FIXED INTERVAL AND PEAK TRIAL PERFORMANCE OF THE RELN DEFICIENT MOUSE
Rachael L. Cushing
A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Masters of Arts
Department of Psychology University of North Carolina Wilmington 2009
Approved by
Advisory Committee
______Dr. Christine Hughes______Dr. Ruth Hurst______
______Dr. Raymond Pitts______Dr. Mark Galizio_____ Chair
Accepted by
______Dean Roer, Graduate School
TABLE OF CONTENTS
ABSTRACT...... v
ACKNOWLEDGEMENTS...... vi
DEDICATION...... vii
LIST OF TABLES...... vi
LIST OF FIGURES ...... ix
INTRODUCTION ...... 1
Behavioral Assessment of the Reln Deficient Mouse...... 5
Summary of Literature Review...... 33
Continuing the Examination of Reln Deficient Mice ...... 35
Operant Methods that Assess Control of Behavior by Time ...... 40
METHODS ...... 59
Subjects...... 59
Apparatus ...... 60
Behavioral Procedures ...... 60
Data Analysis...... 64
RESULTS ...... 68
iii
Fixed Interval 40 s ...... 68
Peak Interval 120 s...... 78
DISCUSSION...... 115
Fixed Interval Procedure...... 115
Peak Interval Procedure ...... 117
LITERATURE CITED ...... 122
APPENDIX...... 142
iv
ABSTRACT
It has been proposed that the Reln gene, which encodes the Reln protein, plays a role in the
etiology of schizophrenia. There are three Reln genotypes of mice available for examinations, the +/+ mice (100% Reln expression); the +/rl mice (50% Reln expression); and the rl/rl mice
(0% Reln expression). Postmortem studies on the brains of individuals with schizophrenia found that they were haploinsufficient for the Reln gene. Of the three Reln genotypes, the +/rl mouse is of interest because its brain abnormalities resembles those of individuals with schizophrenia. All three genotypes of mice were used to study the effects of Reln on behavior as it conformed to the contingencies of time. To assess temporal control of behaviors, mice were trained to lever press in standard operant chambers then exposed to a Fixed Interval (FI) 40 s schedule of reinforcement and the Peak Interval Procedure (PI) 120 s. Results revealed that Reln deficient mice differed from controls in responses occurring after the usual time of reinforcement. They had significantly higher relative frequencies of responding during that time and larger peak spreads, indicating that deficiencies in Reln may cause behavioral abnormalities with respect to temporal environmental contingencies.
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ACKNOWLEDGEMENTS
The writing of my master’s thesis has been the most significant academic challenge I have
had to face. This is a great opportunity to express my respect and gratitude to those that showed
me support, patience and guidance through the whole process.
My mentor, Dr. Ruth Hurst has provided me with endless guidance and encouragement,
despite her many other academic and professional commitments. I thank her for introducing me
to the experimental analysis of behavior and for inspiring and directing me to constantly better
myself.
I would like to thank my committee, Dr. Mark Galizio, Dr. Christine Hughes, and Dr.
Raymond Pitts for all of their knowledge and assistance with this project. I have enjoyed their
enthusiasm for the field of behavior analysis, and have learned much from them.
I am pleased to thank Dr. Shafer from the UNCW Biology department for his contribution to
my study. Dr. Shafer was responsible for the DNA analysis of all mice in our lab and I am very
grateful for all of his hard work.
Most importantly I would like to thank my family who will never know how much their love and support have meant to me. I owe my deepest gratitude to my mother and best friend, Beth,
for always wanting the best for me, bringing out the best in me, and believing the best of me.
Thanks to my father, Michael, for teaching me the value of hard work, the importance of family over everything else, and the places I can find beauty and peace in this world. I want to thank my step-father, Jay, for always encouraging me to follow my dreams and for cheering me on every step of the way. To finish, I owe many thanks to Adam for his unconditional patience and love. He has taught me so much about sacrifice and compromise.
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DEDICATION
I would like to dedicate this thesis to my sister, Catherine, whose struggle with autism has been
my inspiration. She’s proven that with hard work and perseverance any obstacle can be
overcome and any goal achieved. She is truly my hero!
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LIST OF TABLES
Table Page
1. Review of Fixed Interval Findings ...... 69
2. Review of Peak Interval Findings...... 81
viii
LIST OF FIGURES
Figures Page
1. Relative Frequency for the Beginning of FI Training ...... 71
2. Relative Frequency for Beginning of FI Training Compared to +/+...... 72
3. Relative Frequency for the End of FI Training...... 73
4. Relative Frequency for the End of FI Training Compared to +/+ ...... 74
5. IOC for FI, Across All Sessions ...... 76
6. IOC for FI for End of FI Training Compared to +/+ ...... 77
7. IOC for FI from Beginning to End of Training ...... 79
8. IOC for FI, Asymptote...... 80
9. Relative Frequency for PI for Average of First 5 Sessions ...... 85
10. Relative Frequency for PI for Average of Last 5 Sessions...... 86
11. Relative Frequency for PI for 1st Interval for Average of First 5 Sessions...... 87
12. Relative Frequency for PI for 2nd Interval for Average of First 5 Sessions...... 89
13. Relative Frequency for PI for 3rd Interval for Average of First 5 Sessions ...... 90
14. Relative Frequency for PI for 1st Interval for Average of Last 5 Sessions...... 91
15. Relative Frequency for PI for 2nd Interval for Average of Last 5 Sessions ...... 93
16. Relative Frequency for PI for 3rd Interval for Average of Last 5 Sessions...... 94
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17. Max Response Rate for PI Across All Sessions ...... 95
18. Max Response Rate for PI for First 5 Sessions...... 97
19. Max Response Rate for PI for Last 5 Sessions...... 98
20. Mean Number of Bins with Max Responses for PI ...... 99
21. IOC for PI for 1st Interval Across All Sessions...... 101
22. IOC for PI for 2nd Interval Across All Sessions...... 102
23. IOC for PI for 3rd Interval Across All Sessions ...... 104
24. PI Relative Frequency for +/+ as a Proportion of +/+ Max...... 105
25. PI Relative Frequency for +/rl as a Proportion of +/rl Max ...... 106
26. PI Relative Frequency for rl/rl as a Proportion of rl/rl Max ...... 107
27. PI Relative Frequency for All Genotypes as a Proportion of Genotype Max ...108
28. Average PI Peak Time ...... 109
29. Average PI Peak Spread...... 110
30. Mean Time for 1st Part of Peak Spread for PI...... 112
31. Time for 2nd Part of Peak Spread for PI...... 113
32. PI Spread with 1st and 2nd Parts of Spread ...... 114
x
INTRODUCTION
The Reln gene, located on chromosome 7q22, encodes Reln protein (D’Arcangelo, Miao,
Chen, Soares, Morgan, & Curran, 1995) The Reln protein is found in the brain, the spinal cord, blood, and other body organs and tissues of mammals (Smallheiser, Costa, Guidotti, &
Impagnatiello, 2000). During neural development Reln acts on migrating neuronal precursors and controls proper cell positioning in the cortex and other brain structures (Hack, Bancila,
Loulier, Carroll, & Cremer, 2002). The normal migration of neurons in the cerebral cortex follows an “inside out” migratory path, in which later generated neurons pass by the earlier-born neurons in their migration to their final destination (Hattan, 1999). The Reln protein guides neurons as they migrate into the cortex and cerebellum. Absent or deficient Reln expression obviates the cues needed for proper cell migration (Lombroso, 1998).
The Reln protein was named after the peculiar reeling gait of reeler mice (rl/rl). The rl/rl mice, homozygous for a null Reln gene mutation, are completely absent of the Reln protein. With rl/rl mice, the normal migratory path of neurons is reversed, consistent with the absence of the functional role of Reln. Accordingly, the earliest-born neurons migrate to the surface, the next generation settles immediately below, and the final neurons form the deepest cortical layer. The rl/rl mouse has been used as a biological and structural model for investigating the means by which the neuronal network develops during gestation (Hack, Bancila, Loulier, Carroll, &
Cremer, 2002; Hatten, 1999).
Disruption or lack of Reln expression has been associated with several brain anomalies, some of which have been related to specific human neurodevelopmental disorders and psychopathologies. Lissencephaly, a human neural cell migration disorder, is characterized
by low or undetectable amounts of Reln protein (Kato & Dobyns, 2003). Also known as
“smooth brain” lissencephaly is characterized by severe delays in neurological and cognitive development such as little or no language, the inability to stand or sit unsupported, seizures, hypotonia, myopia, and nystagmus (Hong et al., 2000).
Reduced expression of Reln has been reported and confirmed in postmortem studies of schizophrenia and bipolar disorder patients (Grayson et al., 2005; Impagnatiello et al., 1998;
Tamura, Kunugi, Ohashi & Hohjoh, 2007). One research group found the extent of Reln deficiency similar in the same brain areas of all schizophrenic patients studied when compared to nonpsychiatric patients (Impagnatiello et al., 1998). The same group also found that the Reln reduction was not related to sex, age of schizophrenia onset, or time between death and sample collection (Impagnatiello et al., 1998).
An association between the Reln gene and autism has been found (Fatemi, Kroll, & Stary,
2001; Fatemi, Stary, Halt, & Realmmuto, 2002; Keller et al., 2000; Persico et al., 2001). Using case-control and family-based designs, Persico et al. (2001) showed a significant association between autism and Reln gene variants. The authors identified a repeated GGC sequence in the gene encoding Reln that might affect gene expression. This GGC stretch is “polymorphic” meaning it differs in length between different individuals. Twenty percent of Persico et al.’s
(2001) patients with autism were found to have longer variants of the GGC repeats. Persico et al. (2001), based on their sample, concluded that inheriting this “long” allele, leads to a three- fold increased risk in developing autism. In post mortem studies of autistic brains, Fatemi et al.
(2001 a, b) found a 43% reduction in Reln levels compared to non-autistic controls. Fatemi
(2002) followed up this study by testing the blood levels of unprocessed Reln (410 kDa) in autistic twins, their normal siblings and parents in comparison to controls. Results indicated a
significant reduction in 410 kDa Reln protein in autistic twins (-70%), their fathers (-62%), their mothers (-72%), and their phenotypically normal siblings (-70%) versus controls. Their results indicated Reln 410 deficiency as a vulnerability factor in the pathology of autism. Chromosome
7q has been implicated as one that likely contains an autism susceptibility locus, and again, the
Reln gene is located there (IMGSAC, 1998; IMGSAC, 2001; Lamb, Parr, Bailey, & Monaco,
2002). However, in an independent German autism sample, Bonora et al. (2003) analyzed the
Reln gene for relevance of susceptibility for autism. The group found only a “low frequency” of mutations in the Reln gene and concluded that Reln may not play a role in the linkage between autism and chromosome 7q.
Whereas the association with autism is uncertain, Reln deficiency continues to be of interest in this and other disorders. For example, Reln has been implicated as a possible causal variable in Alzheimer’s disease (Bothwell & Giniger, 2000; Helbecque & Amouyel, 2000). Further, though findings have been inconsistent, the possible association between Reln and certain psychopathologies suggests that the behavior of the Reln deficient mouse should be assessed to determine whether or not the model could serve a useful role as a behavioral model for human psychopathology.
The three genotypes of mice available for study are the wild type control mouse (+/+) which expresses 100% Reln; the heterozygous mouse (+/rl), which expresses ~50% Reln; and the reeler mouse (rl/rl) which has no Reln expression. The +/rl mouse, due to its partial and abnormal gene expression, has been suggested by some to be the model of interest in the exploration of psychopathologies such as schizophrenia, and as noted above, there are similar abnormalities in the brains of +/rl mice and people with schizophrenia (Ballmaier et al. 2002; Grayson et al.,
2005; Impagnatiello et al. 1998; Roberts et al., 1998; Tamura et al. 2007) However, reports of
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behavioral abnormalities of +/rl mice have been inconsistent, and thus the +/rl mouse has not been clearly validated as a behavioral model of psychopathology (Goldowitz & Koch, 1986;
Groves, O’Meara, Handford, Smith, Dawson & Reynolds, 2003; Krueger, Howell, Hebert,
Olausson, Taylor & Nairn, 2006; Podhorna & Didriksen, 2004; Salinger, Ladrow & Wheeler,
2003; Tueting et al., 1999).
Because post-mortem studies on the brains of individuals with schizophrenia have detected
Reln deficient, a Reln hypothesis for this disorder and others has been put forward (Costa, Davis,
Pesold, Teuting & Guidotti, 2002; Fatemi, 2001a, b; Fatemi, Stary, & Egan 2002; Keller et al.,
2000; Persico et al., 2001; Tueting, et al., 1999). More specifically, the Reln hypothesis states that Reln deficiency can create a vulnerability to psychopathologies. A way to assess the validity of the Reln hypothesis is to assess the behavior of the +/rl mouse. If the hypothesis is valid, the
+/rl mouse should exhibit brain and behavioral anomalies consistent with those found in schizophrenia. Consistent with the hypothesis, it has been shown that brain abnormalities found in schizophrenia are similar to those found in the +/rl mouse (Ballmaier et al., 2002; Costa et al.,
2002; Impagnatiello et al., 1998; Pesold et al., 1999). These shared neurochemical and neuroanatomical abnormalities in the brain of the schizophrenia patients and +/rl mouse include: decreased GAD67 expression, decreased Reln expression, disrupted correlation between GAD67 and Reln, decreased neuropil expression, increased neuronal packing density, reduced dendritic spine density, and altered disruption of NADPH-diaphorase-positive cells (Costa et al., 2002).
Therefore, the +/rl mouse which has been proposed to be a model for schizophrenia (Costa et al.,
2002; Fatemi 2001; Lui et al., 2001; Rowling, Bristow & Hutson, 2001) needs further assessment to determine its validity as a model. While brain abnormalities in the +/rl mouse have been found to be consistent with the Reln hypothesis, and support the argument that the +/rl mouse is
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a potential model for human psychopathology, the behavioral findings related to this model have been inconsistent.
Behavioral Assessment of the Reln Deficient Mouse
Crawley (1999) and Crawley and Paylor (1997) have reported that animal assays such as sensorimotor gating, Morris water maze and spatial maze, fear conditioning, operant, rotarod, and anxiety related tasks should be used when determining whether or not a particular animal model is suitable as a model for schizophrenia relevant behaviors. A complete list of tasks that have been used to assess the behavior of +/rl mice and the results are available in Appendix A.
While there have been some results suggesting abnormal behavior in the +/rl mice (Tueting,
Costa, Dwivedi, Guidotti, Impagnatiello, Manev, & Pesold, 1999) other results have reported the
+/rl mouse to be no different from +/+ mice (Salinger, Ladrow, & Wheeler, 2003) and some findings of abnormality have failed to replicate (Podhorna and Didriksen, 2004). Further, it is noteworthy that to date that there have been no published findings with respect to operant schedule controlled behavior of +/rl mice, leaving a gap in the behavioral assessment of +/rl mice based on the Crawley (1999) and Crawley and Paylor (1997) recommendations for assessment. The sections below summarize what is known about the behavior of +/rl mice on the behavioral assessments that have been reported to date.
Startle reflex. Startle reflex assays assess the transmission of sensory information to a motor system. The startle reflex is the physical response elicited from an organism when presented with a startling stimulus. It can be in the form of (but not limited to) a muscle twitch, jump, blink, and movement away from stimulus.
Tueting et al. (1999) measured the startle response of 13 male mice (6 +/+, 7 +/rl), aged 40-63 days old, on their reactions to a startle stimulus. Tueting et al. (1999) used an acoustic startle
5
apparatus (SR-LAB, San Diego Instruments) which consisted of a 5.1 cm Plexiglas cylinder
resting on a Plexiglas frame within a ventilated, soundproof enclosure. A speaker placed 24 cm
above the mouse was the source of the acoustic noise bursts. Within the cylinder, motion
(flinch) was detected using a piezoelectric device. These researchers used 16 presentations of a
115 dB startle stimulus to elicit the measured startle response and found no differences between
the genotypes.
Salinger, Ladrow & Wheeler (2003) also measured the startle response of +/rl mice compared
to +/+ mice with an added rl/rl mouse comparison group. Salinger et al. (2003) used 22 mice
approximately 70 days old. There were 6 +/+ (5 M, 1 F), 9 +/rl (all male), and 7 rl/rl (2 M, 5F).
These researchers used a slightly smaller apparatus and different startle stimulus (acoustic
amplitudes) than Tueting et al. (1999). A 3.9-cm acrylic cylinder for holding each mouse was
set on a piezoelectric accelerometer unit similar to Tueting et al.’s (1999) study, attached below
the cylinder. The piezoelectric unit converts vibrations of the body movements of each mouse into a stored signal, relative to response amplitude. Each mouse was placed in the cylinder
individually for a 5 min acclimation period followed by blocks of startle response trials. Each subject was presented with four blocks of six different intensities (72, 84, 90, 100, 110, and 120) over a white noise background. The maximum startle amplitude was measured as the startle response amplitude measured by the SR-LAB software. A main effect was found for genotype in which +/rl mice produced larger startle response amplitudes than +/+ mice when the startle stimulus intensity was increased. Compared to +/+ mice, the rl/rl mice were more responsive to a startle stimulus intensity increase from 90 to 100 dB than were +/+, but less responsive to an increase from 100-110 dB than were +/+. Salinger et al. (2003) concluded that the differences
6
between the +/rl and +/+ mice be discounted due to inconsistencies with the remainder of the
findings of no difference from +/+ mice on various other behavioral assays.
Podhorna and Didriksen (2004) examined male and female +/+ and +/rl mice, young (67-71
days old) and fully adult (102-105 days old) on the startle response assay. Refer to Appendix A
for specifics on sample characteristics. The startle apparatus, an MPOS 2b, was very similar to
the SR-Lab device used by Tueting et al. (1999). It consisted of a 5 cm Plexiglas cylinder
mounted on a Plexiglas platform placed in a sound attenuated cubicle with a high frequency
speaker. Movements were detected and transmitted by a piezoelectric accelerometer attached to
the apparatus. Each mouse was presented with a 77 dB and a 105 dB acoustic stimulation and startle response to each of the two stimuli were recorded. This procedure used 8 blocks of 6 trial
types for a total of 48 trials. No significant differences between +/rl and +/+ mice, on acoustic
startle response, were found in either young adult or fully adult mice. Likewise, no sex
differences were detected with this assay.
Qiu, Korwek, Pratt-Davis, Peters, Bergmann, & Weeber (2006) tested 18 mice (9 +/+, 9 +/rl)
approximately 6 weeks old on the acoustic startle response measure. Male and female mice were
used but the number for each genotype was not given. Qiu et al. (2006) did not give information
on the apparatus used or other specifics of the procedure such as number of trials. What is known
is that the mice were presented with a 120 dB startle stimulus and the startle amplitude of each
mouse was recorded. Researchers reported that the +/rl mice did not differ from the +/+ mice on
this measure.
On the test of sensorimotor gating based on startle reflex only, the only research group to
have found a startle amplitude difference in the +/rl mice when compared to +/+ mice was
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Salinger et al. (2003). However, Salinger et al. (2003) decided that those results should be
overlooked because they did not coincide with the rest of their data.
Startle response habituation. Startle response habituation refers to the suppression of the
startle response of the organism when it has been repeatedly presented with the same startling stimulus. Salinger et al. (2003) used the same apparatus used in the startle response procedure to measure the habituation of responding based on repeated presentations of 120 dB on 63 mice.
There were 20 +/+ (9 M, 11 F), 22 +/rl (11M, 11 F), and 21 rl/rl (8 M, 13 F) used in this measure, each approximately 70 days old. The 120 dB startle stimulus was presented randomly over a 12.39 min session. Results indicated that when compared to the +/+ mice, the +/rl mice did not differ in habituation responses to the same stimulus, but the rl/rl mice demonstrated less startle response habituation than +/+ and +/rl mice. That is, the rl/rl mice did not reduce their startle response amplitude after repeated presentations of the startle stimulus, compared to the
+/+ and +/rl.
Pre-pulse inhibition of the startle reflex. Pre-pulse inhibition (PPI) is the suppression of the startle reflex when an intense startling stimulus is preceded by a weaker pre-stimulus (Swerdlow
& Geyer, 1998). The startle response is said to be a defensive response (Swerdlow & Geyer,
1998) that is dependent on the interaction of sensory information with the motor system
(Nusbaum & Contreras, 2004). The typical reaction to a pre-pulse inhibition paradigm is for the body’s startle response to become smaller in amplitude following a startle stimulus that has been preceded by the pre-pulse than the startle response amplitude following only the startle stimulus.
PPI deficits have been observed in psychotic patients and their close relatives (Braff et al., 1978;
Braff & Geyer, 1990; Braff et al., 1992; Dawson, Hazlett, Filion Nuechterlein & Schell, 1993;
Elvevag et al., 2003; McDowd, Filion, Harris & Braff, 1993). This is especially relevant since
8
PPI is one of the few measures used to examine similar phenotypic variations between species
(Tueting, Doueiri, Guidotti & Costa, 2006).
Tueting et al. (1999) reported abnormalities in sensory motor gaiting, related to PPI, in 7 male +/rl mice when compared to 6 male +/+ mice. All mice were between the ages of 43 and 60 days old. The same startle response apparatus used in the acoustic startle response procedure was used in this measure. The stimulus trial types included an 80 dB pre-pulse followed by a 115 dB stimulus, and a 75dB pre-pulse followed by a 115 dB stimulus. There were 16 trials of each stimulus trial type. Pre-pulse inhibitions were measured using ratios, calculated by dividing the difference between the mean amplitudes for startle and pre-pulse trials by the mean amplitude of the startle trials. PPI deficits, defined by lower PPI ratios, were present in the +/rl mice compared to the +/+ mice, for both the 75 dB and 80 dB trials. These deficits were present during the early trials as well as the late trials, after the basic startle response had habituated.
The presence of the pre-pulse did not inhibit the responding of the +/rl mice to the second pulse
(following the pre-pulse), compared to the +/+ controls.
Following Tueting et al.’s (1999) original report of PPI abnormality in +/rl mice, Salinger et al. (2003) measured the pre-pulse inhibition of 63 mice; 20 +/+ (9 male, 11 female), 22 +/rl mice
(11 male, 11 female), and 21 rl/rl mice (8 male, 13 female) all approximately 70 days old. For
Salinger et al.’s (2003) PPI assay a 120dB acoustic noise was used as the startle stimulus and the pre-pulse stimuli consisted of a 72dB, 76dB and 82dB acoustic noise. One session lasted 12.39 min and included 50 trials of random pre-stimulus/stimulus presentations (i.e., one trial type might include a 76 dB pre-stimulus followed by a 120 dB). The results indicated that there were no measurable differences between the +/+ and +/rl mice on the measure of PPI. However, there
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was a main effect for genotype in which the rl/rl mice differed significantly from both the +/+ and +/rl mice in that they produced a weaker PPI.
Podhorna and Didriksen (2004) used the same mice and apparatus used in their acoustic startle response assay to assess for possible PPI genotypically based behavioral differences.
Mice were presented with three different PPI trials types and the number of trials for each trial type was unclear. The three trial types included a pre-pulse followed by the startle pulse (67 +
105 dB; 72 +105 dB; 77 + 105 dB). No significant differences between +/rl and +/+ mice, on the pre-pulse inhibition ratio, were found in either young adult or fully adult mice. Likewise, no sex differences were detected with PPI. Researchers noted that there was a slight trend toward decreased PPI in fully adult +/rl mice.
In summary, there were mixed results based on the examination of the +/rl mice on the sensorimotor gating assay measuring PPI. The results are hard to interpret due to the failure of indirect replications with respect to Tueting et al.’s (1999) original findings. Qiu et al. (2006) like Tueting et al. (1999) found that the +/rl mice differed significantly in PPI startle amplitude when compared to +/+ mice. That is, when the strong startle-eliciting stimulus was preceded by a mild similar stimulus, +/rl mice did not suppress their responding as much as the controls.
Salinger et al. (2003) and Podhorna et al. (2004) were unsuccessful at finding the same results as
Tueting et al. (1999) and Qiu et al. (2006) on PPI examinations. The Salinger et al. (2003) and
Podhorna et al. (2004) groups did not find any behavioral differences in PPI startle amplitude between the +/rl mice and +/+ mice. The inconsistency in these PPI findings compared to others is of particular importance because of the status of PPI as being a possible bio-behavioral marker for psychosis (Tueting et al., 2006).
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Elevated plus maze. High anxiety has been noted as a hallmark in individuals with
schizophrenia (Emsley, Oosthuisen, Joubert, Roberts, & Stein, 1999). The elevated plus maze is
said to measure the “emotional states” of an animal, anxiety in particular (Biobserv, 2001-2007).
The elevated plus maze has four arms organized in a plus shape. Two of the arms are enclosed
with walls and the other two are open. Each of the two similar arms faces each other. The maze
is elevated some distance off the floor. The number of entries into the open or closed arms and the time spent in each arm of the maze are the main measures with this assay.
Tueting et al.’s (1999) examined 12 male mice (8 +/+, 4 +/rl) aged 40-63 days old on an
elevated plus maze. Each arm of the maze was 50 cm long and 10 cm wide. The walls that were
enclosed were 5 cm high and the maze was suspended 50 cm from the floor. Each mouse
received one session that lasted for 5 min. The +/rl mice explored the open arms significantly
less than +/+ mice and had significantly lower percentages of time spent in the open arms
compared to +/+ mice.
Krueger, Howell, Hebert, Olausson, Taylor, & Nairn (2006) tested 9 male +/+ and 10 male
+/rl mice on the elevated plus maze. Krueger et al.’s (2006) group used Montgomery’s elevated
plus maze. This maze is made of stainless steel, has four arms and is elevated 40 cm. Two of the
arms are open without walls, and the other two walls are enclosed by 15.25 cm high walls. The
four walls are 30 cm long and 5 cm wide. The mice were placed in the center of the maze and
allowed to freely explore for 5 min. Data were expressed as percentage time spent in each arm.
There were no significant differences noted between the +/+ mice and the +/rl mice in time spent
in the open arm and entries into the open arm of the elevated plus maze or in the number of total
arm entries.
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Qui et al. (2006) used an elevated plus maze to asses 9 +/+ and 9 +/rl mice aged approximately 6 weeks. Their subjects’ sexes were not made clear nor were the number and length of trials used. Their maze consisted of two open arms (30 x 5 cm) and two enclosed arms
(30 x 5 x 15 cm). The maze was elevated 40 cm off the floor. Researchers measured percent rest time in open arms, percent entries into the open arms, total time spent in the open arms, and closed and central areas. Results indicated that the +/rl mice demonstrated no significant differences from the +/+ mice on any of these measures.
Overall findings for the elevated plus maze were inconsistent. Krueger et al. (2006) and Qiu et al. (2006) did not report the same behavioral deficits in +/rl mice as reported by Tueting et al.
(1999). Salinger et al. (2003) suggested that the Tueting et al. (1999) findings were spurious.
More specifically, Salinger et al. (2003) criticized Tueting et al.’s work citing their elevated plus
maze data were collected from only 4 +/rl mice, no rl/rl mice were used in the study, and there
were only a small number of observations.
Olfactory learning assays. Olfactory learning assays check for possible differences between
organisms in their ability to use their olfactory senses for guidance. Reln is expressed in
olfactory areas of the adult mouse brain and schizophrenia is often associated with alterations in
olfactory perception (Larson, Hoffman, Guidotti, & Costa, 2003). For these reasons, researchers
have been interested in detecting differences between Reln deficient mice using olfactory assays.
Larson, Hoffman, Guidotti, & Costa (2003) examined 17 male mice (8 +/+, 9 +/rl) on an
olfactory task using an olfactory discrimination chamber. Water deprived +/rl and +/+ mice were
trained to a criterion of 90% correct in a block of 20 trials on eight distinct simultaneous-cue,
two-odor discriminations. The same odorants were always paired together, S+ and S-. The
testing chamber had walls that were 60 cm long, 10 cm wide and 30 cm high. At each end of the
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alley (East and West) were two round ‘sniff ports’ for nose poke responses that were detectable via photo beam breaks. Both ends (East and West) had a single small cup, placed in the floor, for water delivery. At the West end of the alley, air-dilution olfactometers delivered the odor stimulus, from the two sniff ports. The mice were trained to initiate the trial with a nose poke, in either sniff ports of the East end. This would activate the delivery of the two discriminative odors at the West sniff port, S+ and S-. Nose pokes in the port containing the S+ stimulus were reinforced with a drop of water; nose pokes in the S- port or no-response trials were not reinforced with water. The mice were trained to criterion on eight different two-odor discrimination problems. Researchers discovered abnormalities in the +/rl mice, compared to
+/+ mice, in learning the first olfactory discrimination. The +/rl mice required significantly more training sessions and made more mistakes on the first discrimination. However, after learning the first discrimination, +/rl learned future discriminations at an equal rate to their wild type controls and when tested one week after training, both groups performed equally well.
Salinger et al. (2003) assessed olfactory guidance using a nose-poke floor plate inserted into a
TruScan apparatus. Four centrally positioned nose-poke holes were present in the apparatus, of which only the left rear well contained bait. The researchers measured the number of snout touches on each of the four holes and olfactory guidance was recognized as the greater proportion of snout touches of the single baited hole, relative to others. Salinger et al. (2003) used 44 male mice (18 +/+, 13 +/rl, and 13 rl/rl) each aged approximately 70 days old.
Statistical analyses revealed that all three genotypes could use olfactory cues for guidance and that rl/rl mice were superior to +/rl and +/+ in responding to the olfactory stimuli from the baited wells.
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Salinger et al. (2003) used a TruScan apparatus modified for the nose-poke task to assess spatial learning, memory and navigation within the context of the olfactory guidance task. A
TruScan sensor detected each nose poke and locomotor activity for search path of each mouse.
The nose-poke task required the mouse to collect bait from each baited cup and the trial ended when all baits were retrieved or when 5 min had elapsed. Spatial working memory ratio was evaluated as the proportion of food-rewarded visits to the number of total visits to the baited holes until all baited holes were visited or 5 min had passed. For this task, researchers used 20
+/+ (10 males, 10 females), 21 +/rl (11 males, 10 females), and 19 rl/rl (11 males, 8 females) mice, all aged around 70 days old. Once mice were acclimated, they were given seven once-daily
5 min duration training trials on the 16 hole version of the nose-poke task, followed by seven once-daily 5 min trials in which only four centrally located bait cups were baited. Reference memory ratio was calculated as the number of entries and reentries into baited holes divided by the total number of entries into baited and non-baited holes. There was a main effect of genotype for rest time; the rl/rl mice spent a significantly greater percentage of trials at rest rather than on task compared with the other genotypes. However, genotypes did not differ in terms of the reference memory ratio.
In summary, it appears that the only differences noted in +/rl mice compared to +/+ mice on the olfactory assays occurred in Larson et al.’s (2003) study. Here, the difference occurred only at the beginning of training and researchers concluded that the results may be representative of a learning deficit in +/rl mice during the acquisition of learning a new task.
Set-shifting and reversal learning. Set-shifting and reversal learning assays are used as a measure of behavioral flexibility. The ability to shift sets or learn reversals is the ability to change one’s behavior to match the changes taking place in their environment.
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Brigman, Padukiewicz, Sutherland & Rothblat (2006) used an assay similar to the Wisconsin
Card Sorting Task (WCST), to assess set-shifting and reversal learning in +/rl and +/+ mice,
using visual stimuli. The WCST is used in humans to assess the flexibility of behavior to a
change in environmental contingencies and schizophrenia patients have difficulty on this task,
compared to controls, showing significant difficultly in shifting sets (Cannon, Glahn, Kim, Van
Erp, Karlsgodt, Cohen, Nuechterlein, Bava & Shirinyan, 2005). Brigman et al. (2006) trained
+/rl and +/+ mice (no reference to age, number or sex of subjects) on a series of visual
discriminations to assess reversal learning and set-shifting, using a computer-automated touch
screen. A lever to initiate the session and a food well, were placed at the rear of the chamber. On
the other end of the chamber a touch screen unit was attached to the front of a computer monitor
where stimuli were presented. After pre-training to press a lever, trials were initiated by a lever
press which was followed by the onset of stimuli. After stimulus presentation, mice were
required to nose poke the appropriate stimulus to receive a 20mg food pellet. Mice were first trained on a simple line discrimination where one of two stimuli (one horizontal line and two
intersecting lines) when poked, led to reinforcement. After mice met the criterion of 80% correct
on two successive sessions (minimum of five sessions) for the single-pair discrimination they
were presented with a reversal problem. The reversal problem used the same simple lines in the
first problem but the reinforcement contingencies were reversed. After criterion was met for the
reversal task, a compound discrimination task was presented. The compound discrimination task used the same line stimuli previously used but for this task they were superimposed over two white distracter shapes. The previously reinforced line was superimposed on various white shapes throughout the session. Mice received a reinforcer when they continued to respond to the same black line stimulus, despite the white shape (an irrelevant dimension). The fourth task of
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the discrimination series introduced a third cue in which a third line shape and a third white shape appeared. Three different line-shape combinations were used in pairs to create a Set
Formation problem. The originally trained line shape stimulus again served as the reinforcing
stimulus on every trial. The set shift problem was the final training paradigm used in this study.
With this problem the same stimulus combinations as the set formation problems were presented.
However, the reinforcement contingencies were changed so that reinforcement was contingent
on one of the white shape stimuli (former irrelevant stimuli). This set shift problem was
described as resembling the human set-shifting task, assessed by the WCST.
The measures recorded to assess Brigman et al.’s (2006) mice involved a) sessions to criterion, b) total errors to criterion, and c) errors (non-correction) to criterion. Brigman et al.’s
(2006) results indicated that the +/rl mice displayed a modest deficit that “may reflect a genetic vulnerability” (Brigman et al., 2006, p. 984) on tasks that assess set shifting and reversal learning. The +/rl mice learned simple and complex visual discriminations and were able to perform compound visual discriminations as well as the +/+ controls. However, the +/rl mice required more sessions to reach criterion on the reversal task. They also made more errors on that task than the controls. Additional testing revealed that the discrimination performance of the
+/rl mice did not differ from the +/+ mice when irrelevant stimuli (white shapes) were introduced, and that +/rl mice learned to shift sets (black lines to white shapes) as well as controls.
Krueger et al. (2006) tested 7 +/+ and 9 +/rl, 3 month old male mice on the reversal learning task using operant chambers with three nose poke apertures. Mice were trained to perform, a food reinforced, operant nose poke response. All three nose poke holes were illuminated and one was designated as active. A nose poke in the active nose poke hole resulted in delivery of a
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food pellet. After 10 sessions, the position of the active nose poke hole was switched. On the
16th session, mice were subjected to a second reversal. Mice with impairments would be expected to show increased perseverative responding. No differences in correct or perseverative responses between genotypes were observed for either reversal tasks. However, there was a trend towards a deficit in the initial acquisition of the task for the +/rl mice. It is noteworthy that two of the +/rl mice were excluded from the reversals because they failed to acquire the task at all over 10 days.
In summary, Krueger et al.’s (2006) study was similar to Larson et al.’s (2003) study in that they both reported a trend towards deficit in the behavior of the +/rl mice during the initial training of a reversal task. Unlike Larson et al.’s (2003) study who found deficits during the acquisition of learning in the +/rl mouse, the +/rl mice in Brigman et al.’s (2006) study differed from the controls during the later stages of training. That is, group differences were more apparent later in the sessions of the reversal learning than the beginning, indicating that they did not display a typical perseverative impairment. Brigman et al.’s (2006) results, a +/rl deficit in reversal learning, indicate that the +/rl mice showed a selective deficit in reversal learning and made errors similar to those made by individuals with schizophrenia on similar tasks.
Gait. Gait refers to the physical mobility of an organism on foot. Gait assays are often used to assess the functional differences in movements in various organisms. Significant deficits in gait have been associated with schizophrenia, in stride length regulation in particular
(Putzhammer, Heindl, Broll, Pfeiff, Perfah & Hajak, 2003).
To assess gait in +/rl mice, the Salinger (2003) group used a 35-cm-long acrylic tunnel with a rectangular cross section apparatus. Salinger et al. (2003) used 35 male mice (13 +/+, 10 +/rl, and 12 rl/rl) aged approximately 70 days. The mouse’s hind paws were painted with black ink
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and placed in the tunnel. After the mouse made its way toward the opposite end, the position of
the centers of each hind paw print was measured for each of the first five strides of each subject.
The difference between the paw-to-right-wall distance for right and left paw prints determined stride width. The results indicated a significant genotype difference, the rl/rl mice had
significantly wider strides compared with +/+ mice. Salinger et al.’s (2003) report was unclear as to the performance of the +/rl mice compared to that of the other two genotypes, however visual examination of data presented graphically suggest that the +/rl mice did not differ significantly from either +/+ or rl/rl mice, suggesting an intermediate effect for genotype.
Podhorna and Didrikson (2004) assessed spontaneous locomotor activity by placing 35 +/+ and 31 +/rl mice (see Appendix A for genotype, sex, and age details) into motor activity cages equipped with infrared light sources to measure horizontal movements of the animal for one hour. Researchers reported that there were no significant behavioral differences in gait between
+/rl and +/+ mice and no significant effects for sex or age were detected.
Podhorna and Didrikson (2004) tested motor coordination further by using a Rotamex 4/8 rotorod apparatus. Thirty-three 33 +/+ and 31 +/rl mice (see Appendix A for genotype, sex, and age details) were placed on the rod that revolved at a speed of 17 rotations per min. The latency between placement on and falling off of the rotorod was measured. This assay was completed 6 times and average latencies to fall off the apparatus were calculated. Performance on the
Rotorod was not significantly affected by sex or genotype. However, performance did deteriorate with age as fully adult mice showed significantly shorter latency to fall off than young adult mice, regardless of sex or genotype.
Qiu et al. (2006) assessed the motor movement of 9 +/+ and 9 +/rl mice (sex not clear) aged
approximately 6 weeks, using an accelerating rotorod. The rotating rotorod accelerated from 4 to
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40 rpm over a 3 min period. The +/rl mice performed significantly better over a 2 day period of testing compared to +/+ mice but both animal groups performed equally on the last two trials.
All researchers concluded that when comparing +/rl mice with +/+ mice on tests of gait and locomotor function there were no significant differences between the genotypes. This is consistent with what is seen in casual observation of locomotion by +/rl mice. The empirical evidence presented here support no difference between +/rl and +/+ mice in locomotor activity and gait.
Depth perception. Depth perception refers to the ability of an organism to organize its behavior based on the different visual dimensions in their environment. That is, appropriate depth perception is seen in behaviors that indicate the organism can move accurately based on how objects are placed in its space. Significant defective performance on tasks measuring depth perception in individuals with schizophrenia has been reported (Johannsen, Friedman &
Liccione, 1964).
To assess depth perception in Reln deficient mice, Salinger et al. (2003) used a TruScan apparatus with a fabricated insert that consisted of a clear, flat, acrylic sheet that served as a visual cliff over a black and white checkerboard pattern. A TruScan sensor beam recorded movements for the duration of a 60 s trial (1 trial per mouse). For this assay, these researchers used all male mice, 22 +/+, 13 +/rl, and 14 rl/rl. A mouse was considered to have detected differences in depth between the upper and lower horizontal surfaces if it paused before crossing the apparent cliff from the visually shallow side to the visually deeper side. The three genotypes did not differ significantly in latency to cross over from the high side of the apparatus. The observed path of all mice revealed a tendency to spend more time on the shallow side as opposed to the deep side of the apparatus.
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Social dominance. Social dominance assays are used to assess the level of dominance one organism demonstrates over another. These assays are usually done by placing two organisms of the same sex in close proximity to one another to observe the behaviors exhibited by each. The organism that demonstrates more socially aggressive behaviors (i.e., sniffing, chasing, biting, etc.) is considered the more socially dominant of the pair. Behavioral abnormalities, aggression in particular, have been reported in individuals with schizophrenia (Cheung & Schweitzer,
1998).
Salinger et al.’s (2003) group used a social dominance tube to test for social dominance. The acrylic tube was 2.54 cm in diameter and 55 cm long with a holding chamber attached to either end. Eighty six adult male mice were used, 33 +/+, 27 +/rl and 26 rl/rl. Each male mouse was paired with a male mouse of a different genotype and each mouse participated in one trial.
During the task, a mouse either retreated back to its own starting box or advanced to the end box on the other side of the tube or the two mice went back and forth for the 3 min duration of the assay. The mouse that entered the opponent’s start box was scored as the dominant member of the pair, whereas the other was scored as the submissive member. The outcome was scored a tie if neither mouse entered a goal box within 3 min. Researchers found a significant difference between genotypes in the measure of social dominance in that rl/rl mice were scored as the dominant member of the pair significantly more often when paired with both the +/+ and +/rl mice. However, +/+ and +/rl mice were relatively evenly matched and did not differ significantly.
Podhorna and Didriksen (2004) examined the social behavior of +/+ and +/rl mice by examining the social interactions between the subject and an NMRI (albino lab mouse) partner.
Thirty-two +/+ (17 male, 15 female) and 31 +/rl (15 male, 17 female) mice were used in this
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assay. The apparatus consisted of a clear transparent cage measuring 25 cm by 40 cm by 15 cm.
Behavioral activity was defined by frequency, total duration and latency to the first occurrence.
Social activities included social sniffs, climbing on the partner, following the partner, and partner grooming. Aggressive activities were defined as attacks and threats. Timid activities were defined as escape, defensive posture, and alert posture. Walking and rearing were also recorded.
There were no significant differences between age groups or genotypes on individual behavioral measures indicative of social investigation. However, males engaged in more instances of social activities than females.
In summary, as with the measure of gait, no differences were observed between +/rl and +/+ mice on the social dominance assays. Empirical evidence available thus far suggests the +/rl are no different from +/+ mice on measures of social behavior, including social dominance.
Novel object detection. Novel object detection tasks are used to investigate the ability of an organism to detect a novel object in an otherwise familiar environment. The first step is to familiarize the animal to an environment that is usually an open field with two objects. After the animal has been familiarized with this environment the second step usually involves removing one of the familiar objects and replacing it with a novel object. During the second phase, the usual dependent measure is the time spent exploring each of the objects. It is expected that the typical organism will approach the novel object more frequently than the more familiar object.
Individuals with schizophrenia have shown impairment in the ability to recognize visual objects to which they have had previous experience (Vianin, Posada, Hugues, Franck, Bovet, Parnas, &
Jeannerod, 2002).
For the novel object detection task, Salinger et al. (2003) used a TruScan apparatus with a computer controlled sensor beam placed at the level of the floor plate to record the mouse’s
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movements. Fourteen +/+ (8 male, 6 female), 13 +/rl (5 male, 8 female), and 9 rl/rl (5 male, 4 female) mice were used in this study. A quarter and dime were secured into the arena with
Velcro patches and the mice were placed into the box for a 30 min exploration period.
Immediately after the exploration interval, either the quarter or the dime was removed pseudo- randomly, and replaced with a brass key, which served as the novel object. Measurements during the 5 min task interval included latency to approach the novel object and the number of snout touches directed at the familiar object compared to the novel object. A significant difference was found between genotypes in the number of contacts with the objects. The rl/rl mice touched the novel objects more frequently than the +/+ and the +/rl mice. Also, compared to +/+ mice, both +/rl and rl/rl mice approached the novel object significantly sooner than the familiar object.
Open field task. Open field tasks are used to examine the basic behavioral phenotypic actions of the organism being observed. The open field systems usually use photo-beams to measure and track distances and zones traveled by the animal. Individuals with schizophrenia have been reported to demonstrate involuntary abnormal locomotor movements (Quinn, Meagher, Murphy,
Kinsella, Mullaney, & Waddington, 2001).
For the open field test, Salinger et al. (2003) used the TruScan enclosure fitted with a plain floor plate. Computer controlled sensor beam arrays monitored and recorded movements in 5 min intervals for the duration of a single 60 min trial per mouse. One sensor beam recorded movements while the other monitored rearing activity. Measurements included, time in motion
(activity), time spent in the margin zone of the open field, rearing, defecation, and a number of small movements that involved coordinate change of less than 1.52 cm and a return to the original coordinate within 2 s. Twenty +/+ (10 male, 10 female), 21 +/rl (11 male, 10 female),
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and 33 rl/rl (11 male, 12 female) mice were used in this assay. Post hoc analysis revealed that
rl/rl mice produced significantly fewer fecal boli than +/+ mice. During the first 5 min of the
open field exposure, there were significant differences in activity levels (time in motion) between
genotypes, with rl/rl mice spending more time in motion than +/rl and +/+ mice. In addition,
mice housed in isolation spent significantly more time in motion, during the open field test, than
did those housed socially. Reeler mice emitted significantly more non-locomotor movements
than the other genotypes and this effect was especially stronger for rl/rl mice housed in isolation
compared to the +/+ and +/rl mice housed in isolation. Nonlocomotor body movements were
defined as behaviors such as grooming, head extension and retraction, head bobbling, and body
extension and retraction that displaced the center of the mouse no more than 1.52 cm and then
returned it to the starting position. An elevated proportion of the total time spent in the margin of
the open field was considered to be a measure of high levels of anxiety. Individually housed
male mice and rl/rl mice spent a greater proportion of time in the margins of the open field. A
progressive decrease over time in the proportion of total time spent in motion was said to reflect
the rate at which the mice habituated to the novelty of the open field setting. The rl/rl mice exhibited increased overall time spent in motion compared to +/+ and +/rl mice. Rearing activity
was said to reflect the “adequacy of attention control and executive functioning during
exploration” (Salinger et al., 2003, p. 1265). A genotypic difference was found in that rl/rl
reared significantly less often than the +/+ and +/rl mice. This result is not unexpected given the
motor abnormality of the rl/rl mice. However, the +/rl and +/+ mice initially increased and then
decreased the rate of rearing whereas rl/rl mice produced a steady though low rate throughout.
Qiu et al. (2006) used the open field test to assess general locomotor activity and exploratory
behavior, of 9 +/+ and 9 +/rl mice (sex not clear) aged approximately 6 weeks, in a novel
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environment. Their open field chamber measured 27 x 27 cm and each session lasted 15 min
(one session per mouse). These researchers reported no significant behavioral differences between the two genotypes, i.e., there was no significant difference between +/+ and +/rl in total distance traveled in the apparatus.
In summary, Salinger et al. (2003) and Qiu et al. (2006) both reported no significant behavioral differences between the +/rl and +/+ mice on the open field task. Salinger et al.
(2003) did indicate that the rl/rl mice behaved differently compared to +/+ and +/rl mice. The rl/rl mice had significantly fewer fecal boli than +/+ mice and were significantly more hyperactive than +/+ and +/rl mice. There were significantly more nonlocomotor movements
(i.e.: grooming, head extension and retraction, and head bobbing) in rl/rl than +/+ and +/rl and rl/rl spent a significantly less amount of time in the margin field than +/+ and +/rl. Salinger et al. (2003) also reported that rl/rl mice spent significantly less time in motion compared to +/+ and +/rl and rl/rl reared less often than +/+ and +/rl. Qiu et al. (2006) did not make use of this measure (no rl/rl comparison).
Light/dark assays. The light/dark assay uses a light/dark box to assess anxiety like behaviors.
The box is a chamber divided into lightened and darkened halves by inserting an enclosure with an opening for the animal to be able to move from side to side. This assay measures the animal’s preference for the dark enclosed places versus the light exposed side.
Salinger et al. (2003) evaluated the same 64 mice used in their open field test in the light/dark chamber. A TruScan chamber was divided into lightened and darkened halves by inserting a four sided, dark acrylic enclosure with one small opening facing the center of the chamber. The sensory array was in the same position as the open field. The mouse was placed in the rear right corner of the lit half of the chamber at the beginning of the single 5 min trial. Measures included
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numbers of subjects that moved between the lighted and darkened sides, latency to enter the dark
side, and frequency of transitions. Analysis of latency to enter the dark side revealed significant
differences between genotypes, with rl/rl mice exhibiting significantly longer latency than both
+/+ and +/rl mouse genotypes. The rl/rl mice also exhibited significantly fewer transit crossings
between the light and dark side of the chamber compared to +/+ and +/rl.
Podhorna and Didriksen (2003) conducted a light/dark transition test on two genotypes of mice (+/+ and +/rl). Thirty three +/+ (16 male, 17 female) and 31 +/rl (16 male, 15 female) mice were used (see Appendix A for type, sex, and age information). Podhorna and Didrikson (2003) used two connected boxes, one served as the light side (30 cm by 27 cm by 27 cm) and the other as the dark side (15 cm by 27 cm by 27 cm). The researchers used a two-ring photo beam system to measure how much time was spent in the light and dark compartments, as well as to measure the number of crossings, rears, and number of transitions. No significant genotypic differences were found with this assay.
Qiu et al. (2006) used an open field chamber (27 x 27 cm) with a black insert to assess 9 +/+ and 9 +/rl mice (sex not clear), aged approximately 6 weeks, on anxiety like behavior. Mice were placed into the dark side of the chamber for one 5 min session and time spent on each side was analyzed for potential group differences. Results indicated no significant differences between the genotypes on this measure.
For all research summarized here, no behavioral differences between the +/rl and +/+ were observed in the light/dark box. Thus, there is no empirical evidence that +/rl mice differ from
+/+ mice on tasks designed to assess anxiety as measured by light and dark transitions.
Contextual fear conditioning. Contextual fear conditioning assays assess the learning and memory of an aversive event. Subjects are submitted to a neutral stimulus (i.e., tone) that is
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followed by an aversive stimulus (i.e., foot shock). Later, the subject is presented with the
neutral stimulus only and responses considered as “fearful behavior” (i.e., freezing) are measured
in the presence of the newly conditioned stimulus. Individuals with schizophrenia have
demonstrated abnormalities in the use of contextual information based on behaviors that suggest
a misinterpretation of stimuli that evoke emotional responding (Monkul, Green, Barrett,
Robinson, Velligan, & Glahn, 2007).
To test mice on conditioned emotional response (CER), Salinger et al. (2003) used a procedure involving three trials of 6 min duration, one trial per day, using a TruScan chamber with a 2.54 cm opening in the floor plate where a foot shock grid was placed. Twenty +/+ (10 male, 10 female), 21 +/rl (11 male, 10 female), and 19 rl/rl (11 male, 8 female) mice were used.
On Day 1, at the end of a 2 min acclimation period, an 80 dB auditory white-noise stimulus was
presented for 10 s followed by a 2 min no stimulus period after which the 10 s acoustic stimulus
was repeated, followed by a final 2 min no-stimulus period, then the session ended. On Day 2,
the CER trials were carried out. The procedure was identical to the baseline except a .75-mA foot
shock of 2 s duration was presented at the end of a 10 s 80 dB auditory stimulus. Day 3 was the
test day, in which the procedures were identical to the baseline at Day 1. Mice freeze when
frightened, so the effectiveness of the fear conditioning was represented by the increased time
spent without motion as measured for 40 s from the onset of the acoustic stimulus on Day 3,
relative to that following the neutral acoustic stimulus on Day 1. Initially, the rl/rl mice
significantly failed to increase freezing time as compared to +/+ and +/rl mice.
Krueger et al. (2006) used a chamber equipped with an electrified metal floor grid to test
contextual fear conditioning. Krueger et al. (2006) used 18 male mice, 8 +/+ and 10 +/rl.
During training, mice received three foot shocks (1s, 0.4 mA) with an intertrial interval of 30-
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60s, within a 3 min period. Mice were returned to the same box twenty-four hours later, in order to assess contextual fear. This test was repeated again at 48, 72, and 96 hours after training, to assess extinction of the freezing response. No effect for genotype was found with this assay; all mice demonstrated a significant decrease in the percentage of time spent freezing across the 4 days of extinction, following fear conditioning.
Qiu et al. (2006) used a San Diego Instruments conditioning chamber (26 x 22 x 18 cm), made of Plexiglas and equipped with a grid floor, to assess 9 +/+ and 9 +/rl mice aged approximately 6 weeks (sex not clear). During training, each mouse was placed in the chamber for 2 min before the onset of the conditioned stimulus, an 85 dB tone that lasted for 30 s.
Immediately after the tone a 2 s foot shock (0.5 mA) was delivered. A 120 s intertrial interval separated the two pairings of tone and shock. After the second shock was administered, the mouse was kept in the chamber for 2.5 min before being returned to its cage. Twenty four hours later the test for contextual fear conditioning was conducted and researchers measured freezing behavior for a 5 min test in the same conditioning chamber. Freezing behavior was considered lack of movement for 2 s. To assess cued fear memory, mice were assessed under a masked context. This involved changing the size and color of the chamber, using different lighting and the presence of a lemon odor. Each mouse was placed in the novel context for 3 min, approximately 24 hours after training and was exposed to the conditioned stimulus (tone) for another 3 min. Freezing behavior was once again assessed. During training, both genotypes exhibited similar levels of freezing in response to the first and second pairing of tone and shock.
When exposed to the conditioned stimulus in the same context 24 hours later, the +/rl mice showed a significant reduction in the amount of freezing compared to the +/+ mice. However, both groups responded identically in the presentation of the tone in the novel context.
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In summary, Salinger et al. (2003) and Krueger et al. (2006) found no differences in fear conditioning between +/+ and +/rl. Qiu et al. (2006) however found that +/rl mice significantly decreased their freezing behavior when presented with the conditioned stimulus after 24 hours past training. The inconsistency of these results lead this researcher to conclude that more testing may need to be done using contextual fear conditioning assays on Reln deficient mice.
Acute pain response. Nociception or physiological pain is often assessed in mice using a hot plate. It is a measure of how fast the animal responds to a painful event. Subjects are placed on a hot surface and are tested for latency to lift and lick paws. In schizophrenia, it has been shown that many of these individuals have pain insensitivity as demonstrated by a failure to respond to many forms of typically painful stimuli such as electrical or thermal pain (Manpreet, Giles, &
Nasrallah, 2006).
To test for acute pain response, Podhorna and Didriksen (2003) placed 35 (19 male, 16 female) +/+ and 31 (14 male, 17 female) +/rl mice (see Appendix A for sex by type by age break down) were used in this study. The mice were placed into a Plexiglas cylinder (30 cm high, 20 cm in diameter) that was positioned over a metallic plate heated to 55 degrees C for one 60 s session. The latency or time in seconds from placement on the plate to the start of licking or kicking up their hind paws was measured. There was a trend toward a shorter latency in +/rl males than in +/+ males. Fully adult female mice stayed on the hot plate longer than young females.
Qiu et al. (2006) assessed nociception by placing 9 +/+ and 9 +/rl mice aged approximately 6 weeks (sex not clear) on a 55 degree Celsius hot plate. Researchers used latency to paw licks to measure sensitivity to a harmful stimulus. Results indicated no differences between the two groups.
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Based on the findings of Podhorna & Didriksen (2003) and Qiu et al. (2006), +/rl mice did not differ from +/+ mice on assays of acute pain response. This indicates that aversive physical stimuli elicit the same reflex in +/rl and +/+ mice.
Inhibitory Control. Inhibitory control assays tests a subject’s ability to inhibit responding in situations that do not lead to reinforcement. They also test how well a subject can wait, when required, to respond in a way that usually leads to reinforcement. It is also known as a test of impulsivity. Evidence has shown that individuals with schizophrenia have response inhibition impairments and therefore behave in ways we might call impulsive (Enticott, Ogloff, &
Bradshaw, 2008).
Using the same apparatus used in the reversal learning task, Krueger et al. (2006) tested Reln deficient mice on inhibitory control. As before, one nose poke aperture was considered active, the other two apertures were inactive. Six +/+ (4 male, 2 female) and 8 +/rl mice (5 male, 3 female) aged 3 months were trained on a 4 phase task. In the first phase mice were trained on an
FR1 schedule until receiving 25 food pellets in a 30 min period. The schedule was changed to an
FR3 in phase 2. For phase 3, an auditory 3 s stimulus was introduced on a VI 30s schedule. The delivery of a food pellet was contingent on the operant response (nose poke) being emitted during the tone. Ten reinforcers were made available during the 30 min session. In the final phase, mice would only receive reinforcement if they withheld responding for the entire length of a pre-stimulus period (1-10 s) that followed the auditory tone stimulus. A nose poke during this pre-stimulus period resulted in resetting of the pre-stimulus timer. Ten reinforcers were made available per session. Each mouse received a daily 30 min session for 10 days. The efficiency ratio [(number of rewarded trials/ (number of correct nose pokes)] and the percentage of rewarded responses [(number of rewarded trials)/ (total number of trials) * 100] were the
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dependent variables measured in this phase. Heterozygous reeler mice did not differ from wild types in the measure of inhibitory control or total number of active and non-active responses over the 10 days of training. However, the +/rl mice did differ significantly from +/+ mice during the first phase of this task (FR1) but not in any other. As well, they showed a significant deficit in reinforced responses across days.
Krueger et al. (2006) assessed 5 male +/+ and 7 male +/rl mice aged 3 months on attentional function and inhibition further using a three choice serial reaction time task in which mice were trained to attend to and respond to a visual stimulus randomly presented in one of three nose poke apertures in an operant chamber. The visual stimulus remained on for 32 s. A response to the active aperture resulted in the delivery of a food pellet and the beginning of a new trial.
Responses made prior to delivery of the visual stimulus, responses made in either of the non- illuminated apertures, and failure to respond during the stimulus or the subsequent 5 s holding period resulted in an 8 s time out and initiation of the next trial. When a mouse achieved > 80% correct responding and > 50 trials per session the session’s stimulus durations decreased progressively from 32 s to 16, 8, 4, 2, 1 and lastly 0.8 s. Dependent variables included days to reach the above criterion for each stimulus duration, and final performance at the 0.8 s phase.
Heterozygous reeler mice did not show a deficit in acquisition of this task at any stage, nor did they differ from wild-type controls in the measures of attention and impulsivity.
Delayed matching to position. Delayed matching to position tests a subject’s ability to pair stimuli when they are presented at two different times. A stimulus is presented to the subject then a delay occurs in which there are no stimuli. After the delay, two or more stimuli are presented again and responding to the stimulus which was presented before the delay leads to reinforcement. Delayed matching to position/sample impairments have been reported in
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individuals with schizophrenia and are usually referred to as a deficit in working memory
(Pantelis, Stuart, Nelson, Robbins, & Barnes, 2001).
Krueger et al. (2006) used a delayed matching to position paradigm to assess “working memory” on 5 male +/+ and 7 male +/rl aged 3 months old. The nose poke operant chamber was used with two nose poke holes. For the test phase, a delay of 2 s, 5 s, 10 s, or 20 s was introduced between the sample and choice phases. Each phase session consisted of 64 trials pseudo-randomized across the four delay periods and two nose poke positions. No differences between the +/rl and the +/+ mice were found on this test.
Morris water maze. The Morris water maze assesses the spatial learning and memory of a subject. Typically, mice are placed in the water maze and a hidden platform is located in one of the maze’s quadrants. During training, latency to find the platform is measured. After training, the platform is removed and time spent in the quadrant that previously contained the platform is recorded versus time spent in the other quadrants. Again, it has been reported that individuals with schizophrenia show core deficits in visual and spatial working memory (Silver, Feldman,
Bilker, & Gur, 2003).
Krueger et al. (2006) used the Morris water maze to assess 9 male +/+ and 10 male +/rl mice aged 3 months old. This assay was performed in a round water-filled tank (100cm in diameter) containing a hidden platform (12 x 10.5 x 11cm) that was placed 2 cm below water level. Mice were placed in the center of the tank and were required to swim to the platform in order to escape from the water. Latency to reach the platform for each trial was the dependent measure in this assay and the change in escape latency over time was recorded as a measure of spatial learning.
On the 9th session, the platform was removed, and mice were allowed to explore the tank for 1
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min. Time spent searching in the location previously occupied by the platform was recorded.
No differences between genotypes were observed on this task.
Qiu et al. (2006) tested 9 +/+ and 9 +/rl mice approximately 6 weeks old (sex not clear) on the
Morris water maze. Researchers used a 91.5 cm diameter Nalgene pool filled with water kept at
room temperature and made white with non toxic tempura paint. In the pool was an 8 x 8 cm,
submerged, Plexiglas platform. Mice were placed in the pool and given 60 s to find the platform.
Once they found the platform they were allowed to stay there for 20 s before being returned to
their home cages. If mice did not find the platform within 60 s, they were guided there by
researchers and allowed to stay for 60 s before removal. Researchers measured latency to reach
the platform, distance traveled to reach the platform, swim speed, and time spent in each
quadrant. For 8 consecutive days, mice were trained with 4 trials per day with an intertrial
interval of 1 hour. On day 8, the mice were given a probe trial in which they were allowed to swim in the pool with the platform removed. On days 9 and 10, the platform was moved to a
new quadrant of the pool than previously trained and mice had to re-learn the new location of the
platform. Again, on day 10, the probe trial was given again. Percent of time spent in the target
quadrant and the number of platform location crossings were calculated. Results indicated that
both +/+ and +/rl mice showed marked improvement in their ability to find the hidden platform over the first 8 days. There were also no differences in overall swim distance, latencies to reach platforms, searching target quadrant, or number of pseudo-platform crossings.
In conclusion, +/rl mice did not differ in how they behaved when presented with the Morris water maze assay. Heterozygous mice were able to find and remember where the platform was located in the maze, as well as normals, indicating no learning deficiency when these contingencies were in place.
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Summary of literature review.
Tueting et al. (199) found PPI deficits as well as less exploration into the open arms of the elevated plus maze in +/rl mice. The deficits in PPI are indicative of a deficit in sensorimotor gating in +/rl mice. Fewer entries and time spent in the open arms of the elevated plus maze may signify that +/rl mice are more neophobic than +/+ mice. Since individuals with schizophrenia exhibit these same behavioral abnormalities (deficits in PPI and increased neophobia), Tueting et al. (1999) concludes that their findings support the use of the +/rl mouse as a model for testing the Reln hypothesis for vulnerability to psychosis.
Salinger et al. (2003) concluded that the phenotype of the +/rl mice was indistinguishable from that of the +/+ mice. This research group used 257 mice that were tested using high thorough put techniques that required analysis of data using group comparisons. However, the
+/rl mice did differ from the +/+ mice in that the +/rl mice made contact with the novel object earlier and were more sensitive to the increase in startle-stimulus intensity. Salinger et al. (2003) believed that these differences should be discounted due to the fact that they were not consistent with the rest of their data.
Larson et al. (2003) found that +/rl mice needed more training to reach criterion and made more errors than +/+ mice on the first of their olfactory discrimination tasks. Larson et al. (2003) concluded that the +/rl mice were able to perform just as well as controls once the first discrimination problem was acquired, therefore indicating a deficit in the acquisition of olfactory learning. Since olfactory identification deficits are common in schizophrenic patients, Larson et al. (2003) supported the +/rl as a mouse model to psychosis.
In an attempt to investigate whether the +/rl mouse could be used as a genetic model of schizophrenia, Podhorna and Didriken (2004) used an array of complex behavioral tests on +/rl
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and +/+ mice. Two age groups of each genotype were used: young adult (50-61 days) and fully
adult (> 75 days). Overall, Podhorna and Didriksen (2004) did not find any behavioral
abnormalities in the +/rl mice. The lack of detectable deficits led the researchers to conclude
that the +/rl mouse is not a suitable animal model of schizophrenia.
Qiu et al. (2006) assessed +/rl mice on a variety of behavioral assessments and found differences in PPI and contextual fear conditioning. However, these researchers did not find any differences in overall activity, coordination, thermal nociception, startle responses, shock threshold, anxiety-like behavior, and behaviors in relation to the Morris water maze. Therefore, they concluded that Reln haploinsufficiency does not result in expected global behavior abnormalities and thus suggest researchers put more focus on the many neuronal commonalities that +/rl and individuals with schizophrenia share.
Brigman et al. (2006) found that the +/rl mice required more sessions to reach criterion on the
reversal task and that they made more errors. Unlike Larson et al.’s (2003) study who found
deficits during the acquisition of learning in the +/rl mouse, the +/rl mice in Brigman et al.’s
(2006) study differed from the controls during the later stages of training. That is, group
differences were more apparent in the later sessions of reversal learning than the beginning,
indicating that they did not display a typical perseverative impairment but rather a transient one.
This transient change in behavior led Brigman et al. (2006) to conclude that +/rl mice do share
“cognitive impairments” similar to those with schizophrenia. Therefore, Brigman et al. (2006)
suggested the use of more behavioral testing to better examine the +/rl mice as a potential model
of schizophrenia.
Krueger et al. (2006) found no differences between the +/+ and +/rl mice on the behavioral
measures used. However, like Larson et al. (2003), they found that the +/rl mice showed deficits
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in the acquisition of several of the tasks. It was speculated that “it may be that heterozygous
reeler mice are a better model of learning deficits that are also associated with many psychiatric
disorders than…more specifically to schizophrenia” (Krueger et al., 2006, p. 103).
Continuing the Examination of Reln deficient Mice
The literature is not consistent when it comes to conclusions about the adequacy of the +/rl
mouse as a potential behavioral model for psychopathology, as is proven by the inconsistent
results on behavioral tests. It is not clear as to why behavioral discrepancies across labs are evident. It is hard to believe that there are no behavioral abnormalities in the +/rl mouse
considering its brain structural and functional abnormalities. It may be that researchers have not
yet tested for all the appropriate behaviors that could be affected by the consequences of reduced
Reln expression. It is also possible that the assays previously used were not sensitive enough at
detecting slight but important deficits. Further, many labs did not use the rl/rl genotype and
failure to use this genotype denied the opportunity for researchers to assess for the relation
between all three genotypes. If Reln expression is to be implicated in behavioral abnormality,
the degree of abnormality should be in relation to degree of expression. While subtle, it is
possible that +/rl mice might not differ from +/+ mice while also not differing from rl/rl mice.
Should that be the case, and if it were also the case that +/rl mice and rl/rl mice did differ, this
may expose a subtle yet important effect of gene expression on behavior that would not
otherwise be revealed. Regardless, the literature on behavioral experiments involving Reln
deficient mice is not worthless. It has allowed us the opportunity to understand what direction
researchers have taken and suggests future directions.
It may be useful to analyze the behavior of Reln deficient mice with a different type of assay.
More specifically, perhaps by examining Reln deficient mice for the presence of a previously
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unassessed behavioral anomaly, significant though possibly subtle differences due to Reln deficiency could be uncovered. A noted behavioral abnormality with schizophrenia (Elvevag, et al., 2003), autism spectrum disorders (Gowen & Miall, 2005), attention-deficit/hyperactivity disorder (Barkely, 1997; Toplak, Dockstader & Tannock, 2006), Parkinson’s disease (Hellstrom,
Lang, Portin & Rinne, 1997; Pastor, Artieda, Jahanshahi & Obeso, 1992), depression (Bschor et al., 2004; Rammsayer, 1990), and major affective disorder (Penny, Meck, Roberts, Gibbon, &
Erlenmeyer-Kimling, 2005) are behaviors that suggest abnormal time perception. These behaviors involve substandard abilities to organize behavior with respect to temporal properties of the environment. This has received some attention (Hurst, 2005, unpublished data) with respect to Reln deficient mice and it is of particular interest because, although interesting
preliminary results have been obtained, temporal control of behavior has not been thoroughly
examined thus far in Reln deficient mice. Since this behavioral dimension appears to be
defective in multiple psychopathologies and interesting preliminary results have been obtained,
further investigation of the temporal control of behavior of Reln deficient mice appears
warranted.
Two temporal assays, the fixed interval (FI) schedule of reinforcement and a modified peak
procedure (described in more detail in the next section) have been used in preliminary research
to evaluate mice with Reln deficiencies (Hurst, 2005, unpublished data). While these data were
inconclusive about abnormality in +/rl mice, they suggested that further use of these or similar
assays with Reln deficient mice may yield interesting results related to temporal control of
behavior. It is noteworthy that the fixed interval and peak trial procedures have proven useful in
detecting behavioral differences in animals with different neurotransmitter abnormalities based
on drug administration or alterations (Branch & Gollub, 1974; Dietrich & Allen, 1998; Lewis,
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Miall, Dann & Kacelnik, 2003; Maricq, Roberts & Church, 1981; Meck, 1996; Morrissey, Ho,
Wogar, Bradshaw & Szabadi, 1994; Olton, 1989; Taylor, Horvitz & Balsam, 2007). These studies, in particular those that assess the behavior of animal models with neurotransmitter problems similar to those found in Reln deficient mice, should inform expectations with respect to behavioral consequences of Reln deficiency in mice.
Schizophrenia has been associated with neurotransmitter abnormalities in the dopaminergic, cholinergic, and serotonergic systems (Carlsson & Lindquist, 1963; Hyde & Crook, 2001;
Lieberman, Mailman, Duncan, Sikich, Chakos, Nichols, & Kraus, 1998, respectively).
Similarly, Reln deficiency has also been associated with the alteration in all three of these neurotransmitters in ways that have been suggested to be parallel to the problems found in individuals with schizophrenia.
More specifically, individuals with schizophrenia have been found to have abnormal levels of dopamine leading to a dopamine hypothesis for schizophrenia (Carlsson & Lindquist, 1963). It has been shown that some parts of the brains of individuals with schizophrenia have higher levels of dopamine and other parts have reduced levels. In the prefrontal cortex of individuals with schizophrenia, there is evidence of decreased levels of dopamine, compared to individuals without schizophrenia (Akil, Pierri, Whitehead, Edgar, Mohila, Sampson, & Lewis, 1999).
Decreased D1, D3, and D4 receptors have been discovered in the prefrontal cortex of individuals with schizophrenia (Meador-Woodruff, Haroutunian, Powchik, Davidson, Davis, & Watson,
1997; Nestler, 1997). However, the limbic system has elevated levels of D3 receptors in individuals with schizophrenia than those without schizophrenia (Gurevich, Bordelon, Shapiro,
Arnold, Gur, & Joyce, 1997). Increased levels of D2 receptors have also been found in the ventral striatum of individuals with schizophrenia (Seeman, Weinshenker, Quirion, Srivastava,
37
Bhardwaj, Grandy, Premont, Sotnikova, Boksa, El-Ghundi, O’Dowd, George, Perreault,
Mannisto, Robinson, Palmiter, & Tallerico, 2005).
Similarly, the dopaminergic systems of Reln deficient mice have been shown to be similarly abnormal. Ballmaier, Zoli, Leo, Agnati, and Spano (2002) were interested in examining whether
+/rl mice possess disturbances in the mesolimbic dopamine system. They found that +/rl mice have an increase in dopamine terminal fields and D3 receptors in the limbic system. They also found increased levels of D2 receptors in the ventral striatum in the +/rl mice but decreased levels of D2 in the limbic system. Ballmaier et al.’s (2002) results based on these examinations of several markers of the mesotelen-cephalic dopamine pathway suggest a relationship between the Reln-dopamine related neuronal pathology and psychosis.
It has also been proposed that serotonin has a role in the pathophysiology of schizophrenia
(Lieberman et al., 1998). It has been reported that individuals with schizophrenia have elevated levels of serotonin (Govitrapong, Chagkutip, Turakitwanakan, & Srikiatkhachorn, 2000).
Pesold, Impagnatiello, Pizu, Uzunov, Costa, Guidotti, and Caruncho (1998) examined the brains of rats and determined that Reln plays a role in the synthesis of neurotransmitters and the regulation of synaptic plasticity. This conclusion came based on the fact that Reln is frequently released by terminals facing the dendritic spines of Purkinje or pyramidal cells, thought to release neurotransmitters. Therefore, Reln could affect any form of neurotransmitter, including serotonin. Heterozygous mice have also been reported to have higher levels of serotonin than controls (Ognibene, Adriani, Caprioli, Ghirardi, Ali, Aloe & Laviola, 2008).
A pathophysiological cholinergic hypothesis for schizophrenia has been proposed (Hyde &
Crook, 2001). A reduction in the cholinergic neurons was found in the ventral striatum of individuals with schizophrenia (Holt, Bachus, Hyde, Wittie, Herman, Vangel, Saper, &
38
Kleinman (2005). Alternatively, Tandon (1999) found increased muscarinic cholinergic activity
in individuals with schizophrenia. Karson, Casanova, Kleinman, and Griffin (1993) found lower concentrations of choline acetyltransferase (ChAT) in the pontine tegmentum of schizophrenic
subjects compared to controls. A decrease in the density and expression of muscarine receptors
in the cortex of individuals with schizophrenia was also reported by Crook, Tomaskovic-Crook,
Copolov, & Dean (2001). However, significantly higher levels of extracellular acetylcholine
(ACh) have been reported in individuals with schizophrenia (Sarter, Nelson, & Bruno, 2005).
Significantly higher levels of choline acetyltransferase (ChAT) were found in the hippocampus,
caudate, putamen, and nucleus accumbens of individuals with schizophrenia (McGeer &
McGeer, 1977).
Sigala, Zoli, Palazzolo, Faccoli, Zanardi, Mercuri, and Spano (2007) examined the
cholinergic system of the basal forebrain of +/rl mice and reported that the level of Reln
influences brain development. Sigala et al. (2007) reported that +/rl mice show evidence of a
marked reduction of choline acetyltransferase (ChAT) immunoreactive (ir) cell bodies, and
therefore their levels of functioning (i.e., production of neurotransmitters) in the septal and
rostral basal forebrain. However, Sigala et al. (2007) found a significant increase in ChAT
neurons in the lateral striatum of +/rl mice compared to controls, revealing an abnormal variation
in ChAT cell migrations resulting in a redistribution of ChAT neurons.
The relevance of the interactions between cholinergic systems and neurotransmitter synthesis
with inconsistent findings in individuals with schizophrenia and +/rl mice make it difficult to
understand the role they play in the behavioral and physiological phenotypes of individuals with
schizophrenia and +/rl mice. However, taken together, past studies have shown that the
neurotransmitter systems in the brains of individuals with schizophrenia and the brains of +/rl
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mice are abnormal in similar ways. The current study sought to determine whether operant
temporal assays, useful at detecting differences in behavior based on biochemical make up,
might also detect behavioral effects of Reln expression. The operant behavior of animal models
have been assessed with respect to all three of the neurotransmitter systems found deficient in
Reln deficient mice. First, the two methods previously mentioned used to assess operant behavior and its control by time (fixed interval schedule of reinforcement and the peak interval procedure) will be described. Then, findings using these procedures to assess behavioral outcomes of neurotransmitter abnormality will be presented.
Operant Methods that Assess Control of Behavior by Time
Several operant procedures have been used to assess the control of behavior by the passage of
time, including the fixed interval schedule of reinforcement (Catania, 1970; Derrene et al., 2007;
Dews, 1966, 1970; Ferster & Skinner, 1957; Fry et al., 1960; Guilhardi & Church, 2004;
Schneider, 1969), the peak interval procedure (Catania, 1970; Kirkpatrick, Miller, Betti &
Wasserman, 1996; Maricq et al., 1981; Matell & Portugal, 2007; Roberts, 1982; Taylor et al.,
2007; Zeiler & Powell, 1994), the differential reinforcement of low rate behavior schedule of
reinforcement, also known as IRT > t (Catania, 1970; & Ferster & Skinner, 1957), and the
temporal bi-section task (MacInnis & Guilhardi, 2006; Orduna, Hong & Bouzas, 2007; Penney,
Meck, Roberts, Gibbon & Erlenmeyer-Kimlinge, 2005). These procedures have produced
similar patterns of behavior across many laboratories and many species. The procedures that are
the focus of this proposal are the fixed interval schedule of reinforcement and the peak interval
procedure.
Fixed interval schedule of reinforcement. The fixed interval (FI) schedule of reinforcement is
one in which the first response, after a specified period of time has passed, is followed by a
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reinforcer (Ferster & Skinner, 1957). It is a free operant procedure, in that the organism is free
to respond throughout the entire interval but reinforcement is not made available until the pre-
defined time has passed. The temporal properties of responding depend on the ability of the
organism to discriminate the amount of time in a specific interval before reinforcement is made
available and to regulate its behavior with respect to those contingencies. The point of highest
rate presumably reflects when the animal anticipates that food is available. An FI schedule
creates the opportunity to assess the functional relation between rate of responding and rate of reinforcement because the schedule is designed to fix the rate of reinforcement while leaving the rate of responding free to vary over a wide, but fixed, time range (Catania, 1970).
The characteristic pattern of responding on an FI, on the cumulative record, is in the form of the scallop. That is, responding after the last reinforcer is null to slow and increases as time
passes and comes closer to the time of reinforcement. The work of a number of researchers has
resulted in detailed descriptions of the observable features of the FI pattern of responding. For
example, Ferster and Skinner (1957) described the overall behavior of responding in the FI
schedule of reinforcement as one that “normally generates a stable state in which a pause follows
each reinforcer, after which the rate accelerates to a terminal (usually moderate) value” (p. 134).
We see higher rates of responding during the time period most closely associated with
reinforcement and lower rates of responding during the time period most closely associated with
non reinforcement. Other researchers have attempted to develop mathematical descriptions of
the FI pattern of responding. For example, Schneider (1969) described the characteristic
behavior of an FI, on a single cycle as that of a step function, with low responses at the
beginning of the cycle and at a fast relatively stable rate at the end of the cycle. Still others
researchers have established the presence of important relationships between the length of FI
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schedules and the allocation of responses across the interval. Dews (1970), MacInnis and
Guilhardi (2006) and Lejeune and Wearden (1991) all demonstrated that the response rate on an
FI is inversely related to the interval duration. For these studies, when relative response rate was plotted as a function of relative time for multiple schedules of different FI values, the curves superposed. Dews (1970) used an FI 30, 300, and 3000 s to prove this relation. MacInnis and
Guilhardi (2006) used data from Guilhardi and Church (2004) that included relative response rate from an FI 30, 60 and 120 s, to prove this superposition of responses. Lejeune and Wearden
(1991) used data obtained from wood mice that were exposed to an FI 60s, FI 120s, FI 180s and an FI 240s schedule of reinforcement to demonstrate the same relative response pattern and response rate under different FI schedules.
It is unclear exactly what controlling stimulus is responsible for the characteristic pattern of responding on the FI schedule of reinforcement. For example, Ferster and Skinner (1957) and
Guilhardi, Keen, MacInnis, and Church (2005) suggested that the reinforcer; e.g., a food pellet, may serve not only as a reinforcer but as a marker of time. That is, after sufficient experience with the time marker and its delivery contingent upon a response, the behavior of the organism changes as the time marker approaches. Alternatively, Dews (1966) explained that the behavior of the mouse acts as a clock by which the mouse organizes its behavior with respect to reinforcement. Catania (1970) also suggested that the behavior that fills the interval serves as the discriminative stimulus by which the animal times its operant response. While the reason for the characteristic pattern of FI responding may be unclear, the characteristic pattern of responding under this schedule is a highly replicated finding, using many species, and thus a common prediction when FI schedules are implemented.
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Summary of FI literature review. The temporal properties of responding in a variety of inbred
and other types of mice on FI schedules have been documented (Burke, Miller &
Moerschbaecher, 1994; Derenne, Arsenault, Austin & Weather, 2007; Glowa, 1993; Lejeune &
Wearden, 1991; Wada, Moizumi & Kitazawa, 2005; Wenger, 1979; Wenger, 1986; Wenger,
McMillan, & Chang, 1982). Fixed interval performance has also been used to study the effects of
brain dysfunctions. One early study reported by Ferster and Skinner (1957) was done using pigeons. The pigeons had a long history on multiple schedules of reinforcement (e.g., FR, FI) prior to being submitted to an operation which caused marked loss of brain tissue. When tested again, the FI performance was greatly disrupted and it was reported that the over-all response rate was low and many intervals showed negative curvature. There was an occasional brief emergence of the terminal rate during portions of the interval but for some pigeons there was a decline in rate before the end of the trials.
Branch and Gollub (1974) used FI schedules of reinforcement to analyze the effects of d- amphetamine (DA agonist) on 9 male White Carneaux pigeons. Three of the pigeons were exposed to an FI 40 s, another 3 on an FI 100 s, and the last 3 on an FI 300 s schedule. The two chambers had an active illuminated center key and a rectangular opening through which a 3 s access to mixed grain, used as the reinforcer, was made available. After the FI 40 s group finished 92 sessions, the FI 100 s group 89 sessions, and the FI 300 s group 190 sessions, d- amphetamine was administered. Researchers injected .5 ml of d-amphetamine into the pectoral muscles of the pigeons 30 min before a session. The results indicated that d-amphetamine reduced the Index of Curvature compared to control amounts, indicating that the scallop became less shallow during the drug condition compared to the non-drug condition. That is, d-
43
amphetamine increased the average response rates early in each interval more than it increased
rates later in the interval.
Peak Interval Procedure. The peak procedure was first described by Catania (1970) as
another way to study temporal control under FI schedules. Catania (1970) stated that with an FI,
only half of the temporal gradient is being observed. With the Peak Interval Procedure (PI),
there is an additional property of time that is added to the interval and behavior is examined with
respect to this addition. The peak procedure exposes the organism to 2 types of cycles. Standard
trials are the same as the cycles in the FI procedure. Peak trials have the same discriminative
stimulus as the FI but responding does not produce reinforcement nor does it terminate the
stimulus that remains on for several times the length of the target duration (MacInnis, 2006). In
a typical session, the peak trials, also known as reinforcement omission trials, probe trials, or
blank trials occur randomly and only a fraction of the time (i.e., 25% of all trials). This method
allows for the assessment of responding that occurs beyond the usual time of reinforcement. By
examining the rate of responding that occurs after the “expected” time of reinforcement, we can better study behavioral gradients that may be evidence of control of behavior by the passage of time. Characteristic behavior on the PI is similar to the FI in that responding slowly increases as
time to reinforcement increases and decreases or stops after the “expected” time of reinforcement
(stop time).
Research using the peak procedure has shown that the schedule can be useful in measuring
the basic temporal properties of behavior (Church, Meck & Gibbon, 1994; Matell & Portugal,
2007; Roberts, 1981). The PI procedure has also been used to study the behavior of different
mouse strains (Carvalho, Silva & Balleine, 2001; Gallistel, King, Gallistel & McDonald, 2001;
King & McDonald, 2004; Meck, 2001), the effects of certain drugs on timing responses of
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various organisms (Maricq, Roberts & Church, 1981; Meck, 1996; Taylor, Horvitz & Balsam,
2007), and the examination of how brain lesions can systematically alter “timing” behavior
(Dietrich & Allen, 1998; Lewis, Miall, Dann & Kacelnik, 2003; Olton, 1989; Morrissey, Ho,
Wogar, Bradshaw & Szabadi, 1994). More specifically, important to the study of schizophrenic
models, it should be noted that the PI procedure has proven useful at detecting behavioral differences induced by drugs that are associated with the induction of schizophrenic symptoms.
Those drugs associated with the reduction of schizophrenic symptoms have also affected peak interval performance.
The PI procedure has been established as a procedure that can detect behavioral differences in mutant mice. Carvalho, Silva and Balleine (2001) examined mice with a threonine286 to alanine
(T286A) mutation in a subunit of Ca2+/CaM-dependent protein kinase II (α−CaMKIIT286A)
using the peak procedure. Carvalho et al. (2001) reported that α−CaMKIIT286A is thought to
play an important role in long-term potentiation and other forms of synaptic plasticity. The
researchers tested 8 wild type (WT) normals and 9 homozygous (T286A) mice in 8 operant
chambers using one retractable lever with food pellets as the reinforcer. Mice were trained for
30 sessions on an FI 40 s PI 80 s schedule in which 40 of the 50 trials were FI 40 s schedules and
the remaining trials were PI 80 s trials. Trials were separated with a variable 45 s intertrial
interval. For the next 30 sessions, mice were trained on an FI 20 s PI 40 s schedule of
reinforcement. Results based on percentage of response rate by time indicated that T286A mice
responded at a higher rate early in both of the temporal conditions (FI 40 s PI 80 s and FI 20 s PI
40 s) and continued to respond more after the usual time of reinforcement, compared to the wild type controls. T286A mice showed less precision in their timing of reinforcement delivery than the wild types.
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Abner, Edwards, Douglas and Brunner (2001) used the PI procedure to examine the basic
timing behaviors in two strains of mice C57 (N=13) and C3H (N=47) and their response to
psychoactive substances. Subjects were kept at 85-90% free feed weight and housed
individually. Researchers used 16 operant mouse chambers with only the right lever available
and condensed milk as the reinforcer. Mice were trained on an FI 30 s PI 120 s schedule of
reinforcement. After training to criterion they were exposed to testing with drugs. Drug testing took place twice a week with non-drug training during the remaining 3 days. Mice were given scopolamine (an anticholinergic drug), physostigmine (a cholinesterase agonist), methylphenidate (a norepinephrine and dopamine reuptake inhibitor), and d-amphetamine (a dopamine, serotonin, and norepinephrine reuptake inhibitor). Results based on the examination of data obtained from further testing on the FI 30 s PI 120 s indicated that scopolamine and high doses of d-amphetamine (4 mg/kg) affected performance by increasing response variability compared to non-drug training data. That is, the drugs impaired performance as seen in the flattening of the response curve before and after reinforcement and in the increase of variable start and stop times. Physostigmine reduced variability in the time to stop responding, indicative of better temporal precision compared to non-drug training. Methylphenidate slightly shifted responding to the left of the PI functions, compared to non-drug training.
Balci, Ludvig, Gibson, Allen, Frank, Kapustinski, Felolak, and Brunner (2008) investigated
87 C3H mice in operant chambers with lever bars (only right lever was active), using 2 s access to evaporated milk as the reinforcer. Mice were trained on an FI 20 s PI 80 s (+/- 20 s) or an FI
30 s PI 90 s schedule of reinforcement. The mice received five drug compounds that Balci et al.
(2008) described as either “cognitive enhancers” (physostigmine and atomoxetine
(norepinephrine reuptake inhibitor)), “cognitive disruptors” (scopolamine and Chlordiazepoxide,
46
aka CDP, a benzodiazepine (a Ca2+ channel blocker)) or DA agonists (d-amphetamine and methamphetamine). All drugs were given 30 min prior to testing. Mice were run Mondays through Fridays but drugs were given only on Tuesdays and Fridays to allow for a wash-out period and a return to baseline before the next drug administration. One mg/kg of physostigmine was given to 27 mice that ran on the FI 30 s PI 90 s. Results, compared to baseline, indicated that physostigmine decreased the stop time of these mice. Balci et al., (2008) defined stop time as the mean of the trial times in which response rate reached 60%, 70%, and 80% of maximum response rate, after the peak time. Peak time was defined as the mean of trial times in which the response rate was over 90% of the maximum response rate. Atomoxetine was given to 21 mice in doses of 1, 3, and 10 mg/kg and these mice were run on the FI 20 s PI 80 s schedule.
Atomoxetine decreased the spread of the peak function in a dose dependent manner, compared to baseline data. The spread of the peak was defined by Balci et al., (2003) as the time between the average of the trial times in which response rate reached 60%, 70%, and 80% before the peak and the mean of those trial times after the peak. Scopolamine was given to 29 mice at 1 mg/kg dose and those mice were run on the FI 30 s PI 90 s schedule. Scopolamine increased the spread of the PI function and increased the stop time, compared to baseline data. CDP was given in 10 and 15 mg/kg doses to 48 mice that were run on the FI 30 s PI 90 s schedule. Like scopolamine,
CDP increased the spread of the PI functions. D-Amphetamine was given to 18 mice in two doses, .3 and 3 mg/kg. These mice were run on the FI 20 s PI 80 s schedule and results indicated that d-amphetamine increased the spread and stop times of the PI functions, compared to baseline data. Methamphetamine was given by 1 mg/kg to 27 mice that were run on the FI 30 s
PI 90 s schedule. Methamphetamine significantly increased the spread, the peak time, and the
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stop times of the PI temporal function compared to baseline data, mostly due to the fact that the animals did not slow their responding after the usual time reinforcement was made available.
Matell, Bateson and Meck (2006) evaluated the effect of 5 doses of methamphetamine on the behavior of 20 male Sprague-Dawley rats using the PI procedure. Researchers used 10 operant chambers, each equipped with a lever and a food magazine that provided reinforcers (food pellets). All doses of methamphetamine (.5, .75, 1.0, 1.25, and 1.50) were given 15 min prior to the start of each session in a pseudorandom order for a total of 3 times. Each dose was separated by two sessions of saline injections. Rats were trained on an FI 30 s PI 90-110 s schedule of reinforcement. The PI probe values in 90 to 110 s were randomly selected with replacement.
All trials were separated with a variable 55s intertrial interval that ranged from 30 to 80 s.
Results indicated that methamphetamine produced a dose dependent decrease in peak time resulting in a leftward shift in the horizontal PI function, compared to non-drug data. There was also a dose dependent decrease in peak rate (the rate of responding at peak time), compared to non-drug data.
Maricq et al. (1981) examined 10 albino rats on the peak procedure on two conditions (5 rats in each condition). One condition involved an FI 20 s PI 80 s; the other involved an FI 40 s PI
80 s. Each conditional group received 1.9 mg/kg methamphetamine administration approximately every 5 sessions which accounted for about 20% of the total sessions. Using operant chambers, Maricq et al. (1981) used the nose poke as the operant response. The results indicated that methamphetamine reduced the peak time for both conditions, compared to non- drug data. That is, on sessions when rats were given methamphetamine, their highest rate of responding occurred sooner in time than sessions with no drug administration.
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Methamphetamine also increased the overall response rate. Visual examination of rate of
responding over time showed a greater spread of responding during drug administration.
Taylor et al. (2007) tested 8 male albino Sprague-Dawley rats in operant chambers using the
lever press as the operant response. Rats were trained on an FI 24 s PI 96 s for 20 sessions
before the administration of d-Amphetamine. For 4 consecutive days, rats received .5 or 1.0
mg/kg/ml of amphetamine injections followed by 5 days of no-drug training then followed by 4
consecutive days of training with the other drug dose not previously given. Results indicated
that both doses of drugs significantly increased the rate of responding compared to non-drug
days. Also, both drug doses significantly decreased the time where the peak responding
occurred. There were no significant differences in measures of behavior between the two doses.
Cheng, Ali, and Meck (2007a) used the peak procedure to examine the effects of cocaine
(DA agonist), ketamine (NMDA antagonist), and varied durations of baseline training. Cheng et
al. (2007) were attempting to study how conditioned responding does or does not change based
on drug administration and extended training. Experiments involved 32 male Sprague-Dawley
rats that were split into two groups, one that received less than or equal to 30 sessions training on an FI 36 s PI 90 s schedule of reinforcement and one that received greater than or equal to 180 sessions before drugs were administered. Operant chambers were used with only the left lever available and food pellets were used as the reinforcer. Cocaine was administered to compare the effects of training on the expected leftward shift of the PI function. It was hypothesized that with more training, cocaine would lose its effect on the location of the peak but when ketamine is combined with cocaine, the usual effects of cocaine (leftward shifts) would be restored. Eight of the rats received minimal baseline training on an FI 36 s (5 sessions) and an FI 36 s PI 90 s (25 sessions). The intermediate group consisted of 8 rats that were trained for 72 sessions on an FI
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36 s PI 90 s. In order to create a baseline of extended PI training prior to drug testing, the extended group (N=8) consisted of an FI 18 s (15 sessions), FI 18 s PI 90 s (50 sessions), FI 36 s
PI 90 s (50 sessions), FI 72 s PI 90 s (36 sessions), and finally FI 36 s PI 90 s (29 sessions). Rats were given 15 mg/kg injections of cocaine, 10 and 20 mg/kg injections of ketamine, a cocaine ketamine cocktail, .12 mg/kg injections of haloperidol (DA antagonist), and a haloperidol (.12 mg/kg) ketamine (10 mg/kg) cocktail. All haloperidol injections were given to the intermediate group. There were at least 3 days in between drug sessions where rats received saline. The results of this study confirmed the researcher’s hypothesis in that cocaine failed to produce a leftward shift in the PI function following extended training. Cocaine produced the usual leftward shift in peak for rats that received minimal training, compared to baseline. One of the most interesting findings was detected under the cocaine ketamine cocktail condition. This condition was able to restore the effects of cocaine, seen in the leftwards shift of the PI function, for the extended group. Also, the haloperidol ketamine cocktail did not reduce haloperidol’s effects of producing a rightward shift in the peak following the intermediate training.
Cheng, Hakak, and Meck (2007b) examined the effects of methamphetamine and length of baseline training on 48 Sprague-Dawley rats. Experiments were conducted in eight operant chambers using only the left lever. Precision food pellets served as the reinforcer. Three different groups of 16 rats served in each condition. Each group varied in the number of FI 50 s training sessions. One group received 5 (minimal), the other 10 (intermediate) and the third group received 20 (extended) training sessions. After training on the FI 50 s, all rats were introduced to the PI baseline training. With the PI training, half of the trials were extended out to at least 150 s plus an additional duration that had a mean of 20 s, ranging from 1.1 to 70.4 s.
The minimal group received 15 sessions training on the PI, the intermediate group received 50
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sessions and the extended group had 100 sessions. After FI and PI training, the rats were given
methamphetamine in doses of .5 mg/kg and 1.0 mg/kg. Each injection session was separated by
3 days off the drug. The results indicated that the amount of baseline training can alter the
effects of methamphetamine on timing behavior. Researchers found that with more baseline
training (extended) methamphetamine produced less of a leftward shift in responding compared to the groups with less baseline training (minimal and intermediate). However, it did create less precision based on peak analysis of response rate as a function of time. That is, bigger doses of
methamphetamine led to higher rates of responding after the time of usual reinforcement
compared to controls, regardless of training.
Meck (2006) examined 60 Sprague-Dawley rats in 10 operant chambers using food as the reinforcer and the lever press as the operant response on an FI 40 s PI 130 s schedule of reinforcement. Each trial type had an equal probability of occurring in the session. The rats were split into 6 groups of 10. The purpose of the experiment was to evaluate the effects of dopamine
(DA) agonist methamphetamine (METH) and DA antagonists haloperidol (HAL) and sulfated cholecystokinin octapeptide (CCK-8S). These drugs were used on rats with lesions of the cholinergic cell bodies in the medial septal area (MSA), excitotoxic lesions of the nucleus basalis magnocellularis (NBM), radio-frequency lesions of the fimbria-fornix (FFx), and aspiration lesions of the frontal cortex (FC). Each lesioned groups consisted of 10 rats (N=40). The other
20 rats received an array of sham lesions that did not result in significant brain damage and were considered the control group (CON). There was a rightward shift in the horizontal placement of the timing function in the FC and NBM groups compared to the CON group based on results from the postoperative peak-interval training. The FFx and MSA groups did not differ significantly in peak times from the CON group. The most interesting results were taken from
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the investigation of the dopaminergic drugs (METH, HAL, and CCK-8S) on all lesioned subjects. The sham, FFx, and MSA lesioned groups produced a dose dependent rightward shift in PI functions when tested under the influence of DA antagonists (HAL and CCK-8S) and a
dose dependent leftward shift in PI functions for the DA agonist (METH), as would be expected.
However, these drugs produced no change in the horizontal placement of PI functions in either
FC or NBM lesioned groups. These results suggests that behavioral alterations caused by lesions in the FC and NBM are relatively permanent and not as easily altered by dopaminergic drugs as the behavioral alterations caused by lesions in the FFx and MSA regions of the brain.
Lewis et al. (2003) examined 14 C57 house mice using operant chambers and a lever press as the operant response for reinforcement (.3 ml portions of condensed milk). Mice were trained on a modified peak procedure where they received reinforcement for responding between 10 and 14 s and unreinforced probe trials lasted 45 s (FI 10-14 s PI 45 s). Peak trials or probes compromised 20% of the total trials. All mice received lesions in the suprachiasmatic nucleus
(SCN) of the hypothalamus and were again subjected to the timing procedure. The suprachiasmatic nucleus is thought to be responsible for controlling endogenous circadian rhythms. The lesions did not lead to any significant changes in peak performance.
Dietrich and Allen (1998) were interested in examining the role the medial prefrontal cortex and the hippocampus play in the acquisition of learning a timing task. These researchers trained
18 Wistar rats kept at 85% free-feed weight in operant chamber to lever press. The 18 mice were separated into 3 groups: the prefrontal cortex group (PFC), the hippocampus group (HIP), and the control group (CON). The six mice in the PFC group received a lesion in the prefrontal cortex. The six mice in the HIP group received a lesion in the hippocampus. Three mice from the CON group received a sham lesion in the prefrontal cortex area with no cut tissue and the
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other three CON subjects received lesions in the parietal cortex but not the hippocampus. Rats were then trained on an FI 40 s PI 130 s schedule of reinforcement in which each condition had the same probability of occurring. Results indicated that the HIP group did not differ in any measure from the CON group. However, the PFC group did differ significantly from the CON and HIP group in acquisition of learning the task. That is, the PFC group took longer to reach criterion. The PFC group also had a less steep and reduced uniform temporal function, compared to the CON and HIP group, indicating less schedule control of behavior in the PFC group.
Olton (1989) examined the effects of lesions of the frontal cortex (FC) and lesions of the nucleus basalis magnocellularis (NBM) on rats (the type and number were not given). Rats were tested in standard operant chambers with one active lever available and one food pellet served as the reinforcer. Rats were trained to press the lever press on a discrete trial signaled FI schedule in which a stimulus, either auditory or visual, would indicate a short (FI 10 s) or long (FI 20 s) schedule. Peak trials extended the signal for 130 s. The FC lesions produced a rightward shift of the peak time compared to the sham lesioned control group. NBM lesions also produced a rightward shift in the peak time. However, after 4 weeks of training, the peak time of the NBM group returned to normal. There was no recovery of the peak time in the FC lesioned group.
Morrissey et al. (1994) investigated the role of the serotonergic pathway in the timing behavior of rats by giving an experimental group (N=12) a lesion in the serotonergic pathway and a control group (N=12) a sham lesion. The experimental group (lesioned 5HT rats) showed a significant reduction in serotonin yet levels of noradrenaline and dopamine were not significantly affected. Rats were trained on an FI 40 s PI 120 s for 60 sessions. Here, half of the trials were baited (FI 40 s) while the other half ended without the presentation of a reinforcer (PI
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120 s). Results indicated that rats with lesions in the serotonergic pathway behaved significantly different than controls on this assay. The response function of the lesioned group showed that they had a significant broader spread than controls. Spread was defined by these researchers as time between the point where 70% of peak rate of responding occurred before and after the usual time of reinforcement.
Summary of literature on FI and PI schedules. In summary, compared to no drug conditions,
DA agonist d-amphetamine caused a significant increase in responding earlier in the interval than later in the interval and significantly lowered IOC values (Branch & Gollub, 1974). On PI schedules compared to no drug conditions, d-amphetamine flattened the response curve before and after the usual time reinforcement was made available (Abner et al., 2001; Balci et al.,
2008), increased the variability of start and stop times (Abner et al., 2001), significantly increased the rate of responding throughout the interval (Taylor et al., 2007), and significantly decreased the peak time, which graphically looked like a leftward shift in the temporal gradient
(Taylor et al., 2007).
The dopamine agonist methamphetamine significantly increased the spread (Balci et al.,
2008; Cheng et al., 2007b; Maricq et al., 1981), increased the peak time due to increased spread
(Balci et al., 2008), and significantly increases the stop times of the PI temporal function mostly due to significantly higher rates of responding later in the interval (Balci et al., 2008).
Methamphetamine significantly decreased peak time resulting in a leftward shift in the horizontal
PI function (Maricq et al., 1981; Matell et al., 2006; Meck, 2006). A significant overall increase in response rate was reported (Maricq et al., 1981). A dose dependent decrease in peak rate (the rate of responding at peak time) was also reported (Matell et al., 2006).
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Dopamine agonists methylphenidate (Abner, 2001) and cocaine (Cheng et al., 2007a) were shown to significantly reduce the peak time, shifting the PI temporal response pattern to the left.
Atomoxetine, a norepinephrine agonist, decreased the spread of the peak function, indicating better temporal control of behavior (Balci et al., 2008). Dopamine antagonists haloperidol
(Cheng et al., 2007a; Meck 2006) and CCK-8S (Meck, 2006) produced a rightward shift in the
PI temporal response gradient. CDP, a benzodiazepine (Ca2+ channel blocker) caused an increase in peak spread, indicative of less temporal control of behavior (Balci et al., 2008).
Cholinesterase agonist physostigmine caused significantly more precise peaks around the usual time of reinforcement with less spread variability than controls (Abner et al., 2001). In contrast, anticholinergic drug scopolamine significantly increased the variability of start and stop times and increased the peak spread (less temporal precision) (Abner et al., 2001).
Lesions in particular areas of the brain resulted in interesting findings based on temporal control of behavior. Aspiration lesions to the frontal cortex (Meck, 2006, Olton et al., 1989) nucleus basalis magnocellularis (Olton et al., 1989), and excitotoxic lesions of the nucleus basalis magnocellularis (Meck, 2006) produced a rightward shift in responding on the temporal gradient of the PI schedule. Lesions of the prefrontal cortex led to longer acquisitions of task learning and less steep and reduced uniform peaks compared to controls (Dietrich & Allen,
1998). Lesions to the serotonergic pathway caused a significant broader spread than controls
(Morrissey et al., 1994). Lesions of the cholinergic cell bodies in the medial septal area and radio-frequency lesions of the fimbria-fornix did not produce any behavioral abnormalities compared to controls on the measure of PI temporal responding (Meck, 2006). Lesions in the suprachiasmatic nucleus did not lead to any behavioral differences based on measures of PI
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performance (Lewis et al., 2003). Lesions to the hippocampus did not lead to any detectable differences on measures of the PI procedure (Dietrich & Allen, 1998).
Based on the review of literature on FI and PI schedules, it is believed that these two procedures are valuable at detecting behavioral differences associated with a subject’s neurobiological make-up. The current study sought to investigate whether or not these two temporal assays could detect any behavioral differences between Reln deficient mice and
controls. It was hypothesized that if +/rl mice were affected by higher levels of dopamine or
serotonin, they would demonstrate a leftward shift in temporal responding on the temporal
gradients of the FI and PI, as shown by other subjects with increased levels of these
neurotransmitters (Abner, 2001; Balci et al., 2008; Cheng et al., 2007a; Maricq et al., 1981;
Matell et al., 2006; Meck, 2006; Taylor et al., 2007). In order to examine for this effect, the end
of training relative frequency values and IOC values for each genotype were examined. It was
expected that +/+ mice would have higher relative frequency values, compared to +/rl and rl/rl
mice, toward the end of the interval when reinforcement was made available. It was also
expected that +/+ mice would have higher IOC values, compared to +/rl and rl/rl mice, by the
end of FI training. Higher IOC values are indicative of a deeper curve with respect to the FI
scallop, signifying better temporal control of behavior. On the PI, a leftward shift in temporal
responding, expected of the +/rl and rl/rl mice, would result in an earlier peak time/location
based on analysis of relative frequency for the whole PI function. Also, independent IOC values
for the 3 equal 40 s time intervals that make up the full PI interval (discussed in more detail
under ‘data analyses’) might reveal a difference in values between the three genotypes. It would
be expected that similar to IOC values for the FI, IOC values for the first PI interval (1-40 s)
would be lower for the +/rl and rl/rl mice than the +/+ mice.
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Instead of a leftward shift it was also hypothesized that +/rl and rl/rl may show a rightward
shift in temporal responding on the temporal gradient. This was expected due to the results
suggested by some that Reln deficient mice have lower levels of dopamine and serotonin in
certain parts of their brains. Other researchers have shown that certain animal given dopamine
and serotonin antagonists, exhibit rightward shifts in temporal responding on temporal gradients,
compared to controls (Cheng et al., 2007a; Meck 2006; Olton et al., 1989). If this is the case, we
might see that the +/rl and rl/rl mice perform normally or even better than +/+ on the measures
of FI performance. That is, if these Reln deficient mice allocate the majority of their responding
until later in time, they may have higher IOC values than +/+ mice and higher relative frequency
values, closer to the time a reinforcer is made available. However on the PI procedure, a
rightward shift in temporal responding by +/rl and rl/rl mice may result in later peak times and possibly longer spreads than +/+, indicating less accuracy (peak responding around the time a reinforcer is usually made available) and less precision (peak spread around the time a reinforcer is usually made available) of behavior that conforms to the temporal stimuli of the environment.
Another possible outcome was that +/rl and rl/rl mice may show a flatter temporal spread, indicative of a less defined peak of temporal gradient, resulting from decreased control of behavior by the passage of time, similar to the behavioral effects of other subjects affected by different manipulations to their neurochemistry (Abner et al., 2001; Balci et al., 2008; Cheng et al., 2007a; Cheng et al., 2007b; Dietrich & Allen, 1998; Maricq et al., 1981, Meck 2006; &
Morrissey et al., 1994). This effect will first be evaluated on the FI. If +/rl and rl/rl mice show evidence of less control of behavior by time than +/+, it may be demonstrated by the relative frequency of responding and IOC values. This may be seen in higher relative frequencies of responding early in the interval or steady rates of responding throughout the interval by +/rl and
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rl/rl mice, compared to +/+ mice. Therefore, +/+ mice would be expected to have higher IOC values on the FI and higher relative frequency values closer to the time a reinforcer is made available, than +/rl and rl/rl mice. A flatter temporal spread on the PI function, by +/rl and rl/rl mice compared to +/+ mice, would resemble a less defined peak, in that responding would remain steady without the typical rise and fall of peak time. The graph of relative frequency might show that when compared to +/+ mice, +/rl and rl/rl mice might respond earlier in the interval, before the time a reinforcer is usually made available, and continue to respond more after that time. It is also possible that +/rl and rl/rl mice will respond similarly to +/+ mice up to the time a reinforcer is usually made available but not slow their responding after that time, as much as +/+ mice. This would indicate a deficit in control of behavior by time for the second part of the temporal gradient. This can be examined further by looking at the IOC values for the second PI interval (41 -80 s). The +/+ mice would be expected to have higher negative IOC values for the second PI interval (41-80 s) than +/rl and rl/rl mice. That is, +/+ mice would be expected to slow their responding sooner after the usual time a reinforcer is usually made available, indicative of better temporal control of behavior.
Lastly, even though +/rl and rl/rl mice are known to have abnormal physiological make-ups they may not be significantly different from +/+ mice based on measures of temporal control, similar to others who have reported no differences between Reln deficient mice and +/+ mice
(Krueger et al., 2006; Podhorna and Didriken, 2004; Qiu et al., 2006; Salinger et al., 2003).
If temporal control of behavior truly is affected by Reln levels, then we would expect a Reln dosage effect on behavior. That is, since +/rl mice express less Reln than +/+ mice, they would be expected to perform less well on these temporal tasks, than +/+ mice. In turn, rl/rl mice expressing no Reln, should perform less well than both +/+ and +/r mice.
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Implications of the findings of this study might suggest that theses assays are useful tools for detecting subtle differences related to the neurochemical make-up of subjects demonstrated by behavior that conforms to the passage of time. If these procedures prove useful at detecting behavioral abnormalities associates with Reln deficiency, they may also prove useful at detecting any differences caused by pharmacological drugs that affect the behavior of Reln deficient mice on temporal assays.
METHODS
Subjects
Twenty four experimentally naïve adult mice (background strain: B6C3Fe a/a-, Jackson
Laboratories, Bar Harbor, ME) bred and genotyped at UNCG animal colony were used in this study. Mouse genotype was later confirmed by Dr. Shafer’s biology lab at UNCW, using polymerase chain reaction analysis (D’Arcangelo, Miao, & Curran, 1996). The mice had a mean age of 428 days at the beginning of training. The 24 subjects consisted of three genotypes.
There were 8 +/+ (4 males, 4 females), 8 +/rl (4 males, 4 females), and 8 rl/rl (4 males, 4 females). Mice were housed individually with a 12 hr light: dark cycle (lights on at 7:00 a.m.).
The mice had ad libitum access to drinking water in their caging unit, but were kept on restricted feeding schedules to maintain their body weights at 85% of free feeding weight. Lab Diet 5P00 pellets were provided 15 min following the daily session. All behavioral testing was conducted during the light phase. Each mouse was run in the same chamber during approximately the same time daily, 5 days per week. The procedures for all paradigms were approved by the Institutional
Animal Care and Use Committee at the University of North Carolina Wilmington.
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Apparatus
Four standard operant mouse chambers (Med Associates), each measuring 24 cm x 18 cm x
20 cm were used in this study. The chambers were equipped with 2 retractable response levers
located on the bottom right side wall. One lever was to the right of the food trough and the other
was to the left. The left lever was the only lever inserted into the box for all procedures. The
response lever required 5 grams of force to operate. A dispenser for delivering food pellets was
situated on the outside of the chamber, attached to the right side wall. The pellet dispenser
delivered 14 mg BioServ pellets to the food trough according to the schedule of reinforcement in
effect. The food trough was located on the right wall centered between the right and left levers.
The water bottle (used only during the first part of FI training) was situated outside the left side
of the chamber and was inserted into the chamber, by a computer controlled system, at the
beginning of the session. A house light was positioned in the rear top left side of the chamber,
away from the hopper and lever. The front and back sides of the chamber were ventilated
Plexiglas. The right and left side walls and ceiling were aluminum. The floor was composed of
24 stainless steel bars spaced approximately 1 cm apart. Each chamber was enclosed within a
sound-attenuating cubicle equipped with an exhaust fan that provided air ventilation and white noise. The experimental sessions were conducted under low room light and quiet conditions.
Programmed procedures and data collection were computer controlled using MedPC software.
Behavioral Procedures
Magazine training. Mice were adapted to the operant chamber and given 3 sessions of magazine training. With this protocol, mice were placed into the operant chamber with the ventilation fan already running and once the researcher started the program the house light came on, the water bottle was inserted into the chamber and pellets were dropped into the hopper (food
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trough) on a variable time schedule of 60 s (VT 60 s). On the VT 60 s, pellets were dropped
(noncontingent to the animal’s behavior) into the hopper at random times that all average to 60 s
for a total of 42 pellets per session. For example, one pellet might drop 1 s from the last pellet
and another might drop 60 s from the last pellet. No response by the mouse was required. At the
end of the session, the water bottle retracted, the pellets stopped dropping and the house light
turned off.
Fixed ratio 1 with shaping. A fixed ratio 1 (FR 1) schedule of reinforcement was presented to
all mice. This schedule required 1 operant response to receive reinforcement. Once mice were in
the chamber, the same environmental stimuli as in the magazine training protocol were presented
but this time the lever was also inserted into the chamber. The lever did not retract at any point
except at the end of the session which also involved the retraction of the bottle and extinguishing of the house light. During the session, every lever press led to reinforcement until the mice received all 49 pellets or 45 min passed, whichever came first. The mice in the current experiment were given 1 session of FR 1 with no shaping. After the first session, researchers
intervened to shape lever pressing for all mice that had not pressed the lever on their own. The
mice in this experiment required 16 sessions of FR 1 lever-press training with shaping. Shaping
involved reinforcing (releasing a pellet into the trough) the mouse’s physical approximations to
the lever until the first lever press occurred. After the mouse had emitted the first lever press,
shaping ended but FR 1 training continued until all mice successfully lever pressed to receive all
possible reinforcers in one session.
During FR 1 training, 1 female rl/rl mouse died unexpectanted. The death appeared to be
from natural causes as the weight and appearance of the mouse was typical under those
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circumstances. The remaining 23 mice [8 +/+ (4 males, 4 females), 8 +/rl (4 males, 4 females) and 7 rl/rl (4 males, 3 females)] carried on to the next protocol.
Variable-interval 10 s. To introduce the mice to a leaner schedule, they were placed on variable interval (VI) schedules of reinforcement. On VI schedules, lever presses are reinforced at variable times (based on an average time) throughout the session so that one lever press may result in a reinforcer right after the last reinforcer or it may not be made available for some time longer. The VI produces a steady state of responding because the time that reinforcement is made available varies so any one response at any time could lead to reinforcement. The mice in this study received 1 session of VI 10 s, and 8 sessions of VI 20 s. The time intervals that allowed reinforcement to be made available, for each VI schedule, were determined based upon an exponential distribution of intervals (Fleshler & Hoffman, 1962). The VI 10 s had VI schedule values of .5, 1, 2, 3, 5, 6, 8, 10, 12, 16, 21, and 36 s, without replacement. The VI 20 s had VI schedule values of 1, 3, 5, 7, 9, 12, 16, 20, 25, 32, 42, and 68 s, without replacement. All sessions for both VI schedules started with the illumination of the house light, the insertion of the water bottle, and the addition of the left lever. Sessions ended after the mice received 36 pellets.
Fixed-interval procedures. A FI 40 s schedule of reinforcement was introduced to the mice to assess temporal control of behavior as it conforms to the contingencies of the FI schedule. The same environmental stimuli (house light, water bottle, and lever) used during VI training were also used with this procedure for the first 55 sessions. With the FI 40 s, the mice were free to lever press throughout the entire session but only the first lever press after 40 s passed, since the last reinforcer, led to reinforcement. Thirty-six reinforcers were made available in each session.
Each session lasted approximately 45 min. After the 55th session, the water bottles were taken
out of the operant chambers and the mice were run for 15 more sessions (total of 70 sessions).
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Water was no longer made available in order to avoid the possible development of schedule- induced polydipsia. Schedule-induced polydipsia is a form of adjunctive behavior that is represented by excessive intake of water when animals are reinforced with food on intermittent schedules of reinforcement (Falk, 1961). For all remaining procedures, the water was not available during experimental sessions. Before the end of FI 40 s training, two more mice died.
One mouse (+/rl male) was euthanized for an eye tumor and the other mouse (+/+ male) died of apparent natural causes. The remaining 21 mice [7 +/+ (3 males, 4 females), 7 +/rl (3 males, 4 females) and 7 rl/rl (4 males, 3 females)] carried on to the next protocol.
Peak-trial procedures. The peak procedure was introduced after all mice had 70 sessions experience on the FI 40 s. With this procedure, the session began with the illumination of the house light and the insertion of the left lever. Two trial types were presented in quasi-random order; trials were separated by an intertrial interval of 15 s. During the intertrial interval, the house light turned off and the lever retracted for the whole duration. After the intertrial interval each trial began with the illumination of the house light and the insertion of the lever. The first trial type was the FI 40 s schedule of reinforcement. The second trial type was the actual peak trial and involved the omission of the pellet after 40 s had passed and the extension of the interval for an additional 80 s, totaling 120 s. These peak trials ended with no reinforcement.
Trials were set up so that 1 out of every 4 trials was a peak trial. Therefore there could be two peak trials in a row, but never more than two. There were a total of 25 trials per session, and the mice in this project were given 70 sessions training on this procedure. If the mice did not lever press to receive all scheduled reinforcers, the session would end after 60 min. Two more mice died before the end of PI training, a female +/rl was euthanized due to a tumor on its chest and a male +/rl mouse died of apparent natural causes.
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Data Analysis
FI 40 s. All data analyzed for the FI 40 s schedule refer to those sessions that occurred before
the introduction of the FI 40s PI 120 s schedule. FI responses for each mouse were grouped in
10 consecutive and equal 4 s time bins, for each session. As a way of comparing data across
genotypes independent of response rate, relative frequency of responding for each of the 10 time
bins was computed by dividing the total responses in any one bin by the total number of
responses that occurred throughout the entire session. Polynomial extraction with sessions
(mean of Sessions 1-5 and mean of Sessions 50-55) and genotypes entered as factors were used to determine whether or not there were training effects under the FI schedule. Sex was controlled for in that each of the mouse groups was originally composed of equal numbers of male and female mice, but sex was not a focus in this experiment. Age was controlled for in that all mice were approximately the same age and considered “old”. Therefore age and sex were not entered as factors in any statistical analyses.
Cumulative relative frequency was computed across each of the 4-s time bins for each FI 40 s session and was used to compute the index of curvature (IOC). The IOC is a quantitative measure of the degree of curvature (scallop) on a graph of cumulative frequency of responding during the FI (Fry, Kelleher, & Cook, 1960). The IOC is calculated as 9 times the cumulative frequency of responses in the 10th bin (4 s each) in the FI, minus 2 times the sum of cumulative
relative frequencies of responses in the first 9 times bins, all divided by 10 times the cumulative
relative frequency of responses in the 10th bin. The following formula was used to determine
the IOC:
()(9 × R10 − ()2 × R1 + R2 + R3 + R4 + R5 + R6 + R7 + R8 + R9 )
10 × R10
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For this formula, R refers to responses in a particular bin and the number that follows R refers to the time bin from which responses were taken. The IOC is a measure of how far the actual curvature of the cumulative frequency of responses deviate from the hypothetical line created as if there is a constant rate of responding throughout the entire interval. This hypothetical line has an IOC of 0. Larger positive IOC values signify FI performance that is closer to the maximum possible value of 0.9 (max value based on 10 bins). Index of curvature values closer to 0.9 indicate that the mice have allocated most of their responding at the time where reinforcement is made available. Repeated measures ANOVA with genotype and sessions entered as factors were used to analyze training effects and to determine whether the mice had reached asymptote by the
55th session.
In order to make sense of the differences between +/rl and rl/rl from +/+ mice, the data from relative frequency and index of curvature, were reformulated to represent the difference between the two Reln deficient groups from the +/+ group by setting the +/+ mouse data to 100
% and comparing the rest based on this value. If the +/+ mouse data are set to 100% we can compare session by session and in 5 session averages how +/rl and rl/rl mice compare. If +/rl or rl/rl had a higher relative frequency or IOC than +/+, data would be scored higher than 100% and vice versa (less than 100%) if they scored lower.
PI 120 s. PI responses for each mouse were recorded in 1-s bins for a total of 120 s bins.
However, for much of the data analysis, responses were summed into 4 s bins, equaling 30, 4-s time bins for a total of 120 s. Relative frequency was calculated for the entire interval (30, 4-s time bins) to compare the beginning of PI training with the end of PI training. Relative frequency was also calculated in 40 s intervals (1-40 s, 41-80 s, and 81-120 s) and used to compare responding in time to and from the usual time that reinforcement is made available.
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Polynomial extraction, with sessions (mean of Sessions 1-5 and mean of Sessions 46-50) and genotypes entered as factors, were used to determine whether or not there were training effects under the PI schedule.
Maximum response rate in responses per second per session were analyzed for each mouse and presented in 5 session averages. Highest maximum response rates were compared between sessions and genotypes using polynomial extraction. Maximum response rate per second was of interest to determine any genotypic differences on this measure.
It was noted that all mice had multiple bins in any given session where the maximum number of responses occurred. Therefore, genotype averages of the number of bins with maximum responding were analyzed across PI training. Polynomial extraction, with 5 session averages and genotypes entered as factors, was used to determine whether the genotypes differed on this measure. Analyses using polynomial extraction with the first 5 sessions and last 5 sessions and genotypes entered as factors, was also conducted to determine whether the genotypes differed from each other, on this measure, during the beginning and/or end of PI training.
In order to evaluate the IOC on the peak interval procedure responses in each bin were summed into 4 s bins and calculated exactly as in the FI. However with the PI, 3 separate IOC values were calculated based on the three 40 s time intervals of the PI 120 s (1- 40s, 41-80s, and
81-120s). Since the IOC is a measure of curvature based on any given time interval, it should be an appropriate measure of PI performance across the three different time intervals. It would be expected that the first interval (1-40 s) IOC value should be comparable to the FI IOC value (as close as possible to an IOC value of 0.9). The second interval (41-80s) IOC value should be a negative number (as close as possible to an IOC value of -0.9) in that the majority of responding should take place during the beginning of the interval and decrease as time passes. It would be
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expected that the third time interval (81- 120 s) IOC values should involve little to no responding as that trial will never lead to reinforcement, which would equal an IOC value close to zero.
Polynomial extraction with sessions (mean of sessions 1-5 and mean of sessions 46-50), and genotypes entered as factors was used to determine any differences in IOC values across training.
The calculation of peak time and peak/response spread was calculated based on relative frequency of responding using the threshold method (Balci et al., 2008). The relative frequency for each mouse was recalculated based on the highest relative frequency point so that all other points were a percentage of the highest point (100%). The peak time was calculated as the average of trial times (bins) in which responding occurred over 90% of the maximum relative frequency of responding (Balci et al., 2008). Genotype averages were simply calculated based on the means of all the peak times of each genotype respectively.
Peak spread was calculated by finding the relative frequency of responding that reached 70% of the maximum relative frequency of responding both before and after the peak time. Peak spread was the time between the 70% of trial time before the peak time and the 70% of trial time after the peak time (Morrissey et al., 1994). Genotype averages were calculated based on the means of all peak spreads of each genotype respectively. In order to determine where on the time line the majority of the spread took place (before or after the peak time), the time between the first 70% of peak time and peak time was evaluated to determine any genotype differences.
The time from peak time to the second 70% of peak time (after peak time) was also evaluated.
Lastly, the difference between these two time intervals (first 70% versus second 70%) was evaluated to determine where on the time line the majority of the spread occurred (before or after the peak).
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RESULTS
Fixed Interval 40 s
The following analyses are based upon the 20 mice that completed both the FI through the
55th session and the PI procedure through the 50th session. Table 1 shows a summary of all the current findings of this research project based on the FI data.
Relative frequency. Relative frequency of responding was used to assess the interval times when the majority of responding occurred. Fixed interval performance represented by mean relative frequency of responding across 4 s time bins is shown for the early part of FI training
(the average of sessions 1-5) in Figure 1. During the first 5 sessions (Figure 1), when mice were new to the FI 40 s schedule, polynomial extraction with time bins (10, 4 s bins), and genotypes as factors revealed a significant Genotype x Linear effect, F(2, 17) = 5.8993, p = .0133. Least squares contrast found a significant difference between the relative frequencies of +/+ and rl/rl,
F(1, 17) = 10.3465, p = .0051, and a significant difference between +/rl and rl/rl, F(1, 17), =
6.7708, p = .0186. There were no significant differences in relative frequency between +/+ and
+/rl, F(1, 17) = .2385, p = .6316. Based on polynomial extraction analysis of relative frequency of responding during the beginning of training, a Genotype x Cubic effect was also found, F(2,
17) = 4.4323, p = .0282. Least squares contrast revealed a significant difference between the relative frequency values of +/+ and +/rl mice, F(1, 17), = 8.8375, p = .0282, but not between
+/+ and rl/rl, F(1, 17) = 1.6475, p = .2165, and not between +/rl and rl/rl, F(1, 17) = 3.0261, p =
.1000. No significant Genotype x Quadratic effect was found, F(2, 17) = 2.1277, p = .1497.
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Result of Results of Results of rl/rl Schedule of +/rl rl/rl compared to Reinforce- Time of compared to compared to +/rl ment Measure Training +/+ +/+ Fixed Relative Beginning * p = .0282 * p = .0051 * p = .0186 Interval Frequency (sessions 1- (C) (L) (L) 5)
Relative Beginning Frequency (sessions 1- expressed as 5) a % of +/+
Relative End frequency (sessions 51- 55)
Relative End frequency (sessions 51- expressed as 55) a % of +/+
IOC All Sessions (5 session averages)
IOC End * p = .0483 * p = .0050 expressed as (sessions 51- (L) (L) a % of +/+ 55) * p = .0053 * p = .0172 (C) (C)
Change in Beginning to * p = .0492 * p = .0065 IOC End (Training) (Training)
Asymptote Last 15 based on Sessions (5 IOC session averages)
Table 1. Results based on the FI 40 s procedure. Horizontal arrows indicate no significant difference. Significant differences are marked with an asterisk along with the p value from least squares contrast. L = Linear effect and C = cubic effect.
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Figure 2 depicts relative frequency for all mice as a function of comparison, where +/+ mice data were set as the standard (100%) to which both other strains were compared for the average of sessions 1-5. Polynomial extraction with the 10, 4 s time bins, and genotypes entered as factors did not reveal a Genotype x Linear effect, F(2, 17) = 3.0473, p = .0740. No significant
Genotype x Quadratic effect was discovered either, F(2, 17) = .3261, p = .7261, and no significant Genotype x Cubic effect was found, F(2, 17) = 3.3876, p = .0578.
The end of training (sessions 51-55), represented in Figure 3, shows that all genotypes exhibited the same pattern of responding based on relative frequency. All genotypes demonstrated the “scalloping” pattern of responding typical to the FI schedule (Ferster &
Skinner, 1957; Lattal, 1991). Polynomial extraction with time bins (10, 4 s bins), and genotypes entered as factors revealed no significant Genotype x Linear effect, F(2, 17) = 1.1613, p = .3367.
No significant Genotype x Quadratic effect was found, F(2, 17) = .9402, p = .4099, and no significant Genotype x Cubic effect, F(2, 17) = 1.4960, p = .2521.
Figure 4 represents comparison data for the average of sessions 51-55 with +/rl and rl/rl mouse data expressed as a percentage of relative frequencies of responding produced by +/+ mice. The +/+ mouse data was set as the standard (100%) by which the other two strains were compared. Polynomial extraction with the 10, 4 s time bins, and genotypes entered as factors did not reveal any significant differences between the genotypes when data was organized in this way. There was no Genotype x Linear effect, F(2, 17) = 1.1390, p = .3434; no Genotype x
Quadratic effect, F(2, 17) = .2438, p = .7863; and there was no Genotype x Cubic effect, F(2, 17)
= .5674, p = .5774.
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0.3
0.25
0.2
+/+ 0.15 +/rl rl/rl Relative Frequency Relative
0.1
0.05
0 4 s 8 s 12 s 16 s 20 s 24 s 28 s 32 s 36 s 40 s Time
Figure 1. Mean relative frequency for each genotype across 10, 4-s time bins for the average of the first 5 sessions of FI training (Sessions 1-5). Error bars represent standard error of the mean.
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160
140
120
100
+/+ 80 +/rl rl/rl Percent of +/+ 60
40
20
0 4 s 8 s 12 s 16 s 20 s 24 s 28 s 32 s 36 s 40 s Time
Figure 2. Relative frequency represented as a percentage of +/+ responding, where +/+ data were set as the comparison (100%) for +/rl and rl/rl data. Percentage relative frequency is presented over 10, 4 s time bins for the first five sessions of PI training. Error bars represent standard error of the mean.
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0.45
0.4
0.35
0.3
0.25 +/+ +/rl 0.2 rl/rl Relative Frequency Relative 0.15
0.1
0.05
0 4 s8 s12 s16 s20 s24 s28 s32 s36 s40 s Time
Figure 3. Mean relative frequency for each genotype across 10, 4 s time bins for the average of the last 5 sessions of FI training (sessions 51-55). Error bars represent standard error of the mean.
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250
200
150 +/+ +/rl rl/rl
Percent of +/+ 100
50
0 4 s 8 s 12 s 16 s 20 s 24 s 28 s 32 s 36 s 40 s Time
Figure 4. Relative frequency represented as a percentage of +/+ responding, where +/+ data was set as the comparison (100%) for +/rl and rl/rl data. Percentage relative frequency is presented over 10, 4 s time bins for the last five sessions (S 51-55) of PI training. Error bars represent standard error of the mean.
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IOC. The index of curvature was used as a measure of FI performance to evaluate the changes in behavior across sessions and to determine whether mice reached asymptote by the end of training. Figure 5 shows data across training (sessions 1-55) in 5 session averages for the average of the 3 genotypes. Polynomial extraction with 5 session averages, and genotypes entered as factors did not expose any differences in IOC values between the mice across all sessions indicating that all mice demonstrated the same behavioral pattern when responding under the FI schedule. There was no significant Genotype x Linear effect found, F(2, 17) =
2.8911, p = .0830; no significant Genotype x Quadratic effect, F(2, 17) = .4655, p = .6356; and no significant Genotype x Cubic effect, F(2, 17) = 2.5571, p = .1069.
In order to examine further the IOC performance of +/rl and rl/rl mice, their data was expressed as a percentage of +/+ mice data. Figure 6 shows IOC data for 5 session averages for all mice as a function of comparison where +/+ mice data were set as the standard (100%) to which both other strains were compared. Interestingly, when data were organized in this way, significant differences between genotypes were detected. Polynomial extraction with sessions and genotypes entered as factors revealed significant differences between genotypes. A significant Genotype x Linear effect was found, F(2, 17) =5.3988, p = .0153. Least squares contrast found a significant difference between +/+ and rl/rl, F(1, 17) = 4.5264, p = .0483; and between +/rl and rl/rl, F(1, 17) = 10.3581, p = .0050; but not between +/+ and +/rl, F(1, 17) =
1.3791, p = .2564. A significant Genotype x Cubic effect was found, F(2, 17) = 5.9063, p =
.0113. Least squares contrast found a significant difference between +/+ and rl/rl, F(1, 17) =
10.2291, p = .0053; and between +/rl and rl/rl, F(1, 17) = 6.9699, p = .0172; but not between
+/+ and +/rl, F(1, 14) = .1873, p = .6706.
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0.7
0.6
0.5
0.4
+/+ +/rl IOC Values 0.3 rl/rl
0.2
0.1
0 S 1-5 S 6-10 S 11-15 S 16-20 S 21-25 S 26-30 S 31-35 S 36-40 S 41-45 S 46-50 S 51-55 Sessions
Figure 5. Five sessions averages of index of curvature by genotype across all 55 session of FI training. Five session averages are represented on the X axis and IOC values on the Y axis.
Error bars represent standard error of the mean
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150
140
130
120 +/+ +/rl 110 Percent of +/+ rl/rl
100
90
80 S 1-5 S 6-10 S 11-15 S 16-20 S 21-25 S 26-30 S 31-35 S 36-40 S 41-45 S 46-50 S 51-55 Sessions
Figure 6. Index of curvature values represented as a percentage of +/+ responding, where +/+ data were set as the comparison (100%) for +/rl and rl/rl data. Percentage IOC are presented over 10, 4 s time bins for the average of the last 5 sessions of FI training (session 51-55). Error bars represent standard error of the mean.
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Figure 7 represents IOC values for the average of the first 5 session versus the average of the last 5 session (sessions 51-55) for all genotypes, to show the change in IOC value from beginning to end of FI training. A repeated measures analysis revealed an effect for training,
F(1,17) = 188.9745, p < .0001, indicating a significant increase in IOC values from beginning to end of training for all genotypes. Additionally, a Time x Type effect was also discovered, F(
2, 17) = 5.0717, p = .0187. Least squares contrast found that +/+ did not differ from +/rl in their change in IOC values from beginning to end of training, F(1, 17) = 1.1410, p = .3004. However,
+/+ differed from rl/rl in their change of IOC values from beginning to end of training, F(1, 17)
= 4.4876, p = .0492. Another significant Time x Type effect was found between +/rl and rl/rl in their change in IOC values from beginning to end of training, F(1, 17) = 9.6314, p = .0065.
In order to investigate whether or not all mice reached asymptote on the FI 40 s, IOC values for the last three 5 session averages (Sessions 41-45, 46-50, and 51-55) were run with a repeated measures ANOVA using sessions and genotypes entered as factors (Figure 8). No significant differences were found between the last three 5 session averages for any of the three genotypes of mice indicating that all strains had reached asymptote and had reached stability on this task.
There were no Genotypic x Time effects found on this measure indicating that all strains were responding in the same manner, as measured by the IOC, F(2, 17) = .6913, p = .5145.
Peak Interval 120 s
The following analyses are based upon the 20 mice that completed both the FI through the
55th session and the peak interval procedure through the 50th session. Table 2 shows a summary of all the current findings of this research project based on the PI data.
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0.7
0.6
0.5
0.4 +/+ +/rl rl/rl IOC Values 0.3
0.2
0.1
0 S 1-5Sessions S 51-55
Figure 7. Index of curvature values for the average of the first and last 5 sessions of FI training.
Sessions are represented on the X axis and IOC values on the Y axis. Error bars represent standard error of the mean.
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0.7
0.6
0.5
0.4 +/+ IOC Values +/rl rl/rl 0.3
0.2
0.1
0 S 41-45 S 46-50 S 51-55 Sessions
Figure 8. Index of curvature values for the last 3, 5 session averages of FI training. Sessions are represented on X axis and IOC values on the Y axis. Data indicate that all mice reached asymptote. Error bars represent standard error of the mean.
80
Results of Results of Result of Schedule of +/rl rl/rl rl/rl Reinforce- Time of compared to compared to compared to ment Measure Training +/+ +/+ +/rl Peak Relative Beginning * p = .0173 * p = .0181 Interval Frequency (sessions 1- (Q) (Q) 5)
Relative End * p = .0115 * p = .0115 Frequency (sessions 46- (L) (L) 50)
Relative Beginning Frequency (sessions 1- for 1st 5) Interval (1 - 40 s)
Relative Beginning Frequency (sessions 1- for 2nd 5) Interval (41- 80 s)
Relative Beginning * p = .0010 * p = .0026 * p = .0050 Frequency (sessions 1- (Q) (C) (Q) for 3rd 5) * p = .0026 Interval (81- (C) 120 s)
Relative End * p = .0010 * p = .0293 Frequency (sessions 46- (Q) (Q) for 1st 50) Interval (1- 40 s)
Relative End * p = .0191 * p = .0014 Frequency (sessions 46- (L) (L) for 2nd 50) Interval (41- 80 s)
Table 2.
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Results of Results of Result of Schedule of +/rl rl/rl rl/rl Reinforce- Time of compared to compared to compared to ment Measure Training +/+ +/+ +/rl Peak Relative End Interval Frequency (sessions 46- for 3rd 50) Interval (81- 120 s)
Response All Sessions Rate (5 session averages)
Response First 5 Rate Sessions
Response Last 5 Rate Sessions
Bins with All Sessions Max (5 session Responses averages)
IOC for 1st All Sessions Interval (1- (5 session 40s ) averages)
IOC for 2nd All Sessions * p = .0095 * p = .0142 Interval (41- (5 session (L) (L) 80 s) averages)
IOC for 3rd All Sessions Interval (81- (5 session 120 s) averages)
Peak Time End (sessions 46- 50)
Table 2.
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Results of Results of Result of Schedule of +/rl rl/rl rl/rl Reinforece- Time of compared to compared to compared to ment Measure Training +/+ +/+ +/rl Peak Relative End * p = .0085 * p = .0057 Interval Frequency (sessions 46- (L) (L) as a 50) Porportion of Genotype Max Value
Peak Time End (sessions 46- 50)
Peak Spread End * p = .0471 * p = .0013 (whole) (sessions 46- 50)
Peak Spread End (1st interval) (sessions 46- 50)
Peak Spread End * p = .0132 (2nd interval) (sessions 46- 50)
Peak Spread End (difference (sessions 46- between 1st 50) and 2nd interval)
Table 2. Results based on the peak interval 120 s procedure. Horizontal arrows indicate no significant difference. Significant differences are marked with an asterisk along with the p value.
The significant effects are labeled by letter; L = linear, C = cubic, and Q = Quadratic.
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Relative frequency. Relative frequency was examined in many ways for the PI data. For all relative frequency graphs, 1 s bins were recalculated into 4 s bins. Figure 9 shows relative frequency data for the average of the first 5 sessions of PI training. Polynomial extraction with
30, 4 s time bins, and genotypes entered as factors revealed a significant Genotype x Quadratic effect, F(2, 17) = 4.4843, p = .0273. Least squares contrast found that +/rl differed from +/+ in
PI relative frequency values for the beginning of PI training, F(1, 17) = 6.9558, p = .0173, and
+/rl differed from rl/rl, F(1, 17) = 6.8416, p = .0181. There was no significant Genotype x
Quadratic effect between the PI relative frequency values of +/+ and rl/rl, F(1, 17) = .0005, p =
.9822. There were no significant Genotype x Linear effects found on this measure, F(2, 17) =
2.5759, p = .1054, and no significant Genotype x Cubic effects, F(2, 17) = .7935, p = .4683.
Figure 10 shows relative frequency data for the average of the last five sessions of PI training
(sessions 46-50). Polynomial extraction with 30, 4 s time bins, and genotypes entered as factors revealed a significant Genotype x Linear effect for the end of PI training, F(2, 17) = 5.5680, p =
.0138. Least squares contrast found that +/+ differed in relative frequency values from +/rl, F(1,
17) = 8.0257, p = .0115, and +/+ differed from rl/rl, F(1, 17) = 7.8273, p = .0124. However,
+/rl did not differ in relative frequency values from rl/rl, F(1, 17) = 0.0814, p = .7788. No significant Genotype x Quadratic effect was found, F(2, 17) = 1.0920, p = .3580; and no significant Genotype x Cubic effect was found, F(2, 17) = 2.8751, p = .0840.
Relative frequency was also calculated for 3 individual intervals of the entire peak trial, 1-40 s, 41-80 s, and 81-120 s, for the average of sessions 1-5 and the average of sessions 46-50.
Figure 11 shows the relative frequency for the first interval (1-40 s) for the average of sessions 1-
5. Polynomial extraction with 10, 4 s time bins, and genotypes entered as factors did not reveal a
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0.06
0.05
+/+ 0.04 +/rl rl/rl
0.03 Relative Frequency
0.02
0.01
0 4 s 8 s 12 s16 s20 s24 s28 s32 s36 s40 s44 s48 s52 s56 s60 s64 s68 s72 s76 s80 s84 s88 s92 s96 s100 s104 s108 s112 s116 s120 s Time
Figure 9. Relative frequency of responding (ordinate) for the average of the first 5 sessions of PI training. Responses were collected in 4 s time bins (abscissa) equaling 30 time bins in all (120 s). Error bars represent standard error of the mean.
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0.08
0.07
0.06 +/+ +/rl 0.05 rl/rl
0.04
Relative Frequency 0.03
0.02
0.01
0 4 s 8 s 12 s16 s20 s24 s28 s32 s36 s40 s44 s48 s52 s56 s60 s64 s68 s72 s76 s80 s84 s88 s92 s96 s100 104 108 112 116 120 s s s s s s Time
Figure 10. Relative frequency of responding (ordinate) for the average of the last 5 sessions of
PI training (sessions 46-50). Responses were collected in 4 s time bins (abscissa) equaling 30 time bins in all (120 s). Error bars represent standard error of the mean.
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0.2
0.18
0.16
0.14
0.12
0.1 +/+ +/rl 0.08 rl/rl Relative Frequency
0.06
0.04
0.02
0 4 s 8 s 12 s 16 s 20 s 24 s 28 s 32 s 36 s 40 s Time
Figure 11. Relative frequency of responding for the average of the first 5 sessions of PI training for the first time interval (1-40 s) of the PI trial. There are 10, 4 s time bins represented on the abscissa axis and relative frequency on the ordinate axis. Error bars represent standard error of the mean.
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significant Genotype x Linear effect, F(2, 17) = 3.3471, p = .0595. There was also no significant
Genotype x Quadratic effect, F(2, 17) = 0.0226, p = .9777, and no significant Genotype x Cubic effect, F(2, 17) = 1.6748, p = .2168.
Figure 12 depicts the relative frequency by genotype average for the second interval (41-80 s) for the average of the first 5 sessions of PI training. Polynomial extraction with 10, 4 s bins and genotypes entered as factors revealed no significant Genotype x Linear effect, F(2, 17) = 1.0068, p = .3861. There was also no significant Genotype x Quadratic effect, F(2, 17) = 0.0702, p =
.9325, and no significant Genotype x Cubic effect, F(2, 17) = .2176, p = .8067.
The relative frequency for the final interval (81-120 s) is shown in Figure 13 for the first 5 sessions of PI training. Polynomial extraction with 10, 4 s time bins, and genotypes entered as factors revealed a significant Genotype x Quadratic effect, F(2, 17) = 8.7016, p = .0025. Least squares contrast found that the relative frequency values of +/rl differed significantly from +/+ during the 3rd time interval of the PI function, F(1, 17) = 15.7530, p = .0010, and +/rl differed significantly from rl/rl, F(1, 14) = 10.3660, p = .0050. The +/+ mice did not differ significantly from rl/rl, F(1, 17) = .6084, p = .4461. A significant Genotype x Cubic effect was found, F(2,
17) = 6.3952, p = .0085. Least squares contrast revealed that rl/rl differed significantly in relative frequency values from +/+, F(1, 17) = 12.4869, p = .0026, and rl/rl differed significantly from +/rl, F(1, 17) = 4.7554, p = .0435. There was no significant Genotype x Cubic effect found between +/+ and +/rl, F(1, 17) = 1.4747, p = .2412. No significant Genotype x Linear effect was found on this measure, F(2, 17) = 0.2549, p = .7779.
Relative frequencies for the average of the last five sessions (46-50) for the first time interval
(1-40 s) are shown in Figure 14. Polynomial extraction with 10, 4 s time
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0.14
0.13
0.12
0.11 +/+ +/rl 0.1 rl/rl
Relative Frequency 0.09
0.08
0.07
0.06 44 s48 s52 s56 s60 s64 s68 s72 s76 s80 s Time
Figure 12. Relative frequency of responding for the average of the first 5 sessions of PI training for the second time interval (41-80 s) of the PI trial. There are 10, 4 s time bins represented on the X axis and relative frequency on the Y axis. Error bars represent standard error of the mean.
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0.14
0.12
0.1 +/+ +/rl rl/rl Relative Frequency
0.08
0.06 84 s 88 s 92 s 96 s 100 s 104 s 108 s 112 s 116 s 120 s Time
Figure 13. Relative frequency of responding for the average of the first 5 sessions of PI training for the third time interval (81-120 s) of the PI trial. There are 10, 4 s time bins represented on the X axis and relative frequency on the Y axis. Error bars represent standard error of the mean.
90
0.25
0.2
0.15
+/+ +/rl rl/rl 0.1 Relative Frequency
0.05
0 4 s 8 s 12 s 16 s 20 s 24 s 28 s 32 s 36 s 40 s Time
Figure 14. Relative frequency of responding for the average of the last 5 sessions of PI training for the first time interval (1-40 s) of the PI trial. There are 10, 4 s time bins represented on the X axis and relative frequency on the Y axis. Error bars represent standard error of the mean.
91
bins, and genotypes entered as factors revealed a significant Genotype x Quadratic effect, F(2,
17) = 8.0079, p = .0035. Least squares contrast found that the rl/rl differed significantly from
+/+, F(1, 17) = 15.7224, p = .0010, and the rl/rl differed significantly from +/rl, F(1, 17) =
5.6637, p = .0293. There were no significant Genotype x Quadratic effect between +/+ and +/rl,
F(1, 17) = 2.0442, p = .1709. There were no significant effects for Genotype x Linear fit, F(2,
17) = 1.8256, p = .1913, and no significant Genotype x Cubic effects, F(2, 17) = 3.4146, p =
.0567 for this measure.
Figure 15 shows the relative frequency for the average of the last five sessions (46-50) for the second time interval (41-80 s). Polynomial extraction with 10, 4 s time bins, and genotypes entered as factors showed a significant Genotype x Linear effect, F(2, 17) = 7.5863, p = .0044.
Least squares contrast revealed that +/+ differed significantly from +/rl, F(1, 17) = 6.7053, p =
.0191, and +/+ differed significantly from rl/rl, F(1, 17) = 14.4101, p = .0014. The +/rl did not differ significantly from rl/rl, F(1, 17) = 1.1187, p = .3050. No significant Genotype x Quadratic effect was found, F(2, 17) = 1.664, p = .2224; and no Genotype x Cubic effect was found, F(2,
17) = 1.2660, p = .3072.
The relative frequency data for the average of the last 5 sessions (sessions 46-50) for the average of all genotypes for the final time interval (81-120 s) are shown in Figure 16.
Polynomial extraction with 10, 4 s time bins, and genotypes entered as factors revealed no significant Genotype x Linear effect, F(2, 17) = 0.4748, p = .6300, no significant Genotype x
Quadratic effect, F(2, 17) = 0.0674, p = .9351, and no significant Genotype x Cubic effect, F(2,
17) = 0.3656, p = .6991.
Rate of responding. Responses per second for the average of each genotype by 5 session averages are presented in Figure 17. Polynomial extraction using sessions, and
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0.18
0.16
0.14
0.12
0.1
+/+ 0.08 +/rl rl/rl Relative Frequency
0.06
0.04
0.02
0 44 s 48 s 52 s 56 s 60 s 64 s 68 s 72 s 76 s 80 s Time
Figure 15. Relative frequency of responding for the average of the last 5 sessions of PI training for the second time interval (41-80 s) of the PI trial. There are 10, 4 s time bins represented on the X axis and relative frequency on the Y axis. Error bars represent standard error of the mean.
93
0.14
0.13
0.12
0.11
0.1 +/+ +/rl rl/rl Relative Frequency 0.09
0.08
0.07
0.06 84 s 88 s 92 s 96 s 100 s 104 s 108 s 112 s 116 s 120 s Time
Figure 16. Relative frequency of responding for the average of the last 5 sessions of PI training for the third time interval (81-120 s) of the PI trial. There are 10, 4 s time bins represented on the X axis and relative frequency on the Y axis. Error bars represent standard error of the mean.
94
1.2
1
0.8
0.6 +/+ +/rl rl/rl Responses per Second 0.4
0.2
0 S 1-5 S 6-10 S 11-15 S 16-20 S 21-25 S 26-30 S 31-35 S 36-40 S 41-45 S 46-50 Sessions
Figure 17. Maximum rate of responding (responses per second) across all sessions (5 session averages) of PI training for the average of genotypes. Sessions are represented on the X axis and responses per second on the Y axis. Error bars represent standard error of the mean.
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genotypes entered as factors did not reveal a significant Genotype x Linear effect, F(2, 17) =
1.2415, p = .2999. There was also no significant Genotype x Quadratic effect, F(2, 17) = 1.2415, p = .3139, and no significant Genotype x Cubic effect, F(2, 17) = 1.5956, p = .2317.
In order to determine behavioral effects, based on rate of responding between the beginning and end of training, response rates for the first and last five individual sessions (sessions 1-5, and
46-50) of PI training were analyzed. The first five sessions of PI training are shown in Figure
18. Polynomial extraction using the first five sessions, and genotypes as factors revealed no significant Genotype x Linear effect, F(2, 17) =2.3491, p = .1257. There was also no significant
Genotype x Quadratic effect, F(2, 17) = .0765, p = .9266, and no significant Genotype x Cubic effect, F(2, 17) = 1.5836, p = .2341. Data for responses per second for the last 5 sessions are presented in Figure 19. Polynomial extraction using the last five sessions (session 46-50), and genotypes entered as factors revealed no significant Genotype x Linear effect, F(2,17) = 3.0865, p = .0719, no significant Genotype x Quadratic effect, F(2, 17) = 0.4668, p = .6348, and no significant Genotype x Cubic effect, F(2, 17) = .9182, p = .4182.
Number of bins with maximum response. In order to see if there were any differences between genotypes on the number of bins that had the maximum number of responses, data were collected and statistically analyzed. Figure 20 shows this data for 5 session averages for the average of each genotype. Polynomial extraction with sessions, and genotypes entered as factors revealed no significant effect for Genotype x Linear interaction, F(2, 17) = 2.3784, p = .1228 for the number of bins with maximum responses per session, across training. There was also no significant Genotype x Quadratic effect,
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0.25
0.2
0.15
+/+ +/rl 0.1 rl/rl Responses per Second
0.05
0 S 1 S 2S 3S 4S 5 Sessions
Figure 18. Maximum rate of responding (responses per second) across the first 5 sessions of PI training for each genotype. Sessions are represented on the X axis and responses per second on the Y axis. Data points represent the mean for each genotype and error bars represent standard error of the mean.
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1.2
1
0.8
0.6 +/+ +/rl rl/rl Responses per Second 0.4
0.2
0 S 46 S 47 S 48 S 49 S 50 Sessions
Figure 19. Maximum rate of responding (responses per second) across the last 5 sessions of PI training for the average of genotypes. Sessions are represented on the X axis and responses per second on the Y axis. Error bars represent standard error of the mean.
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3
2.5
2 +/+ +/rl rl/rl 1.5 Number of Bins with Responding Max with of Bins Number 1
0.5 S 1-5 S 6-10 S 11-15 S 16-20 S 21-25 S 26-30 S 31-35 S 36-40 S 41-45 S 46-50 Sessions
Figure 20. Mean number of bins, per 5 session average, with the maximum number of responses, per genotype. Sessions are represented on the X axis and the genotype average number of bins with maximum responses are represented on the Y axis. Error bars represent standard error of the mean.
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F(2, 14) = 0.6320, p = .5436, and no significant Genotype x Cubic effect, F(2, 17) = 0.2686, p =
.7676. The overall average number of maximum bins, from the first session to the last (sessions
1-50) is as follows, the +/+ mice had an average of 1.89 bins with maximum responses, +/rl had
1.57, and rl/rl had 1.88.
Index of curvature. To measure the response curvature of each interval, IOC values were calculated based on the same three time intervals (1-40 s, 41-80 s, and 81- 120 s) used to evaluate relative frequency, for the first and last five session averages. Figure 21 represents average IOC data for 5 session averages for the first time interval (1-40 s) of the trial.
Polynomial extraction with sessions, and genotypes entered as factors did not reveal any significant differences. There was no significant effect for Genotype x Linear fit, F(2, 17)
=1.1413 , p = .3421, no significant Genotype x Quadratic effect was detected, F(2, 17) = 1.2196, p = .3199, and no significant Genotype x Cubic effect, F(2, 17) = 3.2872, p = .0621.
Figure 22 shows IOC data for 5 session averages for the second time interval (41-80 s) of the
PI trial. Polynomial extraction revealed a significant Genotype x Linear effect, F(2, 17) =
5.4172, p = .0151. Least squares contrast found that the IOC values of +/+ differed significantly from +/rl, F(1, 17) = 8.5403, p = .0095, and +/+ differed significantly from rl/rl, F(1, 17) =
7.4655, p = .0142. There was no significant difference between +/rl and rl/rl, F(1, 17) = 0.0884, p = .7699. There was no significant effect for Genotype x Quadratic interaction, F(2, 17) =
1.1743, p = .3329, and no significant Genotype x Cubic effect, F(2, 17) = 0.1302, p = .8788.
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0.5
0.45
0.4
0.35
0.3 +/+ +/rl IOC Values rl/rl 0.25
0.2
0.15
0.1 S 1-5 S 6-10 S 11-15 S 16-20 S 21-25 S 26-30 S 31-35 S 36-40 S 41-45 S 46-50 Sessions
Figure 21. Mean Index of curvature values (ordinate) by genotype across all 5 session averages
(abscissa) of PI training for the first time interval of the PI trial (1-40 s). Error bars represent standard error of the mean.
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0 S 1-5 S 6-10 S 11-15 S 16-20 S 21-25 S 26-30 S 31-35 S 36-40 S 41-45 S 46-50
-0.05
-0.1
+/+ -0.15 +/rl
IOC Values rl/rl
-0.2
-0.25
-0.3 Sessions
Figure 22. Mean Index of curvature values (ordinate) by genotype across all 5 session averages
(abscissa) of PI training for the second time interval of the PI trial (41-80 s). Error bars represent standard error of the mean.
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IOC data for 5 session averages for the third and final interval (81-120s) of the PI schedule are presented in Figure 23. Polynomial extraction revealed no significant differences between genotypes. There was no significant Genotype x Linear effect, F(2, 17) = 2.1872, p = .1428, no significant Genotype x Quadratic effect, F(2, 17) = 1.0287, p = .3787, and no significant
Genotype x Cubic effect, F(2, 17) = .1764, p = .8398.
Peak spread and peak time. Data were first analyzed by representing each mouse’s relative frequency value as a proportion of each corresponding genotypes highest relative frequency value by dividing each individual mouse’s relative frequency value by the highest respective genotype relative frequency value. Figure 24 represents averaged data for +/+ mice; +/rl are represented in Figure 25; and rl/rl are in Figure 26. Figure 27 shows all genotypes on one graph.
Polynomial extraction using 30, 4 s time bins, and genotypes entered as factors revealed a significant Genotype x Linear effect, F(2, 17) = 6.6197, p = .0075. Least squares contrast revealed that +/+ differed significantly from +/rl, F(1, 17) = 8.8533, p = .0085, and +/+ differed significantly from rl/rl, F(1, 17) = 9.9708, p = .0057. There were no significant Genotype x
Linear effects for +/rl and rl/rl, F(1, 17) = .0112, p = .9169. There were no significant effects for
Genotype x Quadratic fit, F(2, 17) = 1.3607, p = .2830, and no significant effects for Genotype x
Cubic fit, F(2, 17) = 0.2226, p = .8027.
Peak spread and peak location (time) were analyzed for each mouse and presented as genotype averages. Figure 28 shows peak time data for the average of each genotype.
Multivariate analysis of variance (MANOVA) using genotypes and peak times as factors revealed no significant differences in peak times between the genotypes, F(2, 17) = 0.7393, p =
.4922. Figure 29 illustrates peak spread data for the average of each genotype for the average
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0.15
0.1
0.05
+/+ +/rl
IOC Value rl/rl 0 S 1-5 S 6-10 S 11-15 S 16-20 S 21-25 S 26-30 S 31-35 S 36-40 S 41-45 S 46-50
-0.05
-0.1 Sessions
Figure 23. Mean Index of curvature values (ordinate) by genotype across all 5 session averages
(abscissa) of PI training for the third time interval of the PI trial (81-120 s). Error bars represent standard error of the mean.
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1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 Proportion of Maximum RF of Maximum Proportion
0.3
0.2
0.1
0 4 s 8 s 12 s16 s20 s24 s28 s32 s36 s40 s44 s48 s52 s56 s60 s64 s68 s72 s76 s80 s84 s88 s92 s96 s100 104 108 112 116 120 Time s s s s s s
Figure 24. Relative frequency as a proportion of the maximum +/+ relative frequency value
(ordinate), for the average of the last five sessions of PI training (sessions 46-50).The abscissa represents time for 30, 4 s time bins. Error bars represent standard error of the mean. The dashed horizontal line extending from 0.9 on the ordinate represents the threshold line for determining the peak location. The whole horizontal line extending from 0.7 on the ordinate represents the threshold line for determining the peak spread. The dashed vertical line extending from the abscissa represents the peak time.
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1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 Proportion of Maximum RF of Maximum Proportion
0.3
0.2
0.1
0 4 s 8 s 12 s16 s20 s24 s28 s32 s36 s40 s44 s48 s52 s56 s60 s64 s68 s72 s76 s80 s84 s88 s92 s96 s100 104 108 112 116 120 s s s s s s Time
Figure 25. Relative frequency as a proportion of the maximum +/rl relative frequency value
(ordinate), for the average of the last five sessions of PI training (sessions 46-50).The abscissa represented time for 30, 4 s time bins. Error bars represent standard error of the mean. The dashed horizontal line extending from 0.9 on the ordinate represents the threshold line for determining the peak location. The whole horizontal line extending from 0.7 on the ordinate represents the threshold line for determining the peak spread. The dashed vertical line extending from the abscissa represents the peak time.
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1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 Proportion of Maximum RF Maximum of Proportion
0.3
0.2
0.1
0 4 s 8 s 12 s16 s20 s24 s28 s32 s36 s40 s44 s48 s52 s56 s60 s64 s68 s72 s76 s80 s84 s88 s92 s96 s100 104 108 112 116 120 Time s s s s s s
Figure 26. Relative frequency as a proportion of the maximum rl/rl relative frequency value
(ordinate), for the average of the last five sessions of PI training (sessions 46-50).The abscissa represented time for 30, 4 s time bins. Error bars represent standard error of the mean. The dashed horizontal line extending from 0.9 on the ordinate represents the threshold line for determining the peak location. The whole horizontal line extending from 0.7 on the ordinate represents the threshold line for determining the peak spread. The dashed vertical line extending from the abscissa represents the peak time.
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1.1
1
0.9
0.8
0.7
0.6
+/+ 0.5 +/rl rl/rl 0.4 Proportion of Maximum RF RF of Maximum Proportion
0.3
0.2
0.1
0 4 s 8 s 12 s16 s20 s24 s28 s32 s36 s40 s44 s48 s52 s56 s60 s64 s68 s72 s76 s80 s84 s88 s92 s96 s100 104 108 112 116 120 s s s s s s Time
Figure 27. Relative frequency as a proportion of the maximum genotype relative frequency value (ordinate), for the average of the last five sessions of PI training (sessions 46-50) for all genotypes. The abscissa represents time for 30, 4 s time bins. Error bars represent standard error of the mean.
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50
48
46
44 Time in Seconds
42
40
38 +/+ +/rl rl/rl Genotype
Figure 28. Average peak time in seconds, by genotype, for the average of the last 5 sessions
(sessions 46-50) of PI training. Genotypes are represented on the abscissa and time in seconds is represented on the ordinate. Error bars represent standard error of the mean
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45
40
35
30
25
20 Time in Seconds
15
10
5
0 +/+ +/rl rl/rl Genotype
Figure 29. Average peak spread in second, by genotype for the average of the last 5 sessions
(sessions 46-50) of PI training. Genotypes are represented on the abscissa and time in seconds is represented by the ordinate. Error bars represent standard error of the mean.
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of the last 5 sessions of training (session 46-50). MANOVA using genotypes and peak times as factors revealed a significant Genotype effect, F(2, 17) = 7.5492, p = .0045. Least squares contrast found that +/+ differed significantly from +/rl, F(1, 17) = 4.5841, p = .0471, and +/+ differed significantly from rl/rl, F(1, 17) = 14.7784, p = .0013. There were no significant differences between +/rl and rl/rl, F(1, 17) = 1.7249, p = .2065.
Figure 30 shows data for the mean time (by genotype) from the first 70% of peak time to peak time (first part of spread). This data represents the average of the last 5 sessions of PI training
(sessions 46-50). Statistical analysis using standard least squares did not reveal a genotype difference in the time for the first part of the peak spread, F(2, 17) = 2.6658, p = .0984.
Figure 31 represents data for the mean time (by genotype) from the peak time to the second
70% of peak time. This data represents the average of the last 5 sessions of PI training (sessions
46-50). Statistical analysis using standard least squares revealed a significant effect for genotype, F(2, 17) = 3.8997, p = .0404. Least squares contrast found that +/+ differed significantly from rl/rl, F(1, 17) = 7.6600, p = .0132. However, +/+ did not differ significantly from +/rl, F(1, 17) = 0.6963, p = .4156; and +/rl did not differ significantly from rl/rl, F(1, 17)
= 2.6696, p = .1207.
In order to determine where on the time line the majority of the spread took place (before or after the peak time) for each genotype, the time between the earlier 70% of peak and peak time was subtracted from the time between peak time and the later 70% of peak. Figure 32 represents the data for the peak spread for each genotype with corresponding peak spread intervals. The difference between the first part of the peak spread from the second part of peak spread for each
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25
20
15
Time in Seconds 10
5
0 +/+ +/rl rl/rl Genotype
Figure 30. Mean time in seconds for the first part of the peak spread (time from 70% of peak time to peak time). Data was averaged by genotype for the average of the last 5 sessions of PI training (session 46-50). Genotypes are represented on the X axis and time in seconds on the Y axis. Error bars represent standard error of the mean.
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25
20
15
Time in Seconds 10
5
0 +/+ +/rl rl/rl Genotype
Figure 31. Mean time in seconds for the second part of the peak spread (time from peak time to
70% of peak time). Data was averaged by genotype for the average of the last 5 sessions of PI training (session 46-50). Genotypes are represented on the abscissa axis and time in seconds on the ordinate axis. Error bars represent standard error of the mean.
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40
35
30
25
Spread 20 1st Spread 2nd Spread Time in Seconds
15
10
5
0 +/+ +/rl rl/rl Genotype
Figure 32. Data representing the peak spread using 3 consecutive bars for the average of each genotype to show parts of the spread. The white bars represent the whole spread in seconds.
The light gray bars symbolize the first part of the peak spread in seconds, from the earliest 70% of peak time to peak time. The dark gray bars symbolize the second part of the peak spread in seconds, from the peak time to the later 70% of peak time. Genotypes are represented on the abscissa axis and time in seconds on the ordinate axis. Error bars represent standard error of the mean.
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genotype was statistically analyzed. Standard least squares analysis for the average of the last 5 sessions (sessions 46-50) for the maximum part of spread, by genotype revealed no effect for genotypes, F(2, 17) = .6572, p = .5310.
DISCUSSION
The current study sought to investigate the fixed interval and peak trial performance of Reln deficient mice. The fixed interval procedure and peak interval procedure allow us the opportunity to investigate behavior as it conforms to the contingencies of reinforcement based on the passage of time. These sorts of analyses provide an understanding of an organism’s performance in terms of specific environment-response relations. Typical behavior changes as the schedules of reinforcement change. The behavioral controlling stimulus of interest was time. Temporal properties of the environment can have discriminative properties on behavior and therefore can be differentiated (Catania, 1970).
The fixed interval procedure and peak interval procedure have proven useful at detecting temporal abnormalities of behavior based on different neuroanatomical abnormalities (Dietrich
& Allen, 1998; Lewis et al., 2003; Maricq et al., 1981; Meck, 1996; Morrissey et al., 1994;
Olton, 1989; & Taylor et al., 2007). The types of neurochemical abnormalities used in these studies are similar to the neurochemical abnormalities reported in Reln deficient mice (Ballmaier et al., 2002; Ognibene et al., 2008; & Sigala et al., 2007). It was speculated that Reln deficient mice might also show some of the same behavioral anomalies as the subjects with similar neurotransmitter aberrations have shown on these measures.
Fixed Interval Procedure.
During the beginning of FI training, relative frequencies of +/rl and rl/rl mice differed significantly from +/+ mice; and +/rl differed from rl/rl. Visual examination of the graphs
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indicated that +/+ and +/rl mice had proportionally higher frequencies of responding earlier in the FI interval than rl/rl mice. By the end of the interval, +/rl and rl/rl mice were responding more frequently (proportionally) than +/+ mice. These observations reveal that in the beginning of training, the +/+ and +/rl mice were inferior to rl/rl mice at inhibiting responding early in the interval and +/+ mice were inferior to +/rl and rl/rl mice at responding more frequently at the time when reinforcement was made available. However, by the end of training, there were no differences between the relative frequency values of each genotype. All genotypes inhibited their responding at the beginning of the FI interval and allocated the majority of their responses toward the end of the interval, indicating that all mice learned the schedule.
The index of curvature across all sessions did not reveal any differences between the genotypes. This indicates that all 3 genotypes demonstrated a similar pattern of behavioral change across FI training. All mice significantly increased their IOC values as they received more training. That is, all mice increased the depth of their FI scallop as they had more exposure to the FI schedule. However, when data were set up so that +/rl and rl/rl mice data were compared to +/+ mouse data, where +/+ were presented as the standard (100%) by which to compare, significant differences were detected. This measure found that +/+ and +/rl differed significantly from rl/rl but +/+ and +/rl did not differ. So, +/rl behaved like typical mice (+/+) but rl/rl performed differently than both other genotypes. It appears that this difference occurred during the beginning of FI training, where rl/rl had higher IOC values than +/+ and +/rl and lowered their IOC values to comparable levels later in training. Data based on IOC values also showed that all mice reached asymptote by the end of FI training. If all genotypes had not reached asymptote by the end of FI training, any behavioral differences, or the lack thereof,
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might have been explained by the possibility that they had not finished learning the schedule instead of fundamental differences in how their behavior conforms to the schedule.
The temporal dysregulation of responding by +/rl and rl/rl mice occurred early in FI training.
This is consistent with past research that reported that Reln deficient mice were abnormal in their acquisition of a learning task, compared to +/+ mice (Krueger et al., 2006; Larson et al., 2003).
However, by the end of FI training, all mice were performing equally. To understand better the behavior of Reln deficient mice, all mice were exposed to the peak interval procedure where schedule induced behavior could be better examined with the added contingencies of the PI schedule.
Peak Interval Procedure
Based on relative frequency of responding, +/rl mice differed significantly from +/+ and rl/rl mice at the beginning of FI training. The rl/rl mice did not differ in relative frequency of responding from +/+ mice, during the beginning of PI training. Visual examination of the graph indicates that +/+ and rl/rl mice allocated an increased proportional amount of their responding more at the beginning of the interval, than +/rl mice. This implies that +/+ and rl/rl mice were less able to inhibit their responding early in the interval than +/rl mice, during the beginning of
PI training. The +/+ and rl/rl mice responded proportionally less after the time when reinforcement was usually made available, indicating better discrimination of the temporal contingencies of the schedule once the pellet was omitted.
By the end of PI training, the relative frequency of responding differed between +/rl and +/+ mice and between rl/rl and +/+ mice. However, +/rl mice did not differ from rl/rl mice on relative frequency of responding by the end of PI training. The +/+ mice had a more defined peak than +/rl and rl/rl mice. That is, the majority of +/+ responding took place during the time
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when a reinforcer was usually made available and less during any other time. The +/rl and rl/rl mice also allocated most of their responding during the time a reinforcer was usually made available but did not slow their responding as much as +/+ mice did after that time passed.
Closer examination of relative frequency of responding in each of the 3 intervals of the entire PI showed that rl/rl mice allocated more responding earlier in the interval (before 40 s) than +/rl and +/+ mice, and not as much during the time when a reinforcer was usually made available (40 s). The +/+ mice responded more immediately after the time of usual reinforcement than +/rl and rl/rl mice. However, at approximately 60 s, the +/+ mice allocated less responding than both
+/rl and rl/rl mice, indicating better control of behavior by the passage of time. All genotypes responded similarly in the third time interval (80-120s) in that they all increased their responding towards the end of the interval.
In order to investigate whether differences in genotypes could be explained by motor coordination differences, especially the dyskinesia that typifies the rl/rl mice, rates of responding were examined between the genotypes. It was expected that the abnormal gait of the rl/rl mice would make it incapable of responding at the same rate as the other two genotypes. This explanation was ruled out since at no point during PI training did the rates of responding between the genotypes significantly differ.
While examining PI data for the number of bins in any given session with maximum number of responses, it was noted that most mice had multiple bins throughout all sessions with the maximum number of responses. This data was analyzed to determine if any of the genotypes differed from each other on this measure. Multiple bins in any given session with the maximum number of responses might suggest abnormality. However, statistical analysis revealed no
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differences between the genotypes on this measure. Therefore, it might be concluded that having more than one bin with maximal number of responses in any given session, is a typical finding.
Index of curvature for the 3 time intervals of the PI session were analyzed for genotype differences. The second time interval was the only interval to detect differences between the genotypes. As with the relative frequency data, the interval after the usual time a reinforcer was made available, was the interval that mice differed. As with the findings based on relative frequency, the difference was that +/rl and rl/rl mice did not slow their responding as much as
+/+ mice, after 40 s passed with no reinforcer. Responding after the pellet omission occurred, after 40 s, never led to reinforcement and therefore made responding futile. These results indicate that +/rl and rl/rl mouse behaviors do not conform to the PI schedule as well as +/+ mouse behavior, after the usual time a reinforcer was made available.
Relative frequency and IOC measures revealed that +/rl and rl/rl mice differed from +/+ mice right after the usual time of reinforcement. To understand this difference better, peak time and peak spread were analyzed. There were no genotype differences between when the average genotype’s peak time occurred. However, +/rl and rl/rl differed from +/+ on their peak spreads.
The +/rl and rl/rl mice had significantly longer peak spreads than +/+ mice. Longer spreads might indicate less temporal control of behavior in that responding before or after the usual time of reinforcement is pointless since it will never lead to reinforcement on these schedules, assuming the pellet is the only form of reinforcement. The differences in peak spread were examined further by comparing the first part of the spread (the spread before the peak) with the second part of the spread (the spread after the peak) to see where the majority of the spread occurred in time. It appears that the majority of the peak spread for rl/rl mice occurred during the second part of the peak spread (after the peak) considerably more than the other two
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genotypes. However, data revealed that there were no significant differences between the genotypes on where the majority of the spread occurred mostly due to the variability within subjects.
In summary, the overall evidence of difference between the Reln deficient mice from the +/+ mice occurred during the time after the usual time when a reinforcer was made available. There was evidence that +/rl and rl/rl mice had significantly flatter spreads than +/+ mice, similar to other studies that found the same effect from drugs that altered the neurochemistry of their subjects (Abner et al., 2001; Balci et al., 2008; Cheng et al., 2007b; Maricq et al., 1981;
Morrissey et al., 1994). These finding indicate that Reln may affect the behavior of an organism by influencing the discriminating properties of temporal stimuli and the consequences that follow.
The current results indicate that the behavioral analysis of Reln deficiency has provided some evidence for the behavioral effects of the gene protein’s expression. However, more detailed analysis is necessary to fully understand the effects of Reln deficiency. This study was limited by a small sample of “old” mice. To further understand the effects, larger samples should be used from the same populations. Younger mice should also be evaluated using these procedures to determine whether the effects found were due to the old age of the mice.
The peak interval procedure has proven a useful tool at detecting differences between organisms that have been affected by neurochemical abnormalities. The fixed interval procedure was not as successful at detecting these differences and that may be due to the fact that it only allows us to look at half of the temporal gradient (Catania, 1970). The peak procedure allows us to look at the full temporal gradient therefore allowing us to measure responding based on the time up to the time when a reinforcer is made available and the time that follows. For this study,
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the peak interval procedure proved useful at detecting slight but possibly important differences between the genotypes that may not have been detected using other behavioral assays.
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APPENDIX
Appendix 1. Literature Review of Reln Deficient Mice
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl
Acoustic Tueting et N= 13 SR-Lab Startle Apparatus 16 trials of 115 Startle al. (1999) 6 M +/+, 7 dB. Response M +/rl 40-63 days old
Acoustic Qiu et al. N= 18 Not Clear 120 dB only. Startle (2002) 9 +/+; 9 Number of trials Response +/rl not clear Sex not clear 6 weeks old Acoustic Podhorna et N= 64 MPOS 2b Startle Apparatus 105 dB only Startle al. (2004) 33 +/+ (16 Number of trials Response Y (8M, not clear. 8F); 17 A (8M, 9F)) 31 +/rl (16 Y (8M, 8F); 15 A (8M, 7F)) Y = young (50-70 days old)
142
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl A = Adult (> 75 days old)
Acoustic Podhorna et N= 64 MPOS 2b Startle Apparatus 77 dB only Startle al. (2004) 33 +/+ (16 Number of trials Response Y (8M, not clear. 8F); 17 A (8M, 9F)) 31 +/rl (16 Y (8M, 8F); 15 A (8M, 7F)) Y = young (50-70 days old) A = Adult (> 75 days old)
143
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Acoustic Salinger et N= 22 SR-LAB startle response 4 blocks of 6 Startle al. (2003) 6 +/+ (5M, measurement system different Response 1F); 9 M intensities (72, 84, +/rl; 7 rl/rl 90, 100, 110, 120 More startle (2M, 5F) dB) totaling 24 More startle from 90- ~ 70 days stimulus 100 dB. old presentations.
Less startle from 100- 110 dB
Startle Salinger et N= 63 SR-Lab Startle Apparatus 120 dB only. Response al. (2003) 20 +/+ Occurred Habituation (9M, 11F); randomly 10 22 +/rl times over a 12.39 (11M, min session 11F); 21 rl/rl (8 M, Less 13 F) ~ SRH 70 days old
144
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Prepulse Tueting et N= 13 SR-Lab Startle Apparatus 16 trials of 80 dB Inhibition al. (1999) 6 M +/+, 7 prepulse (115 dB) M +/rl Weaker 40-63 days PPI old
Prepulse Tueting et N= 13 SR-Lab Startle Apparatus 16 trials of 75 dB Inhibition al. (1999) 6 M +/+, 7 prepulse (115 dB) M +/rl Weaker 40-63 days PPI old
Prepulse Qiu et al. N= 18 Not Clear 70 dB prepulse Inhibition (2002) 9 +/+; 9 (120dB) +/rl Number of trials Weaker Sex not not clear PPI clear ~6 weeks old
Prepulse Qiu et al. N= 18 Not Clear 76 dB prepulse Inhibition (2002) 9 +/+; 9 (120dB)
+/rl Number of trials Weaker Sex not not clear PPI clear ~6 weeks old
145
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Prepulse Qiu et al. N= 18 Not Clear 82 dB prepulse Inhibition (2002) 9 +/+; 9 (120dB) +/rl Number of trials Weaker Sex not not clear PPI clear ~6 weeks old
Prepulse Qiu et al. N= 18 Not Clear 88 dB prepulse Inhibition (2002) 9 +/+; 9 (120dB) Weaker +/rl Number of trials PPI Sex not not clear clear ~6 weeks old
Prepulse Salinger et N= 63 SR-Lab Startle Apparatus 72 dB prepulse Inhibition al. (2003) 20 +/+ (120dB). (9M, 11F); Occurred 22 +/rl randomly 10 Weaker (11M, times over a 12.39 PPI 11F); 21 min session rl/rl (8 M, 13 F) ~ 70 days old
146
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Prepulse Salinger et N= 63 SR-Lab Startle Apparatus 76 dB prepulse Inhibition al. (2003) 20 +/+ (120dB). (9M, 11F); Occurred 22 +/rl randomly 10 Weaker (11M, times over a 12.39 PPI 11F); 21 min session rl/rl (8 M, 13 F) ~ 70 days old
Prepulse Salinger et N= 63 SR-Lab Startle Apparatus 84 dB prepulse Inhibition al. (2003) 20 +/+ (120dB). (9M, 11F); Occurred 22 +/rl randomly 10 Weaker (11M, times over a 12.39 PPI 11F); 21 min session rl/rl (8 M, 13 F) ~ 70 days old
147
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Prepulse Podhorna et N= 64 MPOS 2b Startle Apparatus 67 dB prepulse Inhibition al. (2004) 33 +/+ (16 (105dB) Y (8M, Number of trials 8F); 17 A not clear (8M, 9F)) 31 +/rl (16 Y (8M, 8F); 15 A (8M, 7F)) Y = young (50-70 days old) A = Adult (> 75 days old)
Prepulse Podhorna et N= 64 MPOS 2b Startle Apparatus 72 dB prepulse Inhibition al. (2004) 33 +/+ (16 (105dB) Y (8M, Number of trials 8F); 17 A not clear (8M, 9F)) 31 +/rl (16 Y (8M, 8F); 15 A (8M, 7F)) Y = young (50-70 days old) A = Adult (> 75 days
148
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl old)
Prepulse Podhorna et N= 64 MPOS 2b Startle Apparatus 77 dB prepulse Inhibition al. (2004) 33 +/+ (16 (105dB) Y (8M, Number of trials 8F); 17 A not clear (8M, 9F)) 31 +/rl (16 Y (8M, 8F); 15 A (8M, 7F)) Y = young (50-70 days old) A = Adult (> 75 days old)
149
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Elevated Plus Tueting et N= 12 EPM 50cm (l) X 5 min test. Time Maze al. (1999) 8 M +/+; 4 10cm (w) X 5cm (h) and entries into M +/rl open arm were Less 40-63 days measured time in old open arms. More neo‐ phobic
Elevated Plus Krueger et N= 19 EPM 30cm (l) X Same as Tueting Maze al. (2006) 9 M +/+, 5cm (w) X 15.25cm (h) et al. (1999) 10 M +/rl 3 mos old
Elevated Plus Qiu et al. N= 18 EPM Measured % Maze (2002) 9 +/+; 9 30cm(l)x5cm(w)x15cm(h) entries & rest +/rl time in open Sex not arms, total time clear ~6 spent in open weeks old arms and closed and central areas. Trial number and duration not clear
150
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl
Olfactory Larson et N= 17 Olfactory Discrimination Picking one of Learning al. (2003) 8 M +/+, 9 Chamber 60 cm(l) X 10 cm two odors based Assay M +/rl (w) X 30 cm (h) on discriminative More 3-4 mos odor with east and training old west sniff ports. Trained to 90% of 20 trials on eight More 2 odor Errors discriminations
151
Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Olfactory Salinger et N= 45 TruScan App. With nose Olfactory Learning al. (2003) 18 M & F poke floorplate (4 wells, 1 guidance of single Assay +/+; 13 M baited). baited hole. Snout & F +/rl; touches to each 14 M & F well were More rl/rl ~ 70 measured. Trials correct days old not given responses
Olfactory Salinger et N= 60 TruScan App. With nose Seven 5 min Learning al. (2003) 10M, 10F poke floorplate (16 wells, 4 trials. Number of Assay +/+; 11M, baited) entries and 10F +/rl; reentries into Less 11M, 8F baited holes on-task rl/rl ~ 70 divided by the behavior days old total number of entries into baited and non-baited holes
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl
Set Shifting Brigman et +/+ and Med Assoc Chamber. Lever Nose poke and Reversal al. (2006) +/rl No to initiate trial. Nose poke appropriate
Learning reference for O.R. Touchscreen with stimulus based on Took longer to age, visual stimuli given stimulus. to reach number of Reversing criterion subjects or contingencies. sex More errors on reversal
Reversal Krueger et N= 16 Med. Assoc. Oper. Chamber Nose poke in Learning al. (2006) 7 M +/+; 9 with 3 nose poke holes "active" hole led M +/rl to S+. All 3 holes all 3 mos were lit. At old session 10, 16 holes were switched
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Gait Salinger et N = 35 Locally fabricated 35-cm- Measured al. (2003) 13 M +/+; long acrylic tunnel with a positions of left 10 M +/rl; rectangular cross section and right hind 12 rl/rl apparatus. paw prints. Wider ~ 70 days Width of stride strides old was measured. One session per mouse
Gait Podhorna et N= 66 Motor activity cages (20cm Activity was al. (2004) 35 +/+ (18 X 32cm) with 5 X 8 infrared measured for 1h Y (10M, light sources. in 5 min time bins 8F); 17 A (9M, 8F)) 31 +/rl (14 Y (6M, 8F); 17 A (8M, 9F)) Y = young (50-70 days old) A = Adult (> 75 days old)
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Gait Podhorna et N= 64 Rotamex 4/8 rotarod Latency time to al. (2004) 33 +/+ (16 apparatus fall off the rotarod Y (8M, was measured. 8F); 17 A Six sessions per (8M, 9F)) mouse (cut-off 31 +/rl (16 time = 30s) Y (8M, 8F); 15 A (8M, 7F)) Y = young (50-70 days old) A = Adult (> 75 days old)
Gait Qiu et al. N= 18 Rotarod Rotarod (2002) 9 +/+; 9 accelerated from +/rl 4 to 40 rpm over a Sex not 3 min period. clear ~6 Four trials each weeks old day (2 days)
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Depth Salinger et N= 49 Visual cliff apparatus inside One 60 s trial per Perception al. (2003) 22 M +/+; of Tru-Scan arena with mouse. Measured 13 M +/rl; checkerboard inlay. pauses before 14 M rl/rl traversing over ~ 70 days the apparent old depth. Pauses indicated that mouse detected the difference.
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Social Salinger et N= 86 Social Dominance Tube One trial per Dominance al. (2003) 33 M +/+; (2.5cm diameter X 55cm mouse. Socially Task 27 M +/rl; long) Holding chamber on dominant: pass 26 M rl/rl either end into opponent’s ~ 70 days cage (goal box). rl/rl more old Submissive: dominant retreating back into own starting box. Tie: neither enter goal box.
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Social Podhorna et N= 63 Clear transparent cage (25cm NMRI mice used Dominance al. (2004) 32 +/+ (17 X 40cm X 15cm) as partner. 5 min Task M, 15 F); social interaction. 31 +/rl (14 Measured M, 17 F) frequency, duration and latency of defined social behaviors
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Novel Object Salinger et N= 36 TruScan Apparatus 5 min trials. Mice Detection al. (2003) 14 +/+ acclimated to box (8M, 6F); with quarter and 13 +/rl dime. One was Faster More approach touches (5M, 8F); removed and to novel to novel 9 rl/rl (5M, replaced with object object 4F) ~ 70 novel (brass key). days old Frequency and latency measured Faster app- roach to novel object
Open Field Salinger et N= 74 TruScan Apparatus with One 60 min trial. Test al. (2003) 20 +/+ (10 inserted floorplate Movements were M, 10F); recorded by 21 +/rl (11 sensor beam Less fear M, 10 F); arrays measured by 33 rl/rl fecal matter (11M, 12F) ~ 70 days old
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl
Open Field Qiu et al. N= 18 Open Field Chamber (27 x One 15 min Test (2002) 9 +/+; 9 27 cm) session. Measured +/rl locomotor and Sex not exploratory clear ~6 behavior of mice weeks old in novel environment
Light/Dark Salinger et N= 74 TruScan Apparatus divided One 5 min trial. Task al. (2003) 20 +/+ (10 into light and dark halves Measured M, 10F); frequency of 21 +/rl (11 transitions, M, 10 F); latency to enter Longer latency 33 rl/rl dark side, number between (11M, of mice to cross. sides 12F) ~ 70 days old
Less dark side entries
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Light/Dark Podhorna et N= 64 Two connected boxes. Light One 5 min trial. Task al. (2004) 33 +/+ (16 side (2/3 of whole) Measured Y (8M, 30cmX27cmX27cm. Dark frequency of 8F); 17 A box 15X27X27cm transitions, (8M, 9F)) latency to enter 31 +/rl (16 dark side, number Y (8M, of mice to cross. 8F); 15 A (8M, 7F)) Y = young (50-70 days old) A = Adult (> 75 days old)
Light/Dark Qiu et al. N= 18 Open Field chambers (27 x One 5 min Task (2002) 9 +/+; 9 27 cm) with black inserts session. +/rl Analyzed time Sex not spent in each clear ~6 zone. weeks old
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Conditioned Salinger et N= 60 TruScan Chamber with 3 once daily 6 Emotional al. (2003) 20 +/+ footshock grid floorplate min trials. Day 1: Response (10M, precondition (80 10F); 21 dB for 10s then 2 +/rl (11M, min off then 10F); and again). Day 2:
19 rl/rl CER trials; after Initially (11M, 8F) 80 dB, foot shock failed to ~ 70 days given. Day 3: test increase old day . Measured freeze freezing behavior
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Conditioned Krueger et N= 18 Med Associates Chamber Training: 3 (1sec, Emotional al. (2006) 8 M +/+; with metal floor grid for foot .4 mA) Response 10 M +/rl shock footshocks during 3mos old 3 min period. ITI 30-60 s. Tests: 24h, 48h, 72h, & 96h later. Measured freezing behavior
Conditioned Qiu et al. N= 18 San Diego Instruments Training: tone Emotional (2002) 9 +/+; 9 Plexiglas conditioning then 2 s of .5 mA Training Phase Response +/rl chamber (26 x 22 x 18 cm) foot shock. 24 Sex not hrs later, returned
clear ~6 and tone only. 24 24 hrs later. less weeks old hrs later novel freezing context with tone. Measured freezing time 48 hours later
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Acute Pain Podhorna et N= 66 Plexiglass cylinder (30cm Sixty s trial. Response al. (2004) 35 +/+ (18 (h) X 20cm (d)) placed over Measured latency Y (10M, hot plate (55 degrees C) to kicking or 8F); 17 A licking (9M, 8F)) 31 +/rl (14 Y (6M, 8F); 17 A (8M, 9F)) Y = young (50-70 days old) A = Adult (> 75 days old)
Acute Pain Qiu et al. N= 18 Hot plate set at 55 degrees C Measured paw Response (2002) 9 +/+; 9 licking as +/rl sensitivity to Sex not noxious stimulus clear ~6 weeks old
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Tests of Krueger et N= 14 Med. Assoc. Oper. Chamber Phase 1: FR1, 25 Impulsivity/ al. (2006) 6 +/+ (4M, with 3 nose poke holes S+, 30 min. 2: Inhibition 2F); 8 +/rl FR3, 25 S+, 30 Control (5M, 3F) min. 3: FR3 with Phase 1 +/rl took longer 3 mos old 3s tone on VI30s. to reach R during tone led criterion to S+, 10S+, 30 min. 4: After tone, can't respond for 1-10s.
All other phases
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Tests of Krueger et N= 20 Med. Assoc. Oper. Chamber Must respond to Impulsivity/ al. (2006) 10 M +/+; with 3 nose poke holes aperture with 32s Inhibition 10 M +/rl visual stim. After Control 3 mos old meet criterion, stim dur decreases (16, 8, 4, 2, 1, 0.8s)
Delayed Krueger et N= 12 Med. Assoc. Oper. Chamber A delay of 2, 5, Matching to al. (2006) 5 M +/+; 7 with nose poke holes 10, or 20s was Position M +/rl presented in 3 mos old between the sample and choice phases. 64 trials per phase
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Morris Water Krueger et N= 19 Morris Water Maze (100cm Latency to reach Maze al. (2006) 9M +/+; in diameter, 24` C) with platform. Spatial 10 M +/rl hidden platform learning=change 3 mos old (12x10.5x11cm) 2cm under in escape latency Mice water level. over time. Platfom removed in final phase. Final phase a measure of spatial learning
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Results Compared to +/+ Assay Researcher N Apparatus Procedures +/rl rl/rl Morris Water Qiu et al. N= 18 91.5 cm Nalgene pool. 8 x 8 Training: 60s to Maze (2002) 9 +/+; 9 cm platform find platform. (7 +/rl days, 4 trials). Sex not Probe: Platform clear ~6 removed. weeks old Training: Platform moved. Probe: Platform removed
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