0022-3565/99/2903-1337$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 290:1337–1346, 1999
Vol. 290, No. 3
Printed in U.S.A.
Identification of Quantitative Trait Loci for Haloperidol-Induced
Catalepsy on Mouse Chromosome 141
ERIK RASMUSSEN, LAURA CIPP, and ROBERT HITZEMANN
Departments of Psychiatry and Psychology, State University of New York at Stony Brook, Stony Brook, New York (E.R., L.C., R.H.); and
Psychiatry and Research Services, Veterans Affairs Medical Center, Northport, New York (R.H.)
Accepted for publication April 20, 1999
This paper is available online at http://www.jpet.org
Research in our laboratory has focused on the role of genetic factors in haloperidol-induced catalepsy (Hitzemann et
al., 1991, 1993, 1994, 1995; Qian et al., 1992; Kanes et al.,
1993, 1996; Dains et al., 1996; Patel et al., 1997). The murine
catalepsy response is phenotypically similar to the extrapyramidal side effects that complicate the use of haloperidol
and related “typical” neuroleptic drugs in the treatment of
psychosis. Our initial reason for focusing on the catalepsy
phenotype was built on the argument that if we could understand the genetic factors that make some strains of mice
remarkably nonresponsive, it might be possible to use this
information to design a new family or families of extrapyramidal side effect-free antipsychotic drugs. In addition, we
recently observed that animals that differ in their sensitivity
to the catalepsy response also differ markedly in two parameters [prepulse inhibition (PPI) of the acoustic startle re-
Received for publication December 18, 1998.
1
This study was supported in part by Grant MH-51372 from the National
Institute of Mental Health, National Institutes of Health; a grant from the
Department of Veterans Affairs; and a grant from the National Alliance for
Research on Schizophrenia and Depression (NARSAD).
highly significant (x2 5 30, p , .00001). Eight percent of the RR
individuals were piebald compared with 30% of the NN individuals. A genome wide scan confirmed the presence of a QTL
(peak LOD 5 6.4) on chromosome 14 near the piebald (Ednrb)
and 5-hydroxytryptamine2A (Htr2a) loci. Although the parental
BALB/cJ and LP/J strains differed significantly in striatal
5-hydroxytryptamine2A receptor binding, no marked differences were detected between the phenotypic extremes. A
second QTL was detected on chromosome 14 (peak LOD 5
6.9), which was located more proximally and included the Chat
locus. No QTLs were detected on chromosomes 1 and 9, thus
differentiating this cross from previous results obtained for a
C57BL/6J 3 DBA/2J intercross.
sponse (ASR) and latent inhibition] that have been found to
be abnormal in a variety of psychiatric disorders, including
schizophrenia (McCaughran et al., 1997; Kline et al., 1998).
Thus, the detection of genes that regulate catalepsy may be
important for understanding a much broader range of phenotypes. To date, our work has proceeded in three steps. The
first step was to document the dimensions and general characteristics of the genetic effect. It was found that among 40
inbred and recombinant inbred (RI) mouse strains, the range
of ED50 values was 50-fold; the most extreme ED50 values
were 0.2 mg/kg (I/J strain) and 9.5 mg/kg (LP/J strain; Kanes
et al., 1993, 1996; Hitzemann et al., 1995; R.H., unpublished
observations). The differences among the inbred strains were
not the result of pharmacokinetic parameters (Kanes et al.,
1993). Furthermore, the inbred strains showed no differential sensitivity for the catalepsy induced by the D1 antagonist
SCH 23390 (Kanes et al., 1993).
The second step of our analysis focused on finding correlated responses, which were associated with the variance in
haloperidol response (Hitzemann et al., 1991, 1993, 1994;
Qian et al., 1992; Kanes et al., 1993, 1996; Patel et al., 1997).
Using a combination of genetic and phenotypic strategies, it
ABBREVIATIONS: PPI, prepulse inhibition; RI, recombinant inbred; QTL, quantitative trait loci; C, BALB/cJ; LP, LP/J; RR, very responsive; NN,
very nonresponsive; R, responsive; N, nonresponsive; ASR, acoustic startle response; 5-HT, 5-hydroxytryptamine; PCR, polymerase chain
reaction; 7-OH-DPAT, 7-hydroxy-2-dipropylaminotetralin; CPu, caudate-putamen; NAc, nucleus accumbens; B6, C57BL/6J; D2, DBA/2J.
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ABSTRACT
Previous studies have established that neuroleptic-induced
catalepsy in mice is a highly heritable trait. The current study
focuses on the detection of quantitative trait loci (QTL) for
haloperidol-induced catalepsy in a BALB/cJ 3 LP/J F2 intercross. One thousand thirty-seven F2 animals were phenotyped
and divided into four categories: very responsive (RR), responsive, nonresponsive, and very nonresponsive (NN). The RR and
NN phenotypes comprised approximately 18% each of the
total and differed in their haloperidol sensitivity by .10-fold.
Sex differed significantly between the NN and RR groups (x2 5
14.0; p , .0002); females comprised 58% of the RR individuals
but only 38% of the NN individuals. The difference between the
extreme phenotypes in the number of piebald animals was
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Rasmussen et al.
Vol. 290
Materials and Methods
Animals and Sample Size. Male and female C and LP mice were
obtained from the Jackson Laboratory (Bar Harbor, ME). At 8 to 10
weeks of age, C 3 LP and LP 3 C pairs were mated to obtain the F1
animals. Twelve pair each of CLP F1 3 CLP F1, and LPC F1 3 LPC
F1 animals were mated to obtain the F2 animals (both F2 types
herein referred to as CLP). On average, three or four litters were
obtained from each breeding pair. At 8 to 12 weeks of age, the F2
animals were tested for the catalepsy response as described below.
Both males and females were used for all studies. Animals were
maintained on a 7:00 AM/7:00 PM light/dark cycle with food and
water available ad libitum.
The required sample size was estimated as described by Soller et
al. (1976) and Lander and Botstein (1989) from n 5 (Za 1 Zb)2/
(s2QTL/s2RES), where Za and Zb are the normal variates for the
desired values of a and b, s2QTL is the variance associated with or
explained by the QTL, and s2RES is the residual unexplained variance. For this study, the minimum h2QTL was arbitrarily set at 0.06.
It is recognized that the effect size for most QTLs is ,0.06; however,
data from other behavioral phenotypes suggested that some QTLs of
this size or larger were likely to be present (see, e.g., Kanes et al.,
1996). For a 5 0.0001 [Za 5 3.89 (two-tailed)] and b 5 0.2 (Zb 5
1.29), the estimated required sample size is 426. However, because
only the extreme phenotypes (defined in Table 1) were to be genotyped, the sample size was increased by 1.15 to maintain statistical
power (Lander and Botstein, 1989). This correction is relatively
small because most of the genetic information in found in the extreme phenotypes. Finally, it was observed early in these experiments that there was a sex effect on response (Table 1). It was
randomly decided to focus the genomic scan on the male progeny and
to confirm any QTLs detected in the female progeny; therefore, it was
necessary to double the sample size to 1000 animals.
Measurement of Catalepsy. One week before the actual catalepsy evaluation, all animals underwent sham testing procedures.
Saline injection never generated a catalepsy response. On the day of
testing, animals were removed from the home cage and placed in
individual cages for 30 min. Animals were then administered haloperidol by i.p. injection. Fifteen minutes after injection, the animals
were tested for catalepsy as described previously (Hitzemann et al.,
1991). For a positive response, the animal must maintain a fixed
rearing posture against the side of the cage for 30 s. To minimize the
possible effect of differences in metabolism or sensitivity, no subsequent time points were evaluated. In some preliminary studies, the
ED50 value was determined in both the F1 and the F2 crosses using
the “up and down” method (Dixon, 1965). For both crosses, the ED50
value was approximately 4 mg/kg. This dose of haloperidol was then
used to screen the F2 animals as haloperidol responders and nonre-
TABLE 1
Coat color, sex distribution, and haloperidol response among CLP F2 animals
The CLP F2 intercross animals were sequentially phenotyped for haloperidol response, first with a 4 mg/kg challenge, followed 1 week later with either a 0.06 or 7.5 mg/kg
challenge. Previous studies (Kanes et al., 1993) have established that one round of repeated testing has no significant effect on the haloperidol ED50 values for the parental
BALB/cJ (C) and LP/J (LP) strains. Haloperidol ED50 values in the C and LP strains are 0.3 and 9.5 mg/kg, respectively.
Response (M/F)
Coat Color
Albino
Light agouti
Agouti
Piebalda
Total
a
RR
R
N
NN
Total
n 5 54
(31/23)
n 5 32
(15/17)
n 5 107
(65/42)
n 5 15
(9/6)
n 5 193
(111/82)
n 5 86
(49/37)
n 5 58
(30/28)
n 5 189
(102/87)
n 5 34
(18/16)
n 5 333
(181/152)
n 5 69
(32/37)
n 5 76
(38/38)
n 5 178
(82/96)
n 5 54
(27/27)
n 5 323
(152/171)
n 5 35
(17/18)
n 5 46
(14/32)
n 5 107
(41/66)
n 5 56
(18/38)
n 5 188
(72/116)
n 5 244
(129/115)
n 5 212
(97/115)
n 5 581
(290/291)
n 5 159
(72/87)
n 5 1037
(516/521)
Piebald animals are counted as members of the light agouti and agouti groups.
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was found that compared with the more responsive lines,
strains, or F2 intercross individuals, the nonresponding animals have a higher density of D2 dopamine receptors and a
higher number of midbrain DA neurons (reviewed in Kanes
et al., 1996). In contrast, the more responsive animals have a
higher density of striatal cholinergic neurons (Dains et al.,
1996).
The third step of our analysis has focused on establishing
the association between haloperidol-induced catalepsy and
specific gene loci. A quantitative trait loci (QTL) analysis of
the BXD RI series led to the detection of six provisional QTLs
(Kanes et al., 1996). Only two of these QTLs were confirmed
in C57BL/6J (B6)/DBA/2J (D2) F2 intercross animals, phenotyped for haloperidol response, and genotyped for microsatellites near the provisional QTLs; one QTL was near the
brown (b) or Tryp1 locus on chromosome 4, and the other was
near the dopamine D2 receptor locus (Drd2) on chromosome
9.
In the current study, we attempted to confirm and expand
on these genotypic results by turning our attention to a QTL
analysis of an F2 intercross formed from the BALB/cJ (C) and
LP/J (LP) inbred strains. The C and LP strains were chosen
for several reasons. First, the strains differ 30-fold in their
ED50 values for haloperidol-induced catalepsy (0.3 versus 9.5
mg/kg). Second, the C and LP strains are highly polymorphic
(Dietrich et al., 1996). Third, the LP strain provides an interesting opportunity to make an association between coat
color and response. The LP strain is piebald and the s or
Ednrb locus is sufficiently near the Htr2a locus such that if a
polymorphism at Htr2a was affecting the catalepsy response,
it could be detected by a difference in piebald spotting between the responsive and nonresponsive animals. The balance between neuroleptic-induced 5-hydroxytryptamine (5HT)2A and D2 receptor blockade is thought to be important
for the development of extrapyramidal symptoms (Meltzer et
al., 1989). Fourth, the C and LP strains are markedly different in D2 dopamine receptor density (higher in the LP strain;
Kanes et al., 1993), and as noted above, D2 receptor density
has been established as a correlated response to catalepsy.
Fifth, this intercross provides an opportunity to test against
a different genetic background if there is a phenotypic association between the catalepsy response and PPI of the ASR
(McCaughran et al., 1997; Kline et al., 1998).
1999
1339
blocks of six trial types: startle (110-dB SPL white noise) alone;
80-dB SPL prepulse tone delivered at 5, 10, 15, or 20 kHz; and a null
trial. The trial types were delivered in a pseudorandom order, and
the calculation of PPI was identical with that for the standard
paradigm.
One week after completing the PPI test, mice were examined for
locomotor activity using a procedure that paralleled the catalepsy
testing. Mice were removed from the home cage and placed individually in the testing arena; the arena floor was covered with standard
laboratory bedding. Thirty minutes later, the mice were administered saline and returned to the testing arena, and activity was
monitored for 20 min. Activity was assessed in a San Diego Instruments Flex Field locomotor system. The apparatus consisted of a
four-by-eight array of photocells mounted in a 25 3 47-cm metal
frame, situated 1 cm off the floor, and surrounding a 22 3 42 3 20-cm
high plastic arena. Activity was recorded over four 5-min blocks. The
distance traveled during each block was used as the measure of
activity.
Quantitative Receptor Autoradiography. Binding to the
D2/D3 receptor subtypes was determined using quantitative receptor
autoradiography and 125I-labeled epidepride as the ligand. For our
initial studies, the ligand was kindly provided by Dr. Aaron
Janowsky (Portland, OR). More recently, the ligand was synthesized
in our laboratory as described by Clanton et al. (1991). Using the
experimental conditions described below, raclopride (200 nM) completely inhibited the binding of epidepride. This concentration of
raclopride is one that will block binding to D2 and D3 but not D4
receptors (Seeman and Van Tol, 1994). Previously (Kanes et al.,
1996), we examined the binding of [3H]7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT; Levesque et al., 1992) to estimate the regional
localization of D3 receptor binding and the extent to which D3 receptor binding contributes to the overall epidepride binding. Significant
7-OH-DPAT binding was detected only in the ventral striatum and
olfactory tubercle; however, at five times the KD concentration, the
binding of 7-OH-DPAT was never more than 2.5% of the epidepride
binding at a comparable concentration. In other brain areas, including the caudate putamen (CPu), substantia nigra zona compacta,
and the ventral tegmental area, the binding of 7-OH-DPAT was ,1%
of the epidepride binding.
Brains were sliced in 20-mm sections and thaw mounted onto
gelatin-subbed microscope slides into six sets from the most rostral
aspect of the basal ganglia to the retrorubral A8 neurons. Adjacent
sections were used for nonspecific binding. Slides were either used
immediately or stored at 280°C until needed. Frozen slides were
first warmed to room temperature under a gentle stream of air and
then preincubated for 30 min at 4°C in incubation buffer (50 mM
Tris, pH 5 7, 120 mM NaCl) without ligand. These slices were
transferred to fresh buffer at 24°C for 30 min and then incubated
with 125I-labeled epidepride (25, 50, or 200 pM; KD 5 50 pM) in
standard incubation buffer at 30°C for 2 h. After washing four times
in ice-cold buffer, slides were air dried, desiccated overnight at 4°C,
and then exposed to high performance autoradiography film (Hyperfilm 3H; Amersham, Inc., Buckinghamshire, UK) for 4 to 6 h. Specific
receptor binding was defined as the binding of 125I-labeled epidepride in the presence or absence of 10 mM sulpiride. Because 125Ilabeled epidepride also binds appreciably to a2-adrenergic receptors,
100 nM idazoxan was included in all incubation mixtures.
Binding to 5-HT2A receptors was measured essentially as described by Lidow et al. (1989) and followed the general design described above for dopamine receptor binding. The binding of [3H]ketanserin (Amersham, Arlington Heights, IL) was measured at 1.0 nM,
which previous studies have established to be the approximate KD
concentration (e.g., Pazos et al., 1985). Prazosin and amino-6,7dihydroxy-1,2,3,4-tetrahydronaphthalene were added to the incubation mixture to block any binding to D2 and a2 receptors, respectively. Additionally, 10 mm tetrabenazine was added to the
incubation mixture to block ketanserin binding to the tetrabenazine
displaceable sites associated with the synaptic vesicle monoamine
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sponders. One week later, the responders and nonresponders were
challenged with 0.06 and 7.5 mg/kg, respectively. This challenge
provided four phenotypic categories: very responsive (RR), responsive (R), nonresponsive (N), and very nonresponsive (NN). The doses
of haloperidol used in the second challenge were empirically determined to provide RR and NN samples of equal size, with each
containing approximately 18% of the total F2 population.
Measurement of PPI of ASR and Locomotor Activity. A
subset (n 5 250) of the male intercross progeny was also tested for
PPI of the ASR and baseline locomotor activity; only males were used
because of the marked effect of the estrous cycle on PPI (Swerdlow et
al., 1995). Locomotor activity was included in the behavioral testing
battery because data from our laboratory (Kline et al., 1998) suggested that in mice, baseline activity was correlated with PPI and
the catalepsy response. The sequence of testing was PPI to activity to
catalepsy. The parental strains were used as sentinel animals to
determine whether there were carryover effects from the multiple
testing; no carryover effects were detected.
A Coulbourn Instruments ASR test system (Coulbourn Instruments Inc., Lehigh Valley, PA) was used to evaluate the ASR and
PPI of the ASR. Startle platforms were coupled to strain gauge
transducers for detection of the ASR. The signal from each platform
was digitized by a series of AD converters, and a 200-ms portion of
the signal was analyzed starting from the initiation of the startle
stimulus. The strain gauges were calibrated over a 10 to 100g range
with the animal holders in place. Startle amplitude reflected the
animal’s weight plus the weight generated by the animal in response
to the startle stimulus. ASR and prepulse acoustic stimuli were
generated by a voltage-controlled oscillator, amplified by a Coulbourn Instruments acoustic pulse power amplifier, and delivered to
the test chamber by JBL 2425H and JBL 2105H speakers mounted
in the floor and ceiling, respectively. ASR and prepulse stimuli
amplitudes were determined by a Klark-Tecknik DN 60 Real Time
Sound Analyzer and were approximately 0.0002 dyne/cm2. Acoustic
stimuli were shaped with a rise/fall gate to conform to a linear
envelope with a 2.0-ms rise/fall time. The startle platforms and
speakers were housed within a test chamber (50 3 50 3 30 cm high)
lined with 4 cm of acoustic foam. A fan mounted in the floor of the
chamber provided constant ventilation. The background noise level
with the chamber closed was 50 dB. The mouse holders do not
restrain the animal. Four mice were typically tested at one time.
The standard paradigm for the measurement of the ASR and PPI
of the ASR has been described in detail elsewhere (McCaughran et
al., 1997). A startle session consisted of 12 blocks of five trial types.
Each trial type was presented in pseudorandom order and separated
by an intertrial interval of 5 to 20 s (mean, 15 s). The startle stimulus
alone (P-alone trials) consisted of a 60-ms, 110-dB SPL, 10-kHz tone.
Each startle session was initiated by a 5.0-min habituation period
followed by an orienting P-alone trial. This trial was not included in
the statistical analysis of the results. Inhibition of the ASR by a
20-ms white noise burst delivered 100 ms before the startle stimulus
was examined at three different intensities: 56, 68, and 80 dB. A null
trial that consisted of no prepulse or startle stimuli was used as the
baseline for the calculation of the ASR. The null trial was also used
as an indicator of mouse activity within the restrainer. High levels of
activity within the restraining cage produce greater response amplitudes on the null trials because of the increased likelihood of movement at the point that the trial is delivered. ASR amplitude was
defined as P-alone ASR (g) 2 null trial response (g). PPI of the ASR
at each prepulse intensity was calculated as 100 2 [ASR associated
with each prepulse intensity (g)/ASR after the P-alone (g)] 3 100. A
value of 100% would be defined as complete inhibition of the ASR,
whereas a value of 0% would indicate no inhibition. Previous studies
on more than 2000 mice have established that when the startle
response is ,3 g, the reliability of the PPI data drops precipitously.
For this reason, all animals with an ASR of ,3 g (4.5% of the total)
were censored from the analysis.
For the frequency-dependent PPI test, the session consisted of 10
QTLs for Haloperidol-Induced Catalepsy
1340
Rasmussen et al.
Results
Characteristics of Catalepsy Phenotype. One thousand thirty-seven CLP F2 animals were phenotyped for hal-
operidol response as described in Materials and Methods.
The initial and secondary challenge doses of haloperidol were
empirically determined to provide equal numbers of RR (n 5
193) and NN animals (n 5 188). CLP F2 and LPC F2 animals
were equally represented among the phenotypes. Based on
the selection criteria used, the NN and RR animals differed
by at least 10-fold in their sensitivity to haloperidol-induced
catalepsy. The association between haloperidol response, sex,
and coat color is summarized in Table 1. The data analysis
focused on the NN and RR groups because these individuals
contain most of the relevant linkage information. Sex differed significantly between the NN and RR groups (x2 5 14.0;
p , .0002); females composed 58% of the RR individuals but
only 38% of the NN individuals. Albinism differed significantly between the NN and RR individuals, but the effect
was limited to males (x2 5 4.6; p , .03) and was not seen in
females (x2 5 0.42); 28% of the RR males were albino compared with 15% of the NN males. The nonalbino animals
could easily be characterized as light agouti and agouti; genetic marker data revealed that the light agouti phenotype is
associated with the b/b genotype. Compared with the RR
group, the number of light agouti animals was significantly
higher in the NN group (x2 5 4.6; p , .03). The difference
between the RR and NN individuals in the number of piebald
animals was highly significant (x2 5 30, p , .00001). Eight
percent of the RR individuals were piebald compared with
30% of the NN individuals; no sex 3 piebald interaction was
detected.
Before the measurement of catalepsy, a random subgroup
(n 5 250, males only) of the F2 sample was analyzed for their
baseline activity response; data were collected in four 5-min
blocks after placing the animals in the activity apparatus.
The data obtained are presented in Fig. 1, with the animals
categorized according to their haloperidol response. At no
time interval was there a significant group effect (p . .75).
The ASR and PPI of the ASR (Fig. 2) were also measured
in this subgroup of animals. PPI was measured at three
different prepulse intensities (56, 68, and 80 dB SPL); back-
Fig. 1. The relationship between basal locomotor activity and catalepsy
in the CLP intercross. A random subgroup of the animals characterized in
Table 1 (n 5 250, males only) were examined for their basal locomotor
activity before determination of the catalepsy response. Data were collected for 20 min, in 5-min blocks, beginning immediately after placing
the animals in the activity apparatus. The codes refer to the catalepsy
phenotypes (Table 1). The sample sizes per group were 44, 78, 82, and 46
for the RR, R, N, and NN groups, respectively. Data are expressed as the
mean 6 S.E. distance traveled in cm/5-min block.
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transporter. Specific binding was defined as the difference in binding
in the absence and presence of 10 mM methysergide.
All films were calibrated to radioactive standards (125I and 3H
Microscales; Amersham Pharmacia Biotech) and read with a computer-based image analysis system (MCID; Imaging Research, St.
Catherine’s, Ontario, Canada) using landmarks and regional definitions identical with those described previously (Qian et al., 1992;
Kanes et al., 1993). Further details, including drawings of the regions analyzed, are found elsewhere (Qian et al., 1992; Hitzemann et
al., 1993, 1994; Kanes et al., 1993). The data were analyzed for a
particular brain region by a general ANOVA program (CSS; Statsoft
Inc., Tulsa, OK) for the effects of haloperidol response (RR versus
NN), section, sex, and interactions among these effects. The StudentNeuman-Keuls test was used in the post hoc analyses.
DNA Isolation. High-molecular-weight genomic DNA was isolated from liver samples as follows: 250 to 500 mg of liver tissue was
minced with a sterile razor blade, transferred to a 15-ml polypropylene Falcon tube with 5 ml of lysis buffer (100 mM Tris z HCl (pH
8.0), 5 mM EDTA, 100 mg/ml proteinase K, 200 mM NaCl), and
incubated, with rocking at 55°C overnight. After incubation, 20 ml/ml
of 5 M NaCl was added with gentle inversion. The tissue digest was
extracted twice with equilibrated phenol, once with equal volumes of
phenol and chloroform/isoamyl alcohol (chisam; 24:1), and once with
chisam alone. DNA was precipitated with 0.5 volume of 7.5 M ammonium acetate and 2 volumes of ice-cold ethanol. Dried DNA pellets
were resuspended in double-distilled water (ddH2O). Purity and
concentration of the final samples were evaluated by UV spectroscopy, and only samples with a 260/280 ratio of .1.4 were used for
genotyping.
Genotyping Microsatellite Polymorphisms. All of the genotyping involved the -(CA)n- repeating microsatellites first described
by Dietrich et al. (1996). The polymerase chain reaction (PCR)
primer sets were obtained from the MIT/Whitehead catalog (Research Genetics, Huntsville, AL). The phenotypic extremes were
genotyped for the presence of a C and/or an LP allele by PCRmediated amplification of simple sequence repeats (microsatellite)
that are polymorphic for these two strains (Dietrich et al., 1996).
Approximately 100 to 200 ng of the genomic DNA was used as a
template in a 20-ml reaction containing 8 pmol (0.4 mM) of each
primer, 0.5 unit of Taq DNA Polymerase (Perkin-Elmer Cetus, Norwalk, CT, or Boehringer Mannheim, Indianapolis, IN), 37.5 mM
concentration of each nucleotide, and 13 Taq buffer; the final Mg21
concentrations ranged from 1.5 to 3.0 mM. The template was heatdenatured for 5 min at 95°C (one cycle), followed by 30 cycles of
amplification (94°C for 45 s, 55°C for 1 min, and 72°C for 20 s) and
final extension at 74°C for 5 min. The PCR products were separated
by electrophoresis on 3% Metaphor agarose gels (FMC Bioproducts,
Rockland, ME) in 13 Tris-buffered EDTA and visualized by
ethidium bromide staining.
Detection and Mapping of QTLs. Twenty-five of each of the
extreme phenotypes (males only) were randomly selected for the
genome wide scan. For QTL detection, markers spaced at approximately 20 cm were used (Darvasi et al., 1993). The screening threshold for a significant segregation of the alleles was set at p , .1. For
markers meeting the screening threshold, 25 of each of the extreme
phenotypes were again randomly selected and genotyped to confirm
a significant effect. For markers significant at p , .01, the entire
sample of phenotypic extremes (males and females) was genotyped.
For markers with an LOD of $3 (only chromosome 14), additional
markers were added in the region of interest for fine mapping.
Genotypic data were principally analyzed using the x2 statistic; LOD
scores were estimated from LOD 5 0.2173 (x2) for an additive (df 5
1) model (Lander and Botstein, 1989).
Vol. 290
1999
QTLs for Haloperidol-Induced Catalepsy
1341
ground noise was 50 dB SPL. There was no significant group
effect for the ASR (p , .68), for PPI80 (p . .32), for PPI68 (p .
.89), or for PPI56 (p . .21). Overall, these data illustrate that
there was no relationship between haloperidol response and
locomotor activity, the ASR, or PPI.
D2/D3 dopamine receptor binding was measured in the core
and shell of the nucleus accumbens (NAc) and the ventrolateral and dorsomedial CPu using 125I-labeled epidepride in 30
each of the phenotypic extremes, equally divided between
males and females. Three concentrations of 125I-labeled epidepride were examined: 25, 50, and 200 pM (the KD value for
epidepride binding is approximately 50 pM; Kanes et al.,
1996). In each region, data were collected from multiple
sections (see Materials and Methods). Although there are
significant rostral/caudal D2 receptor binding gradients
within the striatum (Qian et al., 1992; Kanes et al., 1993), no
significant group 3 section interactions were detected in any
of the four striatal regions examined. The data obtained were
collapsed across sections, and the results are presented in
Fig. 3. Regardless of region, sex, or concentration, no significant (p . .1) group effects were detected.
Genomewide Scan of CLP Intercross for CatalepsyRelated QTLs. The locations of the Mit microsatellite markers used for the genomewide scan are illustrated in Fig. 4. In
general, the distance between markers was approximately 20
cm. The stepwise approach used for the genomewide scan is
described in Materials and Methods. A summary of the results, presented as a probability histogram derived from the
x2 statistic, is found in Fig. 5. The results indicate that a QTL
or QTLs exceeding the threshold of Lander and Krugylak
(1995; i.e., LOD $ 4.3) were found only on chromosome 14.
The segregation of alleles on chromosome 14 was in the
expected direction; LP alleles were associated with nonresponse, and BALB/c alleles were associated with response.
On chromosome 9 where we previously identified a catalepsy
QTL in the BXD RI series and a B6D2 F2 intercross (Kanes
et al., 1996), only a weak signal was detected despite genotyping the full sample of extreme individuals. However, sim-
Fig. 3. The relationship between striatal D2 dopamine receptor binding
and catalepsy response in the CLP intercross. Thirty of each of the RR
and NN phenotypic extremes (equally divided among males and females)
were used for quantitative receptor autoradiography as described elsewhere (Kanes et al., 1996) with 125I-labeled epidepride as the ligand. Data
were collected in the NAc and CPu at 25, 50, and 200 pM. The estimated
KD is value 40 to 60 pM. Data are expressed as mean 6 S.E. fmol/mg
bound.
ilar to our previous results, it was the alleles from the responding parental strain (C) that segregated with the
nonresponding animals.
Figure 6 expands the results for chromosome 14. The data
are presented in two ways. The unmodified map was obtained by converting the x2 value for a particular marker to
an LOD score. The unmodified map is suggestive of two
QTLs, with one peak at D14 Mit121 (LOD 5 6.9) and another
peak at D14 Mit196 (LOD 5 6.4); these LOD values are
associated with 9.6 and 9.0% of the phenotypic variance,
respectively.
As an alternative to the marker by marker LOD map, a
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 2. The relationships among the ASR, PPI of the ASR, and the
catalepsy response in the CLP intercross. After testing for locomotor
activity, animals were tested for the ASR and PPI of the ASR as described
in Materials and Methods. PPI80, PPI68, and PPI56 refer to the data
obtained for the 80-, 68-, and 56-dB SPL prepulse effect. The ASR data
are expressed as mean 6 S.E. grams of force above the null trial. The PPI
data are expressed as percent 6 S.E. inhibition of the ASR. The question
of whether the various phenotypic categories differed in high-frequency
hearing loss, which can effect the ASR and PPI, was examined as described in McCaughran et al. (1999). No difference was detected among
the groups.
1342
Rasmussen et al.
Vol. 290
Fig. 5. Summary of the genomewide scan for catalepsy related QTLs in
the CLP intercross in the form of a probability histogram. The strategy
for the genomewide scan is described in Materials and Methods. Briefly,
25 to 30 of each of the RR and NN extremes were randomly selected and
genotyped for a particular marker. If a significant effect was detected at
p , .05 or better, an additional set of 25 to 30 of each of the extremes was
genotyped. If the effect was then significant at p , .01 or better, all of the
phenotypic extremes were genotyped. For the initial screening, only
males were used. The entire sample of extremes (males and females) was
genotyped on chromosomes 9 and 14. The data were plotted as the x2
value for a particular marker; four levels of significance are indicated in
the graph and the LOD threshold of 4.3. Only the QTLs on chromosome
14 exceeded the LOD threshold of 4.3.
pseudo composite interval map (Basten et al., 1997) was
constructed. Such a map includes the effects of background
markers; for parametric data, the background data can either make the analysis more sensitive to the effects of the
QTL in the target interval or help to separate closely linked
QTLs. The map built from the knowledge that catalepsy is
Fig. 6. Schematic of the chromosome 14 QTL map. The unmodified map
illustrates the marker by marker LOD scores obtained from the x2 values
(see Materials and Methods). The composite map was obtained by simulating ED50 values for individual animals and then applying the composite interval mapping function of QTL Cartographer (see text). The dotted
line is LOD 5 4.3 threshold for an F2 analysis.
actually a quantitative trait and that the upper and lower
boundaries for haloperidol-induced catalepsy in Mus musculus were 0.2 and 9.5 mg/kg (Kanes et al., 1993, 1996; Hitze-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 4. Positions of the microsatellite markers used in the genomewide scan of the CLP intercross. On chromosomes 9 and 14, additional markers were
added for fine mapping.
1999
mann et al., 1995). Based on this information, a simulated
distribution of ED50 values was created that would produce
the categorical distribution found in Table 1. The simulated
ED50 values obtained for each response category were then
randomly assigned to individuals within that category, and
the data were analyzed using the composite interval mapping module of QTL Cartographer (Basten et al., 1997). The
random assignment and analysis were repeated 1000 times
to generate the composite interval map in Fig. 6. A background map was obtained by complete random assignment of
phenotype to genotype and repeating the analysis 1000
times; the mean LOD score obtained was 1 (data not shown).
The composite map indicated a peak LOD of 8.4 in the proximal region of chromosome 14.
5-HT2A Receptor Binding in Phenotypic Extremes.
The data in Fig. 6 suggest the presence of a QTL (or QTLs)
near the Htr2a locus. To determine whether there was a
difference in 5-HT2A receptor binding between the pheno-
QTLs for Haloperidol-Induced Catalepsy
1343
typic extremes, [3H]ketanserin binding was measured in the
RR and NN phenotypic extremes at 1.0 nM, the approximate
KD concentration (Pazos et al., 1985). Data were also obtained for the parental strains and the C57BL/6J strain for
comparison with previous inbred strain data on 5-HT2A receptor binding (Boehme and Ciaranello, 1983). The results
for the NAc and CPu are presented in Fig. 7. For the NAc
core, the ANOVA revealed significant effects for group (F4,214
5 7.2, p , 2 3 1025), section (F5,214 5 7.5, p , 2 3 1026) but
not the group 3 section interaction (F20,214 5 0.4, p . .99).
Collapsing across sections, binding was marginally higher in
the RR compared with the NN group (12%, p , 3 3 1022);
there was no significant difference between the parental
strains. For the NAc shell, a significant effect was detected
for group (F4,179 5 7.2, p , 2 3 1023) but not the section or
group 3 section interaction; the group difference was associated with a lower binding (215%) in the C57BL/6 strain
compared with the LP strain. There was no significant difDownloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 7. Striatal 5-HT2A receptor binding in the RR and NN phenotypic extremes and the BALB/cJ, LP/J, and
C57BL/6J inbred mouse strains. Data
were collected using quantitative receptor autoradiography in the NAc
and CPu at 1 nM, the estimated KD
concentration. Sections were collected
from rostral to caudal. n 5 10/group.
Data are expressed as the mean 6
S.E. fmol/mg protein.
1344
Rasmussen et al.
Discussion
The present study extends our genetic analyses of the
catalepsy response to a CLP F2 intercross. Previous studies
have largely focused on the B6 and D2 inbred strains, the RI
series derived from these strains, and B6D2 intercross animals (e.g., Dains et al., 1996; Kanes et al., 1996; Patel et al.,
1997). B6D2 genotypes have been widely used for behavioral
QTL analysis, and this emphasis has facilitated the integration of results from different laboratories and the development of consensus QTLs for several behavioral phenotypes.
However, the focus on the B6 and D2 inbred strains and the
crosses derived from these strains may provide a relatively
narrow view of the sources of genetic variation for a particular phenotype. As noted in the introduction, expanding the
analysis to a CLP intercross appeared justified for several
reasons; indeed, the genotypic data suggest that there are
substantial differences in the regulation of the catalepsy
response when comparing the CLP intercross with either the
BXD RI series or a B6D2 intercross. In a previous report, six
candidate QTLs for catalepsy significant at p , .01 or better
were identified from the analysis of the BXD RI strain mean
values (Kanes et al., 1996). The QTLs on chromosomes 4 and
9 were subsequently confirmed in a B6D2 F2 intercross. The
QTL on chromosome 9 was of special interest because it
appeared to be closely linked to the Drd2 locus. Subsequently, a genomewide scan (Patel et al., 1998) in a larger
B6D2 F2 sample again confirmed the QTL on chromosome 9
and identified an additional QTL on distal chromosome 1 in
the same general region where numerous QTLs have been
identified for behavioral phenotypes, including open-field activity (Flint et al., 1995). The data in Fig. 5 illustrate that for
the CLP intercross, no significant catalepsy QTLs were detected on chromosomes 1 and 4 and only a very modest effect
was detected on chromosome 9. Coat color data (Table 1)
suggested the presence of QTLs near the c or Tyr and b or
Tyrp1 loci on chromosomes 7 and 4, respectively; however,
neither of these QTLs were confirmed in the genotypic analysis. The CLP intercross also differed from the B6D2 inter-
cross in that there was a significant sex effect; females were
overrepresented in the RR phenotype, and males were overrepresented in the NN phenotype. However, no significant X
chromosome QTLs were detected, suggesting that the sex
effect is distributed across the autosomal QTLs.
The data in Figs. 5 and 6 illustrate that for the CLP
intercross, a major QTL or QTLs for catalepsy were found on
chromosome 14; not even suggestive QTLs were found on this
chromosome in the BXD RI series and the B6D2 intercross.
The QTL pattern on this chromosome was essentially identical in both sexes, indicating that the source of the sex effect
was not chromosome 14 (data not shown). The data in Fig. 6
may suggest that two QTLs are present; however, this interpretation of the data should be viewed cautiously. Given the
limited resolution of QTL analysis with a moderate-sized F2
sample (Davarsi, 1998), one can only conclude with some
surety that a QTL was present on chromosome 14. Although
recognizing the limitations of the methodology, it is still of
interest to note that the proximal QTL region contains many
potentially relevant candidate genes, including Chat (choline
acetyltransferase at 10.5 cm), Grid1 (glutamate receptor,
ionotropic, d1 at 13.5 cm), Glud (glutamate dehydrogenase at
15.5 cm), hph1 (hyperphenylalaninemia 1 at 19.5 cm), and
Acra2 (acetylcholine receptor, a2, neural at 21 cm).
Previously, we reported a significant difference in the number of cholinergic neurons between the NR and NNR lines of
mice (Hitzemann et al., 1993, 1994); in general, it was found
that the NR line had on average 40% more cholinergic neurons in the rostral-to-medial aspect of the striatum. Similar
results were obtained in the phenotypic extremes of a B6D2
intercross (Dains et al., 1996). Overall, these data were seen
as entirely consistent with the well established interactions
of the striatal cholinergic and dopamine systems (Doshay
and Constable, 1957; Calne, 1978; Stoof et al., 1992). In the
BXD RI series, significant (p , .01) QTLs for the number of
cholinergic neurons were found on chromosomes 1, 6, 9, and
12; these data suggested that at least for the B6D2 genotypes, a polymorphism in Chat was not associated with the
differences in the number of cholinergic neurons. In regard to
the current study, it is of interest to note that in comparison
to the C strain, it is the nonresponsive LP strain that has a
higher number of cholinergic neurons (Dains et al., 1996).
Nevertheless, the data presented here and previously from
our laboratory suggest that some further exploration of the
role of the cholinergic system in the genetics of the catalepsy
response is warranted.
The QTL on the distal region of chromosome 14 was not
only detected through the microsatellite analysis but also
from the difference in piebald spotting between the phenotypic extremes (Table 1). These QTL data prompted an examination of 5-HT2A receptor binding (Fig. 7). With the exception of a small effect in the NAc core, no significant
difference was noted in receptor binding between the RR and
NN extremes. These data suggest that a polymorphism that
has marked effects on 5-HT2A receptor availability is not
associated with the Htr2a locus. However, the data presented
here do not preclude the possibility of a functional change in
the receptor. In this regard, polymorphisms in the human
gene have been reported (Erdmann et al., 1996; Spurlock et
al., 1998), although to our knowledge there have been no
reports of polymorphisms in the coding sequence that would
affect receptor affinity or receptor coupling. It is of some
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
ference between the RR and NN extremes or the C and LP
strains. Similar to the NAc shell in the dorsomedial CPu, a
significant effect was detected for the group interaction
(F4,467 5 7.6, p , 6 3 1026) but not the section effect or the
group 3 section interaction. Post hoc analysis of the group
effect revealed that the RR and NN extremes and the
C57BL/6 and C strains all had significantly lower binding
than the LP strain (p , 1022 or better); the most significant
difference was between the C and LP strain (38%, p , 3 3
1025). The RR and NN extremes were not significantly different (p . 5 3 1021). For the ventrolateral CPu, the ANOVA
revealed significant effects for group (F4,499 5 51, p , 1029),
section (F13,499 5 26, p , 1029), and the group 3 section
interaction (F52,499 5 1.8, p , 9 3 1025). In this region, the
NN and RR extremes and the LP strain had significantly
higher binding than that found in either the C or C57BL/6J
strain; the most marked difference was between the C and
LP strains (27%, p , 8 3 1026). Post hoc analysis of the
strain 3 section interaction revealed that this pattern of
group differences was most marked in the rostral aspect of
the ventrolateral CPu. No significant differences between the
RR and NN extremes were found in the ventrolateral CPu.
Vol. 290
1999
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Send reprint requests to: Dr. Robert Hitzemann, Department of Psychiatry,
State University of New York at Stony Brook, Stony Brook, NY 11794-8101.
E-mail: [email protected]
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