1. Project Leader: Katie Kavanagh
Effects of Lateral Hypothalamic Injections of Glutamate Receptor Antagonist (D-AP5) and Agonist (ATPA) on Food Intake in Rats Under Simulated Microgravity
Thanks Tyler. Hello. I’m Katie Kavanagh and I’ll be presenting the effects of lateral hypothalamic injections of a glutamate
receptor agonist-ATPA- and antagonist-D-AP5- on food intake in rats under simulated microgravity.
Project Leader: Katie Kavanagh

2. Questions:
What brain mechanisms underlie sensory specific satiety in hind limb unloaded rats?
Specifically, are lateral hypothalamic glutamate receptors regulating sensory specific satiety?
The question we’re interested in answering in this study is—what brain mechanisms underlie sensory specific satiety in hind
limb unloaded rats, and more specifically, are lateral hypothalamic glutamate receptors involved? Essentially, we are building on my
advisor’s previous studies that were completed in horizontal rats. She found that it’s expressly the glutamate receptors in the LH that
influence feeding, and we want to know if the results differ in simulated microgravity.

3. Why is this important?
We hope to determine what brain mechanisms regulate SSS and whether they operate similarly in microgravity as they do on Earth.
The question we’re interested in answering in this study is—what brain mechanisms underlie sensory specific satiety in hind
limb unloaded rats, and more specifically, are lateral hypothalamic glutamate receptors involved? Essentially, we are building on my
advisor’s previous studies that were completed in horizontal rats. She found that it’s expressly the glutamate receptors in the LH that
influence feeding, and we want to know if the results differ in simulated microgravity.

4. (via Nucleus Accumbens, etc.) LH Injection of D-AP5 at 1g
Neural Model
Glutamate release
Lateral Hypothalamus
Sensory Inputs
Sight, Smell, Taste
(via Nucleus Accumbens, etc.)
EAT
Motor
Output:
EAT LESS
LH Injection of D-AP5 at 1g X
D-AP5 = glutamate receptor antagonist
Hypothesis 1: D-AP5 will suppress food intake and increase latency to feed during a SSS paradigm in hind limb unloaded rats
This is a model of what we expect will happen in out experiment. Sensory inputs such as sight, smell, and taste reach the
nucleus accumbens (etc.) and are (communicated) to the LH. The neurons in the LH release glutamate which (binds with) the
glutamate receptors. These (saturated/occupied) receptors will send a signal (to the??) which results in the motor output of eating.
My advisor has found that glutamate receptors are the receptors that are mainly involved in eating. Her method was to block
them and see if eating (changed). Based on these studies, we know that D-AP5 blocks the receptors in horizontal rats and suppresses
feeding—but will this continue in hind limb unloaded rats?
My advisor has also found that ATPA stimulates glutamate receptors in horizontal rats by acting as a glutamate amino acid so
that all receptors are (saturated/occupied). This resulted in increased eating.

5. (via Nucleus Accumbens, etc.) LH Injection of ATPA at 1g
Neural Model
Glutamate release
Lateral Hypothalamus
Sensory Inputs
Sight, Smell, Taste
(via Nucleus Accumbens, etc.)
EAT
Motor
Output:
EAT MORE
LH Injection of ATPA at 1g
ATPA = glutamate receptor agonist
Hypothesis 2: ATPA will boost food intake and decrease latency to feed during a SSS paradigm in hind limb unloaded rats
This is a model of what we expect will happen in out experiment. Sensory inputs such as sight, smell, and taste reach the
nucleus accumbens (etc.) and are (communicated) to the LH. The neurons in the LH release glutamate which (binds with) the
glutamate receptors. These (saturated/occupied) receptors will send a signal (to the??) which results in the motor output of eating.
My advisor has found that glutamate receptors are the receptors that are mainly involved in eating. Her method was to block
them and see if eating (changed). Based on these studies, we know that D-AP5 blocks the receptors in horizontal rats and suppresses
feeding—but will this continue in hind limb unloaded rats?
My advisor has also found that ATPA stimulates glutamate receptors in horizontal rats by acting as a glutamate amino acid so
that all receptors are (saturated/occupied). This resulted in increased eating.
How do we target glutamate receptors in the lateral hypothalamus?

6. Getting to the Lateral Hypothalamus: Stereotaxic Surgery
(Paxinos and Watson, 1998)
medial / lateral
dorsal / ventral
anterior / posterior
3-D coordinate system
Based on Atlas of rat brain
Implant guide cannulas aimed at the lateral hypothalamus
Allows access to the LH for repeated injections
The first step we needed to take was to gain access to the lateral hypothalamus using stereotaxic surgery. First, we used an atlas of the rat
brain to determine its x, y and z coordinates. Then, we implanted 2 cannulas (bilateral) into the lateral hypothalamus and (protected) them with
an obdurator and cap to prevent clogging/blockage. Here are a few pictures of the actual surgery.
{point} These are the cannula holders and here are the cannulas that have just been implanted.

7. Methods: Central Microinjections
Procedure= Inject 0.3 mL of:
aCSF (control)
D-AP5 (10 nmol)
n= 9 male Sprague-Dawley rats
D-AP5 Study (bilaterally)
ATPA Study (unilaterally)
aCSF (control)
ATPA (1 nmol)
n= 8 male Sprague-Dawley rats
needle
After the cannulas were implanted we allowed the rats to rest for at least a week before including them in the experiments.
The key part of the experiments was the central microinjections because this is how we deliver the drug to the lateral hypothalamus. For the
ATPA experiment, we injected 0.3 micro-liters of either artificial cerebral-spinal fluid or ATPA unilaterally into 11 male, Sprague-Dawley rats.
Similarly, for the D-AP5 experiment, we injected 0.3 micro-liters of either artificial cerebral-spinal fluid or D-AP5 bilaterally into 10 male rats.

8. Here are a few pictures of us injecting hind limb unloaded rats
Here are a few pictures of us injecting hind limb unloaded rats. Notice how we keep them unloaded even during the injection so we don’t lose the
fluid shift.

9. Experimental Procedures
18 hr. food deprivation
Pre-injection meal – 90 minutes
Rat Chow
Lateral Hypothalamic injection – aCSF, ATPA, or D-AP5
Post-injection meal – 60 minutes
Rat Chow or Froot Loops®
The experimental procedures are very similar to those that Tyler showed you. We gave the 18 hour food deprived rats a pre-injection meal of rat
chow for 90 minutes. Then, here’s where it’s different than before, an injection of either artificial cerebral spinal fluid, ATPA, or D-AP5 was
delivered. The rats were then presented with a post-injection meal that consisted of either rat chow or Froot Loops.

10. Experimental Time Line
MON
Rest
(in standard cages)
TUES
SAT
SUN
Hindlimb
Unload
WED
Food
Deprive
THURS
Experiment
FRI
Counterbalanced 4 times
Repeated 4 times
This is our experimental timeline which will give you a general idea of the schedule of events. All rats had ad libitum access to food and water
except on experimental days and were kept on a 12 hour light/ 12 hour dark cycle. Also, this study was counterbalanced.
We began our experiments on * Wednesdays around 9 am when we hind limb unloaded the rats.
*Thursdays we food deprived them at 3pm and * Fridays we performed our injection study in the morning. The rats were placed back into their
standard cages * after the experiment and were allowed to rest for 4 days * until the following Wednesday.
Rats had ad libitum access to food and water except on experimental days. Light cycle was 12 hr /12hr light/dark.

11. How SSS was measured… Statistical Analysis
Cumulative Food Intake (grams):
pre-injection meal at 60 and 90 minutes
post-injection meal at 30 and 60 minutes
Latency to Eat (minutes):
post-injection meal
Statistical Analysis
Two way repeated measures ANOVA
Student-Newman-Keuls post hoc test
Sensory specific satiety was measured by the food intake of the pre-injection meal at 60 and 90 minutes to establish satiety. Food intake of the
post-injection meal was measured to determine what affect the injection had on SSS. * Also, the latency to eat for the post-injection meal was
measured to see how quickly the drug took effect??.
*The statistical analysis consisted of a two way repeated measures analysis of variants and a student-newman-keuls post hoc test.

12. Results for D-AP5, a glutamate receptor antagonist
EAT LESS
LH Injection of D-AP5 X
Next are the results for the D-AP5 study. Remember that it’s an antagonist so it will block the glutamate receptors and presumably suppress
eating.

13. D-AP5: pre-injection meal data
Rats satiate on chow in pre-injection meal
The results form the pre-injection meal are that there is no significant difference in the chow intake of the rats at 60 and 90 minutes. * This
means that the rats have satiated themselves on chow.

14. D-AP5: post-injection meal food intake at 30 minutes
~99%
The Froot Loop intake for the rats injected with D-AP5 was significantly less than those injected with the control. The chow intakes did not significantly differ.
Injection

15. D-AP5: latency to eat in post-injection meal
The latency to eat the post-injection meal was longer in the rats that had been injected with D-AP5 than in those injected with the control.
Injection

16. Summary : D-AP5 Conclusion
D-AP5, a glutamate receptor antagonist, suppresses Froot Loops ® intake and increases latency to feed when injected into the lateral hypothalamus.
Conclusion
D-AP5 suppresses intake of a highly palatable food suggesting that glutamate receptors in the lateral hypothalamus are part of the neural pathway underlying SSS.

17. Results for ATPA, a glutamate receptor agonist
EAT MORE
LH Injection of ATPA
Next are the results for the ATPA study. Remember that it’s an agonist so it will act as the glutamate and presumably increase eating by saturating
the receptors.

18. ATPA: pre-injection meal data
Rats satiate on chow in pre-injection meal

19. ATPA: post-injection meal food intake at 60 minutes
Animate summary of results
Injection

20. ATPA: latency to eat post-injection meal
~75%
p=0.3
Injection

21. Summary : ATPA Conclusion
ATPA, a glutamate receptor agonist, boosts rat chow but not Froot Loops® intake when injected into the lateral hypothalamus.
Conclusion
Offering Froot Loops® or injecting with ATPA appear to be equally effective in increasing food intake. This suggests that access to a highly palatable food is sufficient to saturate this neural pathway.

22. X Conclusions: EAT EAT LESS EAT MORE RAT CHOW Lateral Hypothalamus
Glutamate release
Lateral Hypothalamus
Sensory Inputs
(via Nucleus Accumbens, etc.)
Sight, Smell, Taste
EAT
Motor
Output:
EAT LESS
LH Injection of D-AP5 in HU X
D-AP5 = glutamate receptor antagonist
EAT MORE RAT CHOW
LH Injection of ATPA in HU
ATPA = glutamate receptor agonist

23. (via Nucleus Accumbens, etc.)
Application:
Although pharmacological interventions are not plausible, it appears that offering astronauts a more palatable food as second course is sufficient to ensure adequate intake.
Sensory Inputs
(via Nucleus Accumbens, etc.)
Sight, Smell, Taste
EAT
Motor
Output:
Glutamate release
Lateral Hypothalamus
Future Directions: Looking at the link between nucleus accumbens and SSS.
As you remember from the 1st model slide….NAC>….

25. Results: DAP-5 Suppresses Overeating During Sensory Specific Satiety
Chow
Froot Loops®
Cumulative Food Intake (grams)

26. Results: Rats are Fed to Satiety on Rat Chow
Cumulative Rat Chow Intake (grams)
@ 60 min
@ 90 min
Meal Duration

28. D-AP5: Pre-injection meal food intake at 60 and 90 minutes
Combined with ATPA stuff

29. ATPA: Pre-injection meal food intake at 60 and 90 minutes

30. D-AP5: post-injection meal food intake at 60 minutes
~75%
The Froot Loop intake for the rats injected with D-AP5 was significantly less than those injected with the control. The chow intakes did not significantly differ.

31. ATPA: Post-injection meal food intake at 30 minutes
“Ceiling effect”

32. Summary : aCSF Conclusion
Our results confirm that rats exhibit SSS during hind limb unloading
Conclusion
The same mechanisms responsible for SSS in horizontal rats appear to be operating in hind limb unloading