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

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.

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.

(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.

(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?

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

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

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

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

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.

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

(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>….

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

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

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

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

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.

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

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