Measuring Striatal Monoamine Changes in R6/2 transgenic Mice During An Operant Task Designed To Challenge Behavioral Flexibility

T.S. Buxton, A. Carrasco, K.D. Fink, D.A. Galinac, N. Jayaprakash, J. Lee, M. Lupo, T.L. Mucha, M.I. Sandstrom*, and S.K. Steffes Dept. of Psychology & Neuroscience Program, Central Michigan University, Mount Pleasant MI 48859 USA

Psychology & Neuroscience atPsychology & Neuroscience at

Central Michigan UniversityCentral Michigan University

INTRODUCTION

Huntington’s disease (HD) results from an autosomal dominant gene mutation of the gene that encodes for the huntingtin protein involving polyglutamine extensions from its 5’ end. Severe motor deficits arise in adulthood, typically around 40-50 years, and patients with larger numbers of polyglutamine repeats experience earlier symptom onset. Motor symptoms are typically preceded by cognitive problems such as decreased behavioral flexibility with planning behavior strategies, and others generally described as hypofrontality. As with movement problems, these cognitive disruptions also seem to exhibit a severity that depends upon the number of the polyglutamine extensions on huntingtin. Our laboratory is interested in first exposing behavior inflexibility during early adulthood prior to the onset of motor deficit expression among transgenic mouse HD models, and subsequently exploring the underlying neurochemistry and neurophysiology of such cognitive disruptions. The R6/2 mouse strain (Jackson Labs: B6CBA-Tg(HDexon1)62Gpb/1J) is a common transgenic Huntington’s disease model harboring an inserted exon 1 of the human huntingtin gene carrying 115-150 CAG (polyglutamine) repeat expansions. Among these mice, while subtle motor abnormalities are detectable within 5-6 weeks (Carter et al., 1999), obvious and overt motor deficits are rare before 8 weeks of age. Prior to this, problems with strategy shifting have been demonstrated using the Morris Water maze and the T-maze among R6/2 mice between 3.5 and 4.5 weeks when compared to wild-type (WT) littermates (Lione et al., 1999). While strategy shifting or attentional set shifting explorations have been a hallmark of clinical HD investigations (Lawrence et al., 1996; Aron et al., 2003), involving tasks such as the Tower of London and Wisconsin Card Sort task for example, these sorts of cognitive challenges have proven difficult to reproduce in experimental animals. It would seem, however, based on the literature regarding preclinical human findings, that a continuous need for shifting behaviors to achieve reward would be more challenging for animal models of HD to learn and maintain if the model validity extends into this cognitive domain. If this were the case, then an operant task requiring constant behavior shifting to acquire reinforcement would be more challenging than a more repetitive task for R6/2 HD+ mice compared with WT littermates. Our first goal was to investigate R6/2 mice using such an operant task. Interesting correlative research has suggested a relationship between frontal cortex and striatal dopamine function and executive planning deficits demonstrated in tasks requiring strategic processing among human Huntington’s patients (Bäckman et al., 1997). Evidence has also been obtained to indicate that R6/2 mice exhibit deficits in dopamine release (Johnson et al., 2007). Our second goal was to characterize monoamine release dynamics during operant contingencies that emphasized behavior shifting to examine whether R6/2 mice exhibit difficulties with such a task in association with any obvious disruption of monoamines. To that end, and to the best of our knowledge, we have engineered the first efforts to evaluate R6/2 operant behavior while taking microdialysis measurements.

METHODS

Male wild-type (WT) and R6/2+ mice (HD, from strain B6CBA, Jackson Labs) were bred from heterozygote ovarian transplanted females at our in-house facilities and weaned on postnatal day 21. Mice were individually housed in 12:12 hr reverse day night with lights cycling from 10:00PM through 10:00AM and were kept at 85% of their body weight with water ad libitum. Custom, automated software programs for each step of progressive training minimized experimenter error and provided a source of documentation for lever pressing and timing. For 22-32 days, starting at 23 days of age, mice underwent daily training to lever press in operant, sound attenuated mouse chambers adapted with open, unrestricted food hoppers. All mice were trained to 90% minimum accuracy on schedules advancing from FR1 to FR4 on a single lever (RPT task). Pseudo-random assignment to one of two groups then occurred beginning three days before surgery. Group one (GRP 1) received 3 days of pre-surgery training on a multiple component schedule comprised of up to one hour of RPT followed by a 10 minute blackout and ending with up to one hour of a variable sequence task (VAR) that required the mice to emit a four-response lever sequence that differed from the immediately preceding one, excluding LLLL or RRRR. A maximum of (30) 20 mg pellets (BioServe, Frenchtown NJ) could be earned during each of the two components during a single day of training with a variable intertrial interval (ITI) of up to 2 minutes was applied after each correct sequence. This training continued for another 3 days following surgery and 48 hours of post operative recovery time. Post surgery training also included acclamation to being attached using a beaded chain between the head stage and swivel connection to simulate microdialysis conditions. Group two (GRP 2) also received 3 days of pre and post-surge training except the same RPT task was presented during both components on a single day. The RPT + VAR tasks were presented during dialysis for both groups; with the VAR task being novel only to GRP 2 at dialysis. A hanging wire test was done on the last day of training to test for motor deficits (See Figure 9). During surgery, a 21-gauge intracerebral guide cannula (C312GS cut 2.0mm below pedestal, Plastics One, Roanoke VA) was implanted and secured with skull screws and dental cement. Stereotaxic surgery was performed under both ketamine & xylazine anesthesia and extended with the use of isoflurane gas. Guide cannulae targeted the striatum at coordinates: +0.8mm anterior, -2.5mm lateral from Bregma. The guide cannula, designed to accept the microdialysis probes made from a 26-gauge infusion cannula (C312IS extending 0.1mm below guide, Plastics One, Roanoke VA), was protected by a dummy cannula inserted prior to experimentation to keep out debris. All animals were surged between 5-6.5 weeks of age. Dialysis during operant RPT and VAR tasks took place during one 140 minute session on the fifth day after surgery. Probes were pre-tested for percentage recovery (see Table 1). A fixed 2 minute ITI was applied after each good sequence in order to provide sufficient time to collect dialysate. To avoid trauma-related neurochemical measurements following insertion of the probe, a one hour discard period was incorporated to allow monoamines to resume physiological levels. A total of (8) 15-min experimental collections were taken; (4) during each of the tasks with a 10 minute break between them. All collections were kept frozen on dry ice during the experiment and then stored in a –80 degree ultrafreezer until processing with high performance liquid chromatography and electrochemical detection (HPLC-ED). Mice were euthanized and transcardially perfused with fixative after both sessions and 40μm brain sections surrounding probe-related damage were stained with Cresyl Violet (see Figure 2).

CONCLUSIONS

This study was originally designed to investigate the neurochemical (monoaminergic) changes resulting from challenges to behavioral flexibility. While we believe this operant approach does present a cognitive challenge during the VAR component of the multiple schedule, maintaining sufficient engagement during this component of the dialysis session has proven difficult. There is clearly an interfering factor introduced by the microdialysis probe attachment that decreases engagement. Another aspect of the dialysis session is the fixed 2-min intertrial interval that we deemed necessary to acquire sufficient collections but likely affected motivation by decreasing reinforcement rate. We believe the critical factor that the microdialysis probe introduces is an increase in effort when increased movement is necessary to achieve success (moving from bar to bar in the VAR component). Thus increased effort and decreased reward during this final dialysis session confounded our original intent to correlate monoaminergic release with behavioral flexibility. Future studies will modify operant contingencies to provide higher reinforcement rates during dialysis to promote engagement during expressions of behavioral flexibility so that dialysis can capture hypothesized monoaminergic distinctions between genotypes.

RESULTS CAG-REPEATS Estimates were made of CAG repeat lengths for the R6/2+ animals throughout the experiment as animals were genotyped based on preliminary results obtained from sequencing at Jackson Labs (standard return = ~164 repeats). Based on these estimates, all animals designated HD harbored approximately 170 CAG repeats consistently, though exact genotyping was not performed.

FIGURE 3. Percentage of total reinforcers earned by each group during each day of multiple component training. Days 1 to 3 are pre-surgery, and days 4-6 are post surgery. The dotted vertical line represents the time of surgery. A total of 30 reinforcers were possible during each component per day. The dialysis occurred on a single day. Decreased success during the VAR for Group 1 could be a result of a much lower rate of reinforcement. Group 1 WT appear to perform more accurately during the VAR task than HD but this difference is not significant. Significantly reduced lever pressing at the time of dialysis accounts for much of the drop in accuracy at that time (see Figure 6).

ACKNOWLEDGEMENTS Our group would also like to thank Gary Dunbar for mouse donations, Ming Lu for the hours of work on genotyping, Mark Reilly his graduate students for their help in planning and programming the operant protocols, and Carol Stevens and the animal care crew for maintaining our colony.

REFERENCES

1) Aron, A.R., et al., (2003), Task set-switching deficits in early stage Huntington’s disease: Implications for basal ganglia function. Journal of Cognitive Neuroscience, 15(5), 629-642.

2)Carter, R.J., et al., (1999), Characterization of progressive motor deficits in mice transgenic for the human Huntington’s disease mutation. Journal of Neuroscience, 19, 3248-3257.

3) Lawrence, A.D., et al., (1996), Executive and mnemonic functions in early Huntington’s disease. Brain, 119, 1633-1645.

4) Lione, L.A. et al., (1999), Selective discrimination learning impairments in mice expressing the human Huntington’s disease mutation, Journal of Neuroscience, 19(23), 10428-10437.

5) Bäckman, L. et al., (1997), Cognitive deficits in Huntington’s disease are predicted by dopaminergic PET markers and brain volumes. Brain, 120, 2207-2217.

6) Johnson, M.A., et al., (2007). Catecholamine exocytosis is diminished in R6/2 Huntington's disease model mice. Journal of Neurochemistry. 103(5), 2102-2110.

FIGURE 1. Med Associates operant mouse chamber in a sound attenuation chamber modified for dialysis . The pump (middle) located outside the box pumps artificial cerebral spinal fluid through PE-10 tubes extending into the box through pre-existing vent slots. A one-inch diameter hole was cut in the acrylic top of the chamber through which the microdialysis probe extends from the fluid swivel head to the animal’s headstage. A connected mouse is pictured on the right.

FIGURE 2. Representative histology sections from mouse subjects of this study. A. Wild-type mouse with good placement; B. R6/2+ mouse with good placement.

FIGURE 4. Good sequences as a percentage of both good and bad sequences emitted during each training component over 6 days of multiple component training, plus dialysis. Days 1-3 are pre-surgery, and days 4-6 are post surgery. The dotted vertical line represents the time of surgery. The simple RPT task resulted in nearly 100% accuracy for both groups across genotype. The VAR task, being more complex, resulted in fewer good sequences overall than during the simpler RPT tasks.

FIGURE 7. Lever presses per good sequence was created as a performance variable. The more accurate the response, the lower the variable would be. Days 1-3 are pre-surgery, and days 4-6 are post surgery. The dotted vertical line represents the time of surgery. The interpretation of this variable is still dependent on engagement (defined by lever pressing activity).

FIGURE 6. Lever presses emitted by each group during each of the training components over 6 days of multiple component training plus dialysis. Days 1-3 are pre-surgery, and days 4-6 are post surgery. The dotted vertical line represents the time of surgery. High lever pressing activity during the VAR component indicates a trial and error approach to the more complex task. In Group 2, the RPT task was well learned across genotype and performed with very little error.

FIGURE 5. Percent change of monoamines 3-4 dihydrophenylacetic acid, homovanillic acid, 5-hydroxyindole acetic acid, and dopamine (DOPAC, HVA, 5-HIAA and DA) of the last RPT collection and 4-6 VAR collections from baseline (the median RPT across 4 RPT collections). Collections taken during VAR 3 are most representative of the monoamine activity occurring at the time strategy switching first needed to occur. Two further collections were taken from a subpopulation of animals (Group 1 WT=1 HD=4; Group 2 WT=1 HD=3 ) following task end to capture delayed changes. No significant genotypic differences were found. It seems clear however that these animals were not sufficiently engaged in the task for the VAR changes to represent the intended cognitive challenge response.

FIGURE 9. The day prior to microdialysis, animals were tested for their ability to hang upside down on a wire screen as a measure of possible motoric dysfunction. No differences were found across genotype for mice tested between 43 and 52 days of age. No notable motoric deficits were deemed present if the mouse could hang on for a minimum of 2 minutes.

FIGURE 8. The amount of time animals spent during each component of dialysis doing the behaviors noted on the x-axis. Manual observations were noted at the time of each dialysis collection. Software data was limited to recording lever presses but we were interested in what a lack of lever pressing might mean. Engagement, or clear attention to levers, seems to compete with a variety of other behaviors during dialysis.

A

B

EARNED REINFORCEMENTS

PROBE PLACEMENTS

A

PERCENT REINFORCED SEQUENCES EMITTED

Group One

1 2 3 4 5 6

RPT D

ial 1 2 3 4 5 6

VAR D

ial

0

200

400

600

800

1000

1200

WT (n=8)

RPT VAR

HD (n=8)

Lever

Pre

sses

Group Two

1 2 3 4 5 6

RPT D

ial 1 2 3 4 5 6

VAR D

ial

0

200

400

600

800

1000

1200

WT (n=4)

RPT RPT

HD (n=10)

Group One

1 2 3 4 5 6

RPT D

ial 1 2 3 4 5 6

VAR D

ial

0

50

100

150

200

250

300

350

WT (n=8)

RPT VAR

HD (n=8)

Lever

Pre

sses/G

oo

d S

eq

uen

ce

Group Two

1 2 3 4 5 6

RPT D

ial 1 2 3 4 5 6

VAR D

ial

0

50

100

150

200

250

300

350

WT (n=4)

RPT RPT

HD (n=10)

DIALYSIS RESULTS

LEVER PRESSES PER GOOD SEQUENCE

LEVER PRESSES

DIALYSIS SESSION OBSERVATIONS

HANGING WIRE TEST Mean SEM DOPAC 7.187 0.529 DA 7.721 0.600 5-HIAA 8.122 0.660 HVA 7.233 0.572

PROBE RECOVERY VALUES

Group One RPT

Clim

bing

Inac

tive

Abse

nt Lev

er

Engaged

Gro

omin

g

Wal

king

@ H

opper

0

20

40

60

80

100WT (n=6)

HD (n=6)

Group One VAR

Clim

bing

Inac

tive

Abse

nt Lev

er

Engaged

Gro

omin

g

Wal

king

@ H

opper

0

20

40

60

80

100WT (n=6)

HD (n=6)

Group One RPT

Clim

bing

Inac

tive

Abse

nt Lev

er

Engag

ed

Gro

omin

g

Wal

king

@ H

opper

0

20

40

60

80

100WT (n=2)

HD (n=4)

Group Two VAR

Clim

bing

Inac

tive

Abse

nt Lev

er

Engag

ed

Gro

omin

g

Wal

king

0

20

40

60

80

100WT (n=2)

HD (n=4)

% o

f D

ialy

sis

Tim

e

0

50

100

150

16-12016-120 31-120 64-120 22-120

GRP 1

WT

GRP 1

HD

GRP 2

WT

GRP 2

HD

Genotype by Group

Gri

p D

ura

tio

n (

secs)

% C

ha

ng

e in

5-H

IAA

fro

m M

dn

RP

T

Group One

-100

-50

0

50

100

150WT (n=6)

HD (n=6)

Group Two

Last R

PT

VAR 1

VAR 2

VAR 3

VAR 4

VAR 5

VAR 6-200

-100

0

100

200

WT (n=2)

HD (n=4)

% C

ha

ng

e in

DO

PA

C f

rom

Md

n R

PT

Group One

-100

0

100

200

300

400

500WT (n=6)

HD (n=6)

Group Two

Last R

PT

VAR 1

VAR 2

VAR 3

VAR 4

VAR 5

VAR 6-150

-100

-50

0

50

WT (n=2)

HD (n=4)

% C

ha

ng

e in

DA

fro

m M

dn

RP

T

Group Two

Last R

PT

VAR 1

VAR 2

VAR 3

VAR 4

VAR 5

VAR 6

-150

-100

-50

0

50

WT (n=2)

HD (n=4)

Group One

-40

-20

0

20

40

WT (n=6)

HD (n=6)

% C

ha

ng

e in

5-H

VA

fro

m M

dn

RP

T

Group One

-40

-20

0

20

40

WT (n=6)

HD (n=6)

Group Two

Last R

PT

VAR 1

VAR 2

VAR 3

VAR 4

VAR 5

VAR 6-150

-100

-50

0

50

WT (n=2)

HD (n=4)

Group One

1 2 3 4 5 6

RPT D

ial 1 2 3 4 5 6

VAR D

ial

0

20

40

60

80

100 WT (n=8)

RPT VAR

HD (n=8)

% o

f R

ein

forc

ers

Earn

ed

Group Two

1 2 3 4 5 6

RPT D

ial 1 2 3 4 5 6

VAR D

ial

0

20

40

60

80

100

WT (n=4)

RPT RPT

HD (n=10)

Group One

1 2 3 4 5 6

RPT D

ial 1 2 3 4 5 6

VAR D

ial

0

20

40

60

80

100

WT (n=8)

HD (n=8)

% G

oo

d S

eq

uen

ces

Group Two

1 2 3 4 5 6

RPT D

ial 1 2 3 4 5 6

VAR D

ial

0

20

40

60

80

100

WT (n=4)

HD (n=10)

443.15