The Basal Forebrain

1. The effect of intrabasalis orexin A infusion on reversal learning performance in rats with 192 IgG-saporin lesions of the nucleus basalismagnocellularisPatrick PiantadosiMentor: Aileen Bailey, Ph. D. St. Mary’s College of Maryland

2. The Basal Forebrain • Provides acetylcholine (ACh) to the cortex, amygdala, hippocampus, and olfactory bulb (Mesulam et al., 1983) • Basal forebrain cholinergic system (BFCS) nBM/SI

3. BFCS and Alzheimer’s Disease • Alzheimer’s (AD) hallmarks • Neuronal death (Ezrin-Waters & Resch, 1989) • nBM/SI degeneration • > 75% cholinergic cell death (Whitehouse et al., 1982) • Pre-symptomatic (Hall, Moore, Lopez, Kuller, & Becker, 2008) • Degree of cholinergic damage = correlated with dementia severity • ↑ Cell death = ↑ Dementia symptoms (Perry, et al.,1978)

4. nBM/SI Functional Importance • Animal “models” of AD • Damage to the nBM impairs: • Learning set acquisition (Bailey, Rudisill, Hoof, & Loving, 2003) • Attention (Lehmann, Grottick, Cassel, & Higgins, 2003) • Feature Binding (Botly & De Rosa, 2009) • Reversal learning (Cabrera, Cortez, Corley, Kitto, & Butt, 2006) • nBM is critical for behavioral flexibility

5. Reversal learning Acquisition Reversal Cues can be from any sensory modality Correct choice! Correct choice! http://cache1.asset-cache.net/xc/872075-001.jpg?v=1&c=IWSAsset&k=2&d=F5B5107058D53DF52E36F7D30C26F3EE63AD1F7CC4B74A89C1A79402D9325B89E30A760B0D811297

6. Acquisition • No difference between nBM lesioned rats and controls (Cabrera et al., 2006) Fig. 1, Cabrera et al., 2006

7. Reversal - Deficit • Animals with nBM lesions perform significantly worse (more errors) than control animals during the first reversal. (Cabrera et al., 2006) Fig. 3, Cabrera et al., 2006

8. The Orexins • Two distinct hypothalamic neuropeptides • Orexin A (OxA) and Orexin B (OxB) • Orexin projections synapse on cholinergic neurons Black = OxA fibers Brown = Cholinergic neuron Sakurai et al., 2005

9. OxA to the basal forebrain… • Increased ACh efflux to the cortex Fig. 2, Fadel & Frederick-Duus, 2008 Fig. 4, Frederick-Duus, Guyton & Fadel, 2007 • Orexin antagonism impairs feeding latency

10. Orexins and the Basal Forebrain • nBM lesions = ↓ ACh to the cortex = Impaired reversal learning • Intrabasalis OxA = ↑ ACh efflux in cortex • Decrease cholinergic neurons in the nBM  Infuse OxA = What effect on cortical / cholinergic dependent behavior?

11. Hypothesis

12. Methods • Subjects • 24 adult male Sprague Dawley rats • Stereotaxic Surgery • 12 animals = 192 IgG-saporin lesions of the nBM (0.2 µl at 0.375 µg/µl) • 12 animals = sham surgery • Bilateral nBM guide cannula

13. Olfactory Discrimination Reversal Learning • Pretraining – 7-8 days • Acquisition – 1-2 days • 50 trials / day • Two olfactory cues • Onion Powder + Sand • Garlic Powder + Sand • One scent was rewarded • Criterion: 8 correct responses in a row • Without a correction trial • Reversal – 1-2 days • 50 trials / day • Same olfactory cues • Reward contingency = reversed • Criterion: 8 correct responses in a row • Without a correction trial Correct choice! Correct choice!

14. Reversal • Prior to reversal animals were infused with: • OxA (n = 12) • 250 nl at 0.25 nmol / 2 min or • aCSF (n = 12) • 250 nl / 2 min

15. ODRL Testing

16. Dependent Measures • Trials to criterion • # of trials to reach criterion • Correction trials • # of correction trials to reach criterion • Error type • Reversal only • Perseverative • < 2/10 correct responses • Non-perseverative

17. Acquisition - Trials to Criterion • No effect of surgical procedure, infusion type, or interaction on trials to criterion during acquisition, all p-values > .05

18. Acquisition - Correction Trials • No effect of surgical procedure, infusion type, or interaction on the number of correction trials during acquisition, all p-values > .05

19. Reversal – Trials to Criterion • Effect of surgical procedure: • F(1,20) = 2.798, p = .11, η2 = .12 • No effect of infusion type or interaction, all p-values > .05

20. Reversal – Trials to Criterion * • Lesion + aCSF animals required more trials to reach criterion during reversal than sham + aCSF animals • t(10) = -2.06, p = .07, η2 = 0.30

21. Reversal – Correction Trials • Effect of lesion: • F(1,19) = 2.876, p = .11 , η2 = .13 • No effect of infusion type or interaction, all p-values > .05

22. Results - Summary

23. Discussion – Surgical Procedure • No robust effect of lesion • Why? • High effect size of surgical procedure on: • Reversal trials to criterion • Reversal correction trials • Nearly significant effect of lesion in aCSF group • Reversal trials to criterion • Histology could not be analyzed • 192 IgG-SAP lesions do not impair reversal learning? • No difference between nBM lesioned rats and sham-operated controls (Tait & Brown, 2008) • Ibotenic acid infusion to the nBM produced reversal learning deficits (Tait & Brown, 2008)

24. Discussion – Orexin A • No effect of orexin infusion • Why? • Cholinergic neurons may be necessary for orexin-mitigated ACh efflux • OxA + few cholinergic neurons = no behavioral effect • Too little OxA infused? • Guide cannula too ventral?

25. Implications • Future research should: • Evaluate the impact of OxA on cortical dependent behavior • AD symptom – weight loss • Highly correlated with death (White, Piper, & Schmander, 1998) • Orexins = located in LHA • Stimulate feeding behavior (Frederick-Duus, Guyton, & Fadel, 2007) • No cholinergic neurons = no effect of orexin = abnormal feeding behavior? • nBM function • Clarify the role of the nBM in reversal learning

26. Acknowledgements • Special thanks to: • Jenn St. Germain • Dr. Aileen Bailey • Dr. Anne Marie Brady • Various neuroscience students!

27. References • Bailey, A.M., Rudisill, M.L., Hoof, E.J., & Loving, M.L. (2003). 192 IgG-saporin lesions to the nucleus basalismagnocellularis (nBM) disrupt acquisition of learning set formation. Brain Research, 969, 147-159. • Botly, L.C.P. & De Rosa, E. (2009). Cholinergic deafferentation of the neocortex using 192 IgG-saporin impairs feature binding in rats. The Journal of Neuroscience, 29, 4120-4130. • Cabrera, S.M., Chavez, C.M., Corley, S.R., Kitto, M.R., Butt, A.E. (2006). Selective lesions of the nucleus basalismagnocellularis impair cognitive flexibility. Behavioral Neuroscience, 120, 298-306. • Ezrin-Waters, C. & Resch, L. (1989). The nucleus basalis of Meynert. The Canadian Journal of Neurological Sciences, 13, 8-14. • Fadel, J. & Frederick-Duus, D. (2008). Orexin/hypocretin modulation of the basal forebrain cholinergic system: Insights from in vivo microdialysis studies. Pharmacology, Biochemistry, and Behavior, 90, 156-162. • Frederick-Duus, D., Guyton, M.F., & Fadel, J. (2007). Food elicited increase in cortical acetylcholine release require orexin transmission. Neuroscience, 149, 499-507. • Hall, A.M., Moore, R.Y., Lopez, O.L., Kuller, L., & Becker, J.T. (2008). Basal forebrain atrophy is a presymptomatic marker for Alzheimer’s disease. Alzheimer’s and Dementia, 4, 271-279. • Lehmann, O., Grottick, A.J., Cassell, C., & Higgins, G.A. (2003). A double dissociation between serial reaction time and radial maze performance in rats subjected to 192 IgG-saporin lesions of the nucleus basalis and/or the septal region. European Journal of Neuroscience, 18, 651-666. • Mesulam, M.M., Mufson, E.J., Wainer, B.H., & Levey, A.I. (1983). Central cholinergic pathways in the rat: An overview based on alternative nomenclature (Ch1-Ch6). Neuroscience, 10, 1185-1201 • Perry, E.K., Tomlinson, B.E., Blessed, G., Bergman, K., Gibson, P.H., & Perry, R.H. (1978). Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. British Medical Journal, 25, 1457-1459. • Sakurai, T., Nagata, R., Yamanaka, A., Kawamura, H., Tsujino, N., Muraki, Y., Kageyama, H., Kunita, S., Takahashi, S., Goto, K., Koyama, Y., Shioda, S., & Yanagisawa, M. (2005). Input of orexin/hypocretin neurons revealed by a genetically encoded tracer in mice. Neuron, 46, 297-308. • Tait, D.S. & Brown, V.J. (2007). Lesions of the basal forebrain impair reversal learning but not shifting of attentional set in rats. Behavioral Brain Research, 187, 100-108

28. Testing Phase • No effect of surgical procedure, infusion type, or interaction on trials to criterion during acquisition, all p-values > .05

29. Reversal – Error Type • No significant effect of lesion type on perseverative errors (p = .17), or non-perseverative errors (p = .77) • Error bars = ±SEM

30. Histology - AChE Sham-operated nBM Lesion Cortical analysis nBM analysis

31. Histology – Nissl Staining

32. Thakkar et al., 2001 Blanco-Centurion et al., 2006

33. Conclusion • No robust effect of lesion • nBM cholinergic neurons not necessary for reversal learning? • Some lesions may not have been effective • No effect of OxA was observed • OxA may not impact behavior without cholinergic neurons • Guide cannula too ventral? • Amount of OxA too small

34. 192 IgG-saporin Lesion • IgG = monoclonal antibody • Saporin = ribosome inactivation protein • Only binds to cells containing the p75 neurotrophin receptor • Cholinergic cell death Cells containing p75 receptors 192 IgG-saporin Dashed lines = cholinergic neurons

35. Results • No significant surgical procedure x infusion type interactions • All figures display error bars representing ± one SD, unless otherwise specified • 2 (surgical procedure) x 2 (infusion type) ANOVA • Multiple independent samples t-tests

36. Acquisition - Correction Trials * • Lesion + aCSF animals trended towards requiring greater amount of correction trials, p = .06, η2 = 0.33 (independent samples t-test) • No other significant differences, all p-values > .05

37. Acquisition Summary • No effect of • Surgical procedure • Infusion type • Interaction • Expected • nBM lesioned animals = no simple discrimination deficit • No difference between animals to receive different infusion types