Sarter Lab

1. Sarter Lab Laxmi Shah, Sarah Williams, Linda Baek, Vivian Chen, Ana Kantorowski

2. Cholinergic System

3. Investigation of sustained attention in Wistar rats with ad libitum access to water Vivian Chen Sarter lab

4. Introduction • Water deprivation • Motivation • Reward • Animal cruelty? • Ad libitum access • Sucrose solution

5. Hypothesis:The presentation of a sucrose reward will not provide the same results as a study conducted with water deprivation.The rats will not perform sustained attentional tasks.

6. Animals & Apparatus

7. Behavioral Training • 3 phases • Recorded number of hits/misses/correct rejections/false alarms, omitted trials • Correction trials, forced-choice trials • 120 total trials run per phase (#3 only 100 are run) • Total time for experiment: 30 days

8. 1st Stage • Trials with light signal • Left lever = “hit” • Right lever = “miss” • Trials with no light • Left lever = “miss” • Right lever = “hit”

9. 2nd Stage • Varied time elapsed between trials • Light signal flashed for 1 second • For every 3rd trial, correct lever was alternated

10. 3rd Stage • Added up to 4 correction trials • Light signal but no reward • Reward only • Rest of protocol remained consistent

11. Anticipated Results • Sucrose reward insufficient to motivate rats to perform • Sucrose solution reward unsuccessful training technique- need to consider designing a different experimental procedure as an alternative method for water deprivation

12. Sustained Attention Task

13. Micro-Dialysis • Cannulation • Measure ACh levels for task blocks • Analysis of samples utilizing HPLC

14. Acetylcholine Efflux Relating to the Prefrontal Cortex and Top-Down Control Functions in Attentional Task-Performing Rats Sarter Lab Rouba Kozak Ana Kantorowski Linda Baek

15. Background • Previously, ACh efflux and performance • Later studies showed dissociation • Further implications for PFC

16. Hypothesis • Prefrontal cortex and control of posterior parietal cortex in top-down functions through cholinergic projections

17. Methods • Fisher-Brown Norway Male Rats • Water-deprived • Training • Operant chambers to learn task

18. Cannula Implantation (PPC) and PFC bilateral infusion 192 SAP or Saline Recovery + habituation Dialysis 1 Dialysis 2 3+ days Discard period Task Pre Post Standard session Distractor session 12:00 9:00 8-min collections

19. Response Signal Non-Signal Incorrect Lever (False Alarm) L Incorrect Lever (Miss) L Correct Lever (Correct Rejection) R Correct Lever (Hit) R

20. Methods Task • Operant Chambers • Signal and non-signal • Distracter • Correct Responses reinforced with water • Trained until 70% accuracy

21. Methods • Surgery • Lesion PFC • Cannula PPC • Recovery • Microdialysis

22. Lesion Non-lesioned Rat Lesioned Rat

23. Cannulae Placement

24. Results • Distracter increase Ach levels • Lesioned animals and distracter • In lesioned animals, Ach levels continue to increase after distracter where as control animals return to baseline

25. Results Standard Task w/o distracter

26. Results Task with Distracter Control Group

27. Results Task with Distracter Lesioned Group

28. Conclusions Prefrontal cortex plays key role in Ach related subcortical control in top-down processes. Alzheimer’s Disease unwanted stimulus = distracter

29. Increases in Cortical Cholinergic Neurotransmission in Rats Performing a Conditioned Appetitive Response and Detected by the Amperometric Measurement of Choline Laxmi Shah, Vinay Parikh Sarter Lab

30. Background • Cortical cholinergic system represents a major component of forebrain circuitry mediating attentional processes • Acetylcholine (ACh) Right Medial Prefrontal Cortex

31. Acetylcholine mediates attentional abilities • Previously: electrical readings of anesthetized rats only • Now: levels of choline uptake in freely moving, awake animals performing a highly attention demanding task= conditioned appetitive response task Microdialysis Showed stable increase in Ach efflux -Took 5-20 minutes to achieve -Low temporal and spatial resolution Evidence linking increases in Ach to stimuli, responses, cognitive operations is still unknown…. • Fixed-Potential Amperometry • -Valid measure of increases in Ach efflux • High temporal/spatial resolution • Fast, real time measurements within seconds

32. Aim and Hypothesis • Determine whether transient changes in choline concentrations can be detected in animals performing a cued appetitive response. • If so, it is hypothesized that choline levels increase during the visual cue and performance stimulus of the attention task.

33. Methods • Fisher Brown Norway Rats • Mildly-food Deprived (85% weight) • Randomly divided into two groups: • Attention demanding task training • Freely moving chamber • Surgery • Lesion Right Medial Prefrontal Cortex (mPFC) • Cannula

34. Conditioned Appetitive Response • Shielded Chamber • CCTV camera installed in ceiling of chamber • Two port holes (left and right) • 25 Trials/day - randomly selected • Successive trials were separated by 90 ± 30 seconds • Trained to associate: Latency Pd: 6±2 seconds Light Stimulus: 1s Food Presentation of 12 mg Fruit Loops

35. Microelectrode and In vivo Recordings • Microelectrode/Micropipette • 4 recording sites • 10, 20, 30, 40 µM choline injected • Cannula attached to microelectrode • Recovery: 48 hours • Electrochemical readings WHILE performing behavioral task • Video tracking of sessions • Choline oxidase • Ensure electrode still working after session

36. Behavioral Analysis • Committed vs. Omitted Trials • Determining if detected cue? • Committed: Noticeable change in behavior 5s before and after light cue • Video

37. Results √ Detection of light cue triggering behavior (committed trials) gives significant increase in choline levels compared to non detected cues

38. Conclusions • Detection of the cue involves attentional resources for cue evoked response processing • Stopped ongoing behavior • Preparation for Response • Reward Anticipation • Distractor Filtering • Executive functions are mediated via the increase in mPF ACh release during cue detection

39. Donepezil-induced augmentation of attentional performance-associated acetylcholine release in rats Sarah Williams Elise Dagenbach, Sarter Lab

40. The Prefrontal Cortex and Theory of Attentional Effort • Disregulations in patients with mild cognitive impairment (CGI) and Alzheimer’s • Cholinergic projections from basal forebrain to prefrontal cortex • Top-down modulation • An increase in attentional effort stimulates efflux of ACh in the prefrontal cortex.

41. Donepezil Hydrochloride • Acetylcholinesterase: enzyme that hydrolyzes ACh in the brain • Acetylcholinesterase inhibitor Increases ACh concentrations in the brain • Recruitment ACh efflux vs. donepezil ACh efflux?

42. Drug-Dose Response • .5 mg/kg – 5 mg/kg; acute i.p. injections, 2 hrs pre-task • Regular sustained attention task • MAIN OBJECTIVE: finding two dosages of donepezil that do not interfere with task completion

43. Effect of donepezil on performance with distracter • Two treatment groups • Acute i.p. injections, 2 hrs pre-task • Comparison of performances: • Sustained attention task • Distracter sustained attention task • Distracter sustained attention task plus treatment with donepezil

44. Effect of donepezil on ACh release • Four treatment-task groups • Sustained-control • Distracter-control • Sustained-donepezil (2) • Distracter-donepezil (2) • Acute i.p injections, 10 min. prior to placement in box • Dialysis

45. Discussion • Expected results? • Donepezil augments levels of ACh and attentional recruitment • Added attentional recruitment rescues performance from distracter impairments • Contrary results? • Ceiling effect on ACh release; “over-recruitment”

46. The Big Picture Implications: • model neurodegenerative diseases • Schizophrenia and dementia • Result of anomalies in cortical cholinergic system