the possibility that the difference is due to random sampling variability. The t-test indicates t=0.0201 with a P = 0.984. The data indicates that inhibition of the AcbSh by muscimol had no effect on salt intake. Our analysis of mean burst size and mean cluster size also shows no difference in the pattern of drinking between the muscimol injected group and the saline injected group that may indicate a difference in motivation. Figure 4 shows the average number of licks per 5 minute periods. Again, we see no difference between the two groups. The results demonstrate no apparent effect due to muscimol in the AcbSh (vs. sterile saline). The next step in the research will be to conduct a histological examination of the brain of tested rats to check for correct placement of cannulae into the AcbSh. Upon these findings, future research should focus on understanding the relationship of the AcbSh with other regions of the brain and the specific effect of muscimol on other parts of the brain. The answer to these findings will help clarify the result of our investigation. Acknowledgements Financial support by NSF RET Grant – EEC 0743068 (Andreas Linninger, PI) is gratefully acknowledged. In addition, I would like to thank the following: Professor Andreas Linninger, Program Director Seon B. Kim, Program Assistant Dr. David Wirtshafter, Research Mentor University of Illinois at Chicago Co-Investigators: Beth Cowgill and William Schulenberg Work Cited 1. Prevalence and trends in obesity among US adults, 1998-2008. Flegel, KM, et al. 3, 2010, Journal of the American Medical Association, Vol. 303, pp. 235-241. 2. World Health Organization. Technical report series 894: Obesity: Preventing and managing the global epidemic. Geneva : World Health Organization, 2000. 3. Stratford, TR. 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