Sarter Lab

Sarter LabSarter Lab

Laxmi Shah, Sarah Williams, Linda Baek, Vivian Chen, Ana Kantorowski

Laxmi Shah, Sarah Williams, Linda Baek, Vivian Chen, Ana Kantorowski

Cholinergic SystemCholinergic System

Investigation of sustained attention in Wistar rats with ad libitum access to

water

Investigation of sustained attention in Wistar rats with ad libitum access to

water

Vivian ChenSarter lab

Vivian ChenSarter lab

IntroductionIntroduction

Water deprivation Motivation Reward

Animal cruelty? Ad libitum access Sucrose solution

Water deprivation Motivation Reward

Animal cruelty? Ad libitum access Sucrose solution

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.

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.

Animals & ApparatusAnimals & Apparatus

Behavioral TrainingBehavioral 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

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

1st Stage1st Stage

Trials with light signal Left lever = “hit” Right lever = “miss”

Trials with no light Left lever = “miss” Right lever = “hit”

Trials with light signal Left lever = “hit” Right lever = “miss”

Trials with no light Left lever = “miss” Right lever = “hit”

2nd Stage2nd Stage

Varied time elapsed between trials Light signal flashed for 1 second For every 3rd trial, correct lever was

alternated

Varied time elapsed between trials Light signal flashed for 1 second For every 3rd trial, correct lever was

alternated

3rd Stage3rd Stage Added up to 4

correction trials Light signal but no

reward Reward only

Rest of protocol remained consistent

Added up to 4 correction trials Light signal but no

reward Reward only

Rest of protocol remained consistent

Anticipated ResultsAnticipated 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

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

Sustained Attention TaskSustained Attention Task

Micro-DialysisMicro-Dialysis

Cannulation Measure ACh levels

for task blocks Analysis of samples

utilizing HPLC

Cannulation Measure ACh levels

for task blocks Analysis of samples

utilizing HPLC

Acetylcholine Efflux Relating to the Prefrontal Cortex and

Top-Down Control Functions in Attentional Task-Performing

Rats

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

Sarter Lab

Rouba Kozak

Ana Kantorowski

Linda Baek

BackgroundBackgroundPreviously, ACh efflux and performanceLater studies showed dissociationFurther implications for PFC

Previously, ACh efflux and performanceLater studies showed dissociationFurther implications for PFC

HypothesisHypothesis

Prefrontal cortex and control of posterior parietal cortex in top-down functions through cholinergic projections

Prefrontal cortex and control of posterior parietal cortex in top-down functions through cholinergic projections

MethodsMethods Fisher-Brown Norway Male Rats Water-deprived Training

Operant chambers to learn task

Fisher-Brown Norway Male Rats Water-deprived Training

Operant chambers to learn task

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are needed to see this picture.

Cannula Implantation(PPC) and PFC bilateral infusion 192 SAP or Saline

Standard session

Distractor session

Dialysis 1 Dialysis 2Recovery +habituation

8-min collections

Discard period Task Pos

t Pre9:00

12:0

0

3+ days



Response

Signal Non-Signal

CorrectLever(Hit)R

Incorrect Lever(Miss)L

CorrectLever(CorrectRejection)R

IncorrectLever(FalseAlarm)L

MethodsMethods

Task Operant Chambers Signal and non-signal Distracter Correct Responses reinforced

with water Trained until 70% accuracy

Task Operant Chambers Signal and non-signal Distracter Correct Responses reinforced

with water Trained until 70% accuracy

MethodsMethods Surgery

Lesion PFC Cannula PPC

Recovery Microdialysis

Surgery Lesion PFC Cannula PPC

Recovery Microdialysis

LesionLesion

Non-lesioned Rat Lesioned Rat



Cannulae PlacementCannulae Placement

ResultsResults

1. Distracter increase Ach levels

2. Lesioned animals and distracter

3. In lesioned animals, Ach levels continue to increase after distracter where as control animals return to baseline

1. Distracter increase Ach levels

2. Lesioned animals and distracter

3. In lesioned animals, Ach levels continue to increase after distracter where as control animals return to baseline

ResultsResults

blocks of trials (8 min each)

T1 T2 T3 T4 T5

attentional performance (VI)

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

sham-lesioned

PFC 192 SAP

blocks of trials/collection intervals (8 min each)

T1 T2 T3 T4 T5

ACh efflux (% change from bsl)50

100

150

200

250

PFC 192 SAP

sham-lesioned

Standard Task w/o distracter

ResultsResults


T1 T2 T3 T4 T5


-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

distractor

distractor task

regular task


T1 T2 T3 T4 T5

ACh efflux (% change from bsl)

50

100

150

200

250

300

distractor

regular task

distractor task

Task with DistracterControl Group

ResultsResults


T1 T2 T3 T4 T5


-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

regular task

distractor task distractor


T1 T2 T3 T4 T5

ACh efflux (% change from bsl)100

200

300

400

500

regular task

distractor task

distractor

Task with DistracterLesioned Group

ConclusionsConclusions

Prefrontal cortex plays key role in Ach related subcortical control in top-down processes.

Alzheimer’s Diseaseunwanted stimulus = distracter

Prefrontal cortex plays key role in Ach related subcortical control in top-down processes.

Alzheimer’s Diseaseunwanted stimulus = distracter

Increases in Cortical Cholinergic Neurotransmission in Rats Performing a

Conditioned Appetitive Response and Detected by the Amperometric

Measurement of Choline

Increases in Cortical Cholinergic Neurotransmission in Rats Performing a

Conditioned Appetitive Response and Detected by the Amperometric

Measurement of Choline

Laxmi Shah, Vinay ParikhSarter Lab

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BackgroundBackground Cortical cholinergic system represents a major

component of forebrain circuitry mediating attentional processes

Acetylcholine (ACh)

Cortical cholinergic system represents a major component of forebrain circuitry mediating attentional processes

Acetylcholine (ACh)

QuickTime™ and aTIFF (Uncompressed) decompressor


Right Medial Prefrontal Cortex

Acetylcholine mediates attentional abilities 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

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

MicrodialysisShowed stable increase in Ach efflux

-Took 5-20 minutes to achieve-Low temporal and spatial resolution

Evidence linking increases in Ach tostimuli, 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

Aim and HypothesisAim and Hypothesis

o Determine whether transient changes in choline concentrations can be detected in animals performing a cued appetitive response.

o If so, it is hypothesized that choline levels increase during the visual cue and performance stimulus of the attention task.

o Determine whether transient changes in choline concentrations can be detected in animals performing a cued appetitive response.

o If so, it is hypothesized that choline levels increase during the visual cue and performance stimulus of the attention task.

MethodsMethods 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

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

6 Control Rats 6 Rats Lesioned:Right Medial Prefrontal Cortex

Conditioned Appetitive ResponseConditioned 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:

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:

Light Stimulus: 1s Latency Pd: 6±2 seconds

Food Presentationof 12 mg Fruit Loops

Microelectrode and In vivo Recordings

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

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

Behavioral AnalysisBehavioral Analysis

Committed vs. Omitted Trials Determining if detected cue?

Committed: Noticeable change in behavior 5s before and after light cue

Video

Committed vs. Omitted Trials Determining if detected cue?

Committed: Noticeable change in behavior 5s before and after light cue

Video

ResultsResults

√ Detection of light cue triggering behavior (committed trials) gives significant increase in choline levels

compared to non detected cues

√ Detection of light cue triggering behavior (committed trials) gives significant increase in choline levels

compared to non detected cues

time (s)

0 5 10 15 20 25 30

cue triggers behavior (detected)cue not detected

choline 1 µM choline (µM)

0

1

2

3

4 cue (detected)cue (not detected)

**

n = 4; P < 0.01 vs cue (not detected)

ConclusionsConclusions 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

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

Donepezil-induced augmentation of attentional performance-associated

acetylcholine release in rats

Donepezil-induced augmentation of attentional performance-associated

acetylcholine release in rats

Sarah Williams

Elise Dagenbach, Sarter Lab

Sarah Williams

Elise Dagenbach, Sarter Lab

The Prefrontal Cortex and Theory of Attentional Effort

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.

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.

Donepezil HydrochlorideDonepezil Hydrochloride

Acetylcholinesterase: enzyme that hydrolyzes ACh in the brain

Acetylcholinesterase inhibitor Increases ACh concentrations in the brain Recruitment ACh efflux vs. donepezil ACh efflux?

Acetylcholinesterase: enzyme that hydrolyzes ACh in the brain

Acetylcholinesterase inhibitor Increases ACh concentrations in the brain Recruitment ACh efflux vs. donepezil ACh efflux?

Drug-Dose ResponseDrug-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

.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

Effect of donepezil on performance with distracter

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

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

Effect of donepezil on ACh releaseEffect 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

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

DiscussionDiscussion 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”

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”

The Big PictureThe Big Picture

Implications: model neurodegenerative diseases

Schizophrenia and dementia Result of anomalies in cortical cholinergic

system

Implications: model neurodegenerative diseases

Schizophrenia and dementia Result of anomalies in cortical cholinergic

system

Sarter Lab

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