Download The nucleus accumbens is a small region in the brain that controls

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. The nucleus accumbens shell as a model of integrative subcortical forebrains systems
regulating food intake. [ed.] SJ Cooper and TC Kirkham. Appetite and Body Weight: Integrative Systems
and the Development of Anti-Obesity Drugs. London : Elsevier, 2006.
4. Corticostriatal-hypothalamic circuitry and food motivation: Integration of energy, action, and reward.
Kelley, AE, et al. 2005, Physiology & Behavior, Vol. 86, pp. 773-795.
5. Amygdaloid connections with posterior insular and temporal cortical areas in the rat. McDonald, AJ
and Jackson, TR. 1, 1987, Journal of Computational Neurology, Vol. 262, pp. 59-77.
6. Neurons containing hypocretin (orexin) project to multiple neuronal systems. Peyron, C, et al. 23,
1998, Journal of Neuroscience, Vol. 18, pp. 9996-10015.
7. Specificity in the projection patterns of accumbal core and shell in the rat. Heimer, L, et al. 1, 1991,
Neuroscience, Vol. 41, pp. 89-125.
8. Excitotoxic lesions of the core and shell subregions of the nucleus accumbens differentially disrupt
body weight regulation and motor activity in rat. Maldonado-Irizarry, CS and Kelley, AE. 6, 1995, Brain
Research Bulletin, Vol. 38, pp. 551-559.
9. GABA in the nucleus accumbens shell participates in the central regulation of feeding behavior.
Stratford, TR and Kelley, AE. 11, 1997, Journal of Neuroscience, Vol. 17, pp. 4434-4440.
10. Interaction between natural motivation systems and those which respond to drugs of abuse.
Bernstein, IL. 3, 2003, Appetite, Vol. 41, pp. 333-334.
11. Induction and expression of salt appetite: Effects on Fos expression in nucleus accumbens. Voorhies,
AC and Bernstein, IL. 1, 2006, Behavioural Brain Research, Vol. 172, pp. 90-96.
12. Evidence of a functional relationship between the nucleus accumbens shell and lateral hypothalamus
subserving the control of feeding behavior. Stratford, TR and Kelley, AE. 24, 1999, The Journal of
Neuroscience, Vol. 19, pp. 11040-11048.
13. Deterministic and probabilistic control of the behavior of rat ingesting liquid diets. Davis, JD. 1996,
Am J Physiol Regul Integr Comp Physiol, Vol. 270, pp. 793-800.