The effect of intrabasalis orexin A infusion on reversal learning performance in rats with 192 IgG-saporin lesions of the nucleus basalis magnocellularis

Patrick Piantadosi

Mentor: Aileen Bailey, Ph. D.

St. Mary’s College of Maryland

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

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)

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

Reversal learning

Acquisition Reversal

Correct choice! Correct choice!

Cues can be from any sensory modality

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Acquisition

No difference between nBM lesioned rats and controls (Cabrera et al., 2006)

Fig. 1, Cabrera et al., 2006

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

The Orexins

Two distinct hypothalamic neuropeptides Orexin A (OxA) and Orexin B (OxB)

Orexin projections synapse on cholinergic neurons

Brown = Cholinergic neuron

Black = OxA fibers

Sakurai et al., 2005

OxA to the basal forebrain…

Increased ACh efflux to the cortex

Orexin antagonism impairs feeding latency

Fig. 4, Frederick-Duus, Guyton & Fadel, 2007Fig. 2, Fadel & Frederick-Duus, 2008

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?

Hypothesis

Group # 1 2 3 4

Treatment Sham-Operated + aCSF

Sham-Operated + OxA

nBM Lesion + aCSF

nBM Lesion + OxA

Acqusition Hypothesis No impairment No impairment No impairment No impairment

Reversal Hypothesis No impairment No impairment

Impaired compared to Group 1,2,4

Similar to 1 & 2

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

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!

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

ODRL Testing

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

Acquisition - Trials to Criterion

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

Lesion Sham0

5

10

15

20

25

30

35

40aCSF

OxA

Surgical Procedure

Acq

uisi

tion

Tria

ls to

Crit

erio

n

Acquisition - Correction Trials

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

Lesion Sham0

5

10

15

20

25

30 aCSF

OxA

Surgical Procedure

Acq

uisi

tion

Cor

rect

ion

Tria

ls

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

Lesion Sham0

10

20

30

40

50

60

aCSFOxA

Surgical Procedure

Rev

ersa

l Tria

ls to

Crit

erio

n

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

Lesion Sham0

10

20

30

40

50

60

aCSF

OxA

Surgical Procedure

Rev

ersa

l Tria

ls to

Crit

erio

n

*

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

Lesion Sham0

20

40

60

80

100

120

aCSFOxA

Surgical Procedure

Rev

ersa

l Cor

rect

ion

Tria

ls

Results - Summary

Group # 1 2 3 4

Treatment Sham-Operated + aCSF

Sham-Operated + OxA

nBM Lesion + aCSF

nBM Lesion + OxA

Acquisition Results No impairment No impairment No impairment No impairment

Reversal Results No impairment No impairment

↑ Trials to criterion

compared to Group 1

No difference compared to Group 1,2,3

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)

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?

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

Acknowledgements

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

References Bailey, A.M., Rudisill, M.L., Hoof, E.J., & Loving, M.L. (2003). 192 IgG-saporin lesions to the nucleus basalis magnocellularis (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 basalis magnocellularis 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

Testing Phase

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

Acquisition Reversal0

10

20

30

40

50

60

Sham+aCSF

Sham + OxA

Lesion + aCSF

Lesion + OxA

Test Phase

Tria

ls to

Crit

erio

n

Reversal – Error Type

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

Perseverative Errors Non-perseverative Errors0

5

10

15

20

25

30

ShamLesion

Types of Error

Num

ber o

f Err

ors

Dur

ing

Rev

ersa

l

Histology - AChE

Sham-operated nBM Lesion

Cortical analysis

nBM analysis

Histology – Nissl Staining

Thakkar et al., 2001

Blanco-Centurion et al., 2006

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

192 IgG-saporin Lesion

IgG = monoclonal antibody

Saporin = ribosome inactivation protein

Only binds to cells containing the p75 neurotrophin receptor Cholinergic cell

death

192 IgG-saporin

Cells containing p75 receptors

Dashed lines = cholinergic neurons

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

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

Lesion Sham0

5

10

15

20

25

30 aCSF

OxA

Surgical Procedure

Acq

uisi

tion

Cor

rect

ion

Tria

ls

*

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