Laszlo Zaborszky1,3 and Csaba Vadasz2,3
1Laszlo Zaborszky, M.D., Ph.D., D.Sc.
Associate Professor
Center for Molecular and
Behavioral Neuroscience
Rutgers University
197 University Ave.
Newark, NJ 07102
Fax: (973)353-1844
E-mail: zaborszky@axon.rutgers.edu
2Csaba Vadasz, Ph.D.
Director, Neurobehavioral Genetic Research Program
New York University Medical Center
The Nathan S. Kline Institute for Psychiatric Research
140 Old Orangeburg Road
Orangeburg, NY 10962
Fax:(845) 398-5531
E-mail:VADASZ@NKI.RFMH.ORG
3Correspondence
can be addressed to both authors
ABSTRACT
The mesotelencephalic dopamine system is
genetically variable and affects motor behavior, motivation, and learning. Here we examine the genetic variation of
mesencephalic DA neuron number in a quasi-congenic RQI mouse strain and its
background partner, and a recombinant inbred strain with different levels
of mesencephalic tyrosine hydroxylase
activity (TH/MES). We used B6.Cb4i5-a6/Vad, C57BL/6By, and CXBI, which are known to express high,
intermediate, and low levels of TH/MES, respectively. Unbiased stereological
sampling with optical disector counting methods were employed to estimate the
number of TH-positive neurons in the A8-A9-A10 cell groups. Morphometric
studies on the mesencephalic dopamine cell groups indicated that male mice of
the B6.Cb4i5-a6/Vad strain
were endowed with a significantly lower number of TH-positive cells than CXBI
mice. In all strains studied, the right
retrorubral field (A8 area) had a higher number of dopamine neurons compared to
the left A8 area. The results suggest an
inverse relationship between TH/MES and number of dopamine neurons in the A9-A10
cell groups, and significant lateral asymmetry in the A8 cell group. A detailed
anatomical atlas of the mesencephalic A8-A9-A10 dopaminergic cell groups
in the mouse is also presented to facilitate the assignment of TH-positive
neurons to specific cell groups.
Key words: complex trait, QTL
introgression, tyrosine hydroxylase, dopamine neuron, stereology, mesencephalon
In the past several decades, it was
hoped that genetic strain differences in neural phenotypes (e.g., in brain morphology
or in neurotransmitter level) would explain associated behavioral differences, and that such discovery would
lead to the development of animal models
for human pathological behavior.
The mesotelencephalic dopamine system has been implicated in several
neuropsychiatric disorders, such as schizophrenia (Meltzer and Stahl, 1976), Parkinson's disease (Hornykiewicz, 1979), attention-deficit hyperactivity disorder (Faraone and Biederman, 1998), substance abuse (Koob,
1999), in the control of motor activity and learning (Beninger, 1983), attention (Yamaguchi
and Kobayashi, 1998), and other behaviors. Although it has been well
established that genetic factors contribute to variations at both the neural
and behavioral levels, it has been difficult to clarify the mechanisms by which
these behaviors are modulated by a specific genetic change in the
mesotelencephalic dopamine system. The
integrated use of more refined genomic, neurobiological, and behavioral
strategies is needed for the better understanding of these mechanisms. Towards
this end, we conceptualized and experimentally tested the quasi-congenic
Recombinant QTL Introgression (RQI) animal models for continuously distributed
quantitative traits by the development of
B6.C and B6.I replicate lines (Vadasz, 1990), and sets of RQI strains (Vadasz et al., 1994;
Vadasz et al., 1996; Vadasz et al., 1998). These lines were constructed by repeated backcrosses
to a background strain C57BL/6By (B6, characterized by intermediate TH/MES)
with concomitant selection for high (B6.C) and low (B6.I) expression of
mesencephalic tyrosine hydroxylase (TH) activity. Subsequently, using B6.C-derived
inbred RQI strains we demonstrated that from the donor BALB/cJ strain
(characterized by high TH/MES) virtually all major QTLs could be introgressed
into a new strain B6.Cb4i5-a6/Vad, which was
about 95% identical genomicly with the B6 background strain (Vadasz et al., 1998). Here, we present data on genetic variation of the
number of TH-expressing neurons in three different mesencephalic cell-groups.
To facilitate the proper cell-group-assignment of the TH-immunostained neurons,
we provide an anatomical atlas1 of the
different TH-positive dopaminergic cell groups in the mesencephalon of the
laboratory mouse.
Animals were
maintained in our Research Colony at The Nathan Kline Institute Animal Facility
on a 12/12 light/dark schedule (lights on at 6 a.m., lights off at 6 p.m.) with
free access to food (Purina #5008). After weaning at the age of 5-6 weeks
(depending on the development of the litter), litter-mate males were housed
together. For immunocytochemistry, 16 adult male mice weighing 22.5-30.4 g at
the time of the perfusion were used. Cell counting was carried out on 4 male
mice per strain. Subsequently, data from one CXBI subject was excluded because
of uneven staining in some of the sections.
To develop QTL Introgression (QI)
lines, we used BALB/cJ (C) and CXBI/By (I) as donor strains because previous
research demonstrated that among several highly inbred strains, C and I had the
highest and lowest TH/MES (Vadasz et al., 1982). I is a recombinant
inbred strain carrying C57BL/6ByJ (B6) and C genes (Bailey, 1981). B6 served as
the background strain, because its TH/MES was intermediate between those of the
donor strains, and it had already been used as a background strain for numerous
congenic lines. QTLs responsible for
the continuous variation of TH/MES were introgressed onto the B6 strain
background from C and I donor strains with high and low TH/MES, respectively. F2
generations were derived from mating B6 females to C or to I males. a and ß closed
replicate lines were created by equal division of each (B6XC)F2 and
(B6XI)F2 litter resulting in four QI lines: B6.C-a, B6.C-ß, B6.I-a, and B6.I-ß.
The QTL transfer was carried out in two directions by four or five
backcross-intercross cycles with concomitant selection for the extreme high and
low expression of TH/MES in replicates, resulting in four QTL introgression
lines. In the B6.C and B6.I introgression lines the top and bottom one-third of
the population were selected, respectively.
A comparison of the population means for TH/MES after five
backcross-intercross cycles (i.e., in the b5i7
generation) indicated that within introgression type (B6.C or B6.I) the
replicate lines were not significantly different; however, B6.C and B6.I
replicate lines expressed significantly higher and lower TH/MES than that of
the B6 background (one-way ANOVA followed by Tukey’s post hoc multiple
comparison tests: B6.C-a, B6.C- ß > B6 > B6.I-a, B6.I- ß; HSD
alpha =0.05, df=272).
In order to drive
the heterozygous genes into a homozygous condition (fixation), the QTL
introgression phase was followed by initiation of bxs mating for at least 30
generations. As to the nomenclature,
the full name of an RQI strain, for instance, B6.Cb4i5-a6/Vad, can be
abbreviated as C4A6. The first letter, I (or C), stands for the donor strain
name; 4 designates the
backcross-intercross series (b4i5); A (or B) indicates
the replicate line a (or b), and the last
character; 6, is the identification number of the strain. Here the RQI strain
is referred to as "quasi-congenic" in similar sense that
"congenic" is used for the Recombinant Congenic (RC) series (Demant and Hart, 1986).
Under deep anesthesia with a mixture of
Diazepam (220 uL) and Innovar (85 uL), the animals were perfused through the
heart with 30 mL of saline followed by 100 mL of an ice-cold solution
containing 4% paraformaldehyde, 15% picric acid, and 0.05% glutaraldehyde in
0.1M phosphate buffer (PB; pH 7.4). This was followed by 100 mL of the same
fixative without glutaraldehyde. Brains
were removed immediately and post-fixed in the second fixative containing 30%
sucrose for 1-2 days, before being cut into three series of 50 µm coronal
sections using a sliding microtome. The first series of sections was stained for tyrosine
hydroxylase (TH), the second series of sections were processed with Cresyl
violet, and the third series of sections were stored in an anti-freeze solution.
Sections were rinsed in 0.1 M PB and incubated for 36 hours at 4oC
in a 1:100 dilution of a mouse monoclonal TH antiserum (Incstar) containing
0.5% Triton X-100 and 0.1% sodium azide. The sections were rinsed three times
in PB before being incubated with biotinylated antimouse IgG (Vector, 1:100)
for 2 hours. Sections were subsequently
treated with the coupled oxidation reaction of Itoh et al. (1979) with
3,3’-diaminobenzidine as chromogen intensified with ammonium nickel sulfate.
The sections were then rinsed in PB, dehydrated, and coverslipped with DPX.
Caudal diencephalic sections, starting where the first TH neurons (Fig. 1) of
the A9 cells appeared, were selected for subsequent morphometric analysis.
All cell counts were carried out
with the help of an interactive computer system (NeurolucidaR,
Stereo InvestigatorR, MicroBrightField, Inc) connected to a Zeiss
Axioplan microscope. The outlines and landmarks of every third section from the
midbrain, including the borders of the A8, A9, and A10 cell groups were drawn with
5 x lens. For cytoarchitectonical
landmarks the adjacent Cresyl violet-stained sections were consulted.
Additionally, several series of TH-stained sections, subsequent to the counting
procedure, were counterstained with Cresyl violet (Figs. 1, 2A-H). The outlined regions containing TH-positive
neurons were scanned under a 100 x objective lens (Zeiss Plan-NEOFLUAR, NA=1.3)
and neurons were recorded using systematic random sampling and the optical
dissector methods (West et al., 1996; West et al., 1991). In all cases
the following stereological parameters were used: sampling grid
area=10,000 :m2;
counting frame=2,500 µm2, height of optical disector=14-15 µm,
depending on the measured section thickness (20-21 µm). In each subdivision of
the ventral mesencephalon the average number of cell bodies sampled in the
disector (per animal and per side) were the following: A8: 118 (52-195); A9:
264 (180-486); A10: 386 (248-687).
For practical
purposes we adopted a simple nomenclature following the original description of
the catecholaminergic cell groups in rat by Dahlstrom and Fuxe (Dahlstrom and Fuxe, 1964).
A9 cell group.
These TH-positive neurons are mainly confined to the pars compacta of
the substantia nigra (SNC, Fig. 1, 2A,B). Although on cytoarchitectonic
grounds, often the lateral part of the
substantia nigra is separated
from the pars compacta (Franklin and Paxinos, 1997), in our study the laterally located TH-positive cells
were included in the SNC. Additionally,
scattered TH-positive cells can be observed, mostly confined to the medial part
of the zona reticulata. The compact
zone consists of 3-6 cell layers, to which dorsally and medially the lateral
wing of the A10 cells joins. However, no sharp border exists rostrally between
the A9 and A10 cell groups. More caudally (Fig. 2C), the dorsomedial border of
the A9 group was defined by the ventral
fibers of the medial lemniscus (ml). The TH-positive cells in the pars
compacta often form pockets containing high density TH-positive groups
separated by TH-free zones. One conspicuous unstained area is represented by
the small-celled medial terminal nucleus of the optic tract (MT, Fig. 2B). The ventrally located pars
compacta cells give rise to dendrites extending ventrolaterally deep into the
pars reticulata. Dendrites of dorsally placed neurons course medio-laterally.
Reticulata TH-positive cells often show random orientation.
A10 cell group. Dahlstrom and Fuxe (Dahlstrom and Fuxe, 1964) described this group as the largest dopaminergic cell
group in rat, situated mainly in the area dorsal to the interpeduncular
nucleus, largely corresponding to the ventral tegmental area (VTA), and
extending caudally into the rostral linear nucleus (RLi). On the basis of
cytoarchitecture, in rat (Gonzalez-Hernandez and Rodriguez, 2000;
Paxinos and Watson, 1998; Phillipson, 1979), mice (Franklin
and Paxinos, 1997), cat (Taber,
1961) monkey, and human (Halliday
and Tork, 1986; McRitchie et al.,
1998; Pearson et al., 1990) several subgroups of the VTA have been
distinguished: the paranigral nucleus,
a compact cluster of bipolar or triangular cells medial to the pars compacta
and dorsolateral to the interpeduncular nucleus; the parabrachial pigmented
nucleus, large cells on the dorsal
aspect of the VTA towards the medial lemniscus; and the interfascicular nucleus
(IF): dense small cell group located on either side of the dorsal aspect of the
interpeduncular nucleus. Dorsal to the interfascicular nucleus are the rostral
(RLi) and caudal linear (CLi) nuclei. TH-positive cells in these aggregates
extend dorsally to the ventral aspect of the oculomotor nuclei (3, Fig. 2F) and the dorsal raphe
nucleus (DR, Fig. 2D-H), respectively. There is a considerable controversy as
to the delineation of the different subdivisions of the A10 group. Some
studies, for example Swanson (Swanson,
1982), did not divide the VTA and use it as a synonym for
the A10 group, others adopted different subdivisions (Deutch et al., 1988;
German and Manaye, 1993; Giolli et al.,
1985; Halliday and Tork, 1986). For cell-counting purposes we did not subdivide the
A10 group.
In our study,
the dorsal border of the A10 group was
defined where the dense TH neuropil staining with a relatively sharp border
blends into an area that contains only a few TH-positive fibers. Except at the
most rostral part of this group, the midline was used as a medial border of the
A10 (Fig. 2A). Cells of the A10 group
show a characteristic position to the medial lemniscus as these fibers pierce
through the ventral portion of the midbrain: rostrally, the medial lemniscus is
dorsal to the A10 group (Fig. 2A), further caudally, (Fig. 2B), the A10 cells
increasingly are admixed with the lemniscal fibers. Finally, at the level of
Fig. 2E, most of the TH-positive cells occupy a position dorsal to the medial
lemniscus. The interfascicular nucleus is characterized by a very dense
terminal staining that can be easily defined at the mid-dorsal aspect of the
interpeduncular nucleus (Fig. 2B-D).
More caudally, with the appearance of the pontine nuclei (Pn, Fig. 2D), the ventromedial part of the A10
group is still occupied by the dense cells of the interfascicular nucleus, but
the rest of the VTA can be subdivided into a ventrolateral zone that contains a few small cells but is occupied
by a dense neuropil and a dorsal area,
which contains larger cells with less dense neuropil staining. These two areas
may correspond to the paranigral and parabrachial pigmented nuclei of rodents
and humans, respectively. Between these two subdivisions the neuropil displays a reticular structure with few or
no TH-positive cells. At level of Fig. 2D, at the dorsolateral portion of the
A10 groups, a densely stained triangular group of large cells lies between the
substantia nigra and the A8 cell group (asterisk, Fig. 2D). Above the interfascicular nucleus, there is an oval-shaped area devoid of cells that is
flanked by the cell-dense rostral linear nuclei. Further caudally, the A10
group is split by the crossing fibers of the pedunculus cerebellaris superior
(xscp, Fig. 2H) into a cell-dense dorsal area, which constitutes the caudal
linear nucleus, and a ventral area, which contains a few elongated cells that
are displaced ventrolaterally by this fiber system. Neither TH-positive cells in the supramammillary region nor those
in the dorsal raphe nucleus are included in the A10 group as defined here.
A8 cell group. In our delineation of the A8 cell
group we followed the description of
Deutch et al. (Deutch et al., 1988). This cell group appears rostrally as a small group
of neurons between the dorsolateral group of the A10 cell group and the A9
neurons of the substantia nigra (Fig. 2D-E). More caudally (Fig. 2G) the
bilateral A8 groups become very prominent and a dorsally situated dense and a
ventrally located more diffuse cell group can be distinguished. At the larger expanse of the crossing of the
pedunculus cerebellaris superior (xscp), only the compact dorsal group remains
(Fig. 2H). At the level of the
parabigeminal nucleus (PB), the most caudal portion of the A8 cell group is
located dorsomedially to the pedunculopontine tegmental nucleus (PPT).
We applied unbiased stereological methods
using optical dissector counting to estimate the number of TH-positive neurons in the A8-A9-A10
dopaminergic cell groups in three mouse strains with different TH/MES. Based on
previous results, the B6, C4A6, and CXBI strains were chosen with intermediate,
high, and low TH/MES, respectively (Vadasz
et al., 1982; Vadasz et al., 1998). The number of TH-positive neurons was counted
separately on the left and right sides of the A8, A9 and A10 cell groups. Preplanned comparison of the strains with
the highest and lowest TH/MES showed that CXBI mice are endowed with a
significantly higher number of TH-positive cells in the A9-A10 cell groups than
C4A6 mice (A9: 14,492 +/- 597 vs. 7,787 +/- 1162, mean +/- SE, p<0.005; A10: 20,720 +/- 1359 vs. 12,102
+/- 1395; mean +/- SE, p<0.01, Fig.
3). However, no significant strain
differences were found in the A8 cell group.
No significant lateral differences were
detected in the number of TH-positive neurons of the A9-A10 cell groups. In contrast, in all three mouse strains
studied, the right A8 area had a higher number of TH neurons compared to the
left A8 area, indicating lateral asymmetry across strains. Combining
individuals from the three strains, the average (±SE) TH neuron number on the left A8 area was 1461 (±221), while on
the right A8 region we counted 1704 (±194) neurons (Paired Samples T-Test, N=11, p=0.01).
Using a simple
and reproducible delineation scheme the main results of this work are the
following: 1) the number of TH-positive cells are significantly higher in the
CXBI strain as compared to the C4A6 strain in the A9-A10 cell groups; 2) there
is a significant lateral asymmetry in the number of TH-positive cells in the A8
area in all strains investigated. Because previous studies established the midbrain
TH activities in these strains (Vadasz
et al., 1998), these results suggest an inverse relationship
between cell number and TH activity in the midbrain.
Although the
exact relationships between regional brain neuron number and behavior is not
known, it has been suggested that quantitative variation in neuron number is
one of the significant variables underlying individual differences in behavior,
or predisposing to neurobehavioral disorders (Reis
et al., 1981). Control of
complex traits, such as neuron number, involves both genetic and environmental
factors, and their interactions in the course of development. Examples of
neuron number variations include mouse strain differences in granule cell
number in the area dentata (Wimer et al., 1978), in 5-HT neurons in the dorsal raphe nucleus (Daszuta and Portalier, 1985), and in retinal ganglion cells (Williams et al., 1998). Genetic
variation in dopamine neuron number was associated with strain-dependent
differences in midbrain TH activity (Baker
et al., 1980; Harris and Nestler,
1996; Ross et al., 1976) and behavior (Reis
et al., 1981).
Strain-dependent variation in brain
dopamine levels and TH activity have been reported by several laboratories (Ciaranello et al., 1972; Kessler et al.,
1971; Ross et al., 1976; Vadasz et al., 1985; Vadasz et al., 1982; Vadasz et al., 1986; Vadasz et al., 1987; Waller et al., 1983). It was
suggested that a 20-50% difference in midbrain TH activity between the mouse
strains, BALB/c and CBA, was consequent to a 20% difference in the number of DA
neurons in the midbrain (Baker et al., 1980; Ross et al., 1976). Also, the
size of the caudate putamen in BALB/c was found significantly greater than that
of CBA (Baker et al., 1980). Examination
of midbrain dopamine cell density in
two mouse lines selected for high (NR) and low
(NNR) cataleptic response to haloperidol suggested that the increase in
midbrain D2 autoreceptor density within the substantia nigra zona compacta
(SNzc), but not the ventral tegmental area (VTA), was associated with a 41% increase in DA neuron number in the NNR
line, while no difference was found in DA neuron number between lines in the
VTA (Hitzemann et al., 1993). These results were confirmed demonstrating that the TH-positive neuron
number was increased in the NNR/Np mice as compared to NR/Np mice, and the
highest difference in DA neuron number between BALB/crl and C57BL/crl was
43% in both rostral SNzc and caudal VTA
(Hitzemann et al., 1994). Using MPTP treatment for modelling Parkinson's
disease, Muthane et al. (Muthane et al., 1994) noted that MPTP treatment dramatically reduced
striatal levels of DA in C57BL/6 mice, while the effect on CD-1 mice was
minimal. In MPTP-treated animals the
reduction in the number of SNzc TH-positive neurons was significantly greater
in C57BL/6 mice, than in CD-1 mice. On the other hand, there was no difference
between the two strains in a subpopulation of dopaminergic neurons that
coexpress TH with calbindin. Studies on the striatal terminal field indicated
that mesostriatal neurons give rise to comparable axonal branching within the
striatum in BALB/c and CBA strains (Mattiace
et al., 1989), even though the BALB/c strain has significantly more
midbrain DA neurons than the CBA (Baker et
al., 1980; German et al., 1983;
Ross et al., 1976). Mice of the BALB/cJ strain were more sensitive to
the action of amphetamine than those of the CBA strain (Reis et al., 1983), suggesting that strain-dependent differences in
TH-positive cell number are associated with differential DA-mediated behaviors
in BALB/c and CBA.
According
to our study, midbrain TH activity was significantly higher in B6.Cb4i5-a6/Vad strain as
compared to CXBI mice (Vadasz et al., 1998), thus suggesting an inverse
relationship between A9-A10 TH activity and dopamine cell number. A similar
inverse relationship was observed between TH activity and cocaine-induced
running (Vadasz et al., 1994). Our findings are in line with those of Harris and
Nestler (1996), who observed that 50% fewer TH-positive neurons in the VTA of
the Lewis rats were associated with a 45% higher level of TH as compared to
Fischer 344 rats (Harris and
Nestler, 1996). Similar
studies on BALB/c and CBA/J mice, however, suggested positive association
between TH activity and cell number (Baker
et al., 1980; Ross et al., 1976). The discrepancies among the reported studies in
regional TH activity vs. TH-positive neuron number may derive from numerous
factors, including the use of different species, strains, measure of TH
(activity vs. amount of protein), maintenance conditions affecting gene
expression, genetic susceptibility to drug-induced TH-mRNA expression (Marcel
et al., 1998), anatomical delineation of dopaminergic cell groups, and methods of counting neurons (West et
al., 1991).
The underlying mechanism for this
inverse relationship is unknown. It is possible that genetically based
variation in DA neuron number can lead to compensatory processes, such as
altering TH activity per DA neuron.
Such genetically initiated plasticity can lead to different behavioral
processes depending on the affected target areas of the mesotelencephalic
dopaminergic system, including the nucleus accumbens, the prefrontal cortex and
the dorsal striatum.
The functional significance of the A8
dopamine neurons is not well known, and this is the first report to demonstrate
lateral asymmetry in this cell group. In the rat, the A8 neurons were shown to
contribute to the dopaminergic innervation of
the hypothalamic median eminence, an area concerned with neuroendocrine
regulation, (Kizer et al., 1976), the
striatum, nucleus accumbens, olfactory tubercle, amygdala, bed nucleus of the
stria terminalis, as well as innervate the pyriform and entorhinal cortices (Deutch et al., 1988). In addition,
via direct connections, the A8
dopamine cells may modulate the functional activity of the A9 and A10 cell
groups (Deutch et al., 1988). Supporting this notion, feline studies have shown
that lesion in the retrorubral area, which includes the A8 cell group, produces
motor programming deficits inherent to a hypofunction of the A9 system (Arts et
al., 1998). In non-human primates, the A8 cell group innervates
the frontal cortex, which is commonly implicated in psychiatric and
neurological disorders (Williams and
Goldman-Rakic, 1998). Also, it was
suggested that the A8-10 projection to the hippocampus may have a role in
metamphetamine-produced hypermotility and modulation of memory processes (Gasbarri et al., 1996; Gasbarri et al.,
1997).
Animal and human
experiments have shown that neurochemical and anatomical asymmetries exist within
the basal ganglia. These asymmetries correlate with preferred direction of
rotation and limb preference (Glick,
1985; Kooistra and Heilman, 1988; Richter et
al., 1999). Recent works
focusing on attention-deficit/hyperactivity disorder (ADHD) suggest a possible
abnormal hemispheric asymmetry of attention functions in boys with ADHD: boys
with ADHD react more slowly to uncued targets in the left visual field (Nigg et
al., 1997).
Electroencephalographic studies also support lateralization and a
gender-specific atypical frontal brain activation in ADHD (Baving et al., 1999). Because
dopamine neurotransmission has been implicated in ADHD and in attention (Swanson, 2000; Swanson et al., 2000; Yamaguchi and Kobayashi,
1998), it possible that the A8 dopamine cell group asymmetry found in our study
plays a role in the lateralized attention deficits in ADHD.
The progress in
understanding the genetic control of quantitative traits has been slow due to
the involvement of multiple genes, the presence of multiple alleles and their
complex interactions. Using the RQI method it is possible to transfer QTL(s) onto the same homogeneous genetic
background (Vadasz et al., 1994) and distribute the QTLs in recombinant strains (Vadasz et al., 1998). After successful intogression of QTLs responsible
for a complex trait, an RQI animal model can be characterized using the
armamentarium of modern neuroscience to elucidate how genetic predisposition
could lead to quantitative differences in neural circuitry and behavior. Phenotyping and genotyping of more than 100
RQI strains will also allow the mapping of relevant QTLs. The joint use of
advanced animal models and molecular genetic manipulation of dopamine
system-specific genes can shed light on questions about genetic differences in
DA neuron number and information processing in circuitries. For example, if a genetically determined
higher number of neurons is detected in one of the nodes of a neural circuitry,
would this gain be expressed in the target area and in each node of a pathway?
What is responsible for cell-number variation in the dopaminergic system:
genetic differences in neurogenesis or in postnatal survival (Baker et
al., 1982)? Answers to
these and similar questions, identification of the involved genes, and further
work on specific genetic manipulation of the mesotelencephalic dopamine system
can provide new insights into mechanisms related to Parkinson's disease (de Silva et al., 2000; Morino et al.,
2000; Polymeropoulos et al., 1997;
Shimura et al., 2000), ADHD (Swanson
et al., 2000), substance abuse (Brodie
and Appel, 2000; Duaux et al., 2000;
Noble, 2000), and other complex diseases (Ciaranello and Boehme, 1981; Dikeos et al., 1999; Ebstein et al.,
1997; Meloni et al., 1995; Serretti et al., 1999; Thibaut et al., 1997).
The work described in this paper was supported
by NIH grants NS23945 (L.Z.) and AA11031 (C.V.) for morphometric studies, and NS19788 (C.V.) for the development of
the RQI system.
Special thanks are due to Mr. Paul
Anderson and Mr. Istvan Kiraly for expert technical assistance. This work could not have been completed
without the enthusiastic and expert contribution of Drs. I. Sziraki, L. Murthy, P. Kabai, Gy. Kobor, A. F.
Badalamenti, M. Juhasz, M. Sasvari-Szekely, B. Juhasz, and I. Laszlovszky,
during the construction and characterization of the RQI strains.
High resolution image to show the rostral part of
the substantia nigra, pars compacta. The dense fiber bundle is the
nigrostriatal tract. The original image was scanned with a Zeiss AxioCam
digital camera with 2600 x 2060 pixel resolution using Zeiss Axioscope microscope with objective
lens: 10 x Achroplan. The image was
resized to 2492 x 1868 using Adobe Photoshop 5.02. cp= cerebral peduncle; LM= lateral mammillary nucleus; SuM=
supramammillary nucleus; VTM= ventral tuberomammillary nucleus
Series of sections showing TH-positive
neurons in the A10 (red), A9 (black) and A8 (green) cell groups. The left side
of the figures depicts the original TH-stained sections counterstained with
Cresyl violet from a CXBI case (1483-1), the right side of the figures shows
the distribution of TH-positive neurons as plotted from the same sections using
20 x objective lens and the Neurolucida mapping software. Sections are
separated by 100 µm, except between A and B and C and D, where the distance is
250 µm.
Abbreviations: A8-A10 catecholaminergic cell groups according to Dahlstrom and Fuxe, 1964; 3=oculomotor nucleus; bp=brachium pontis; df=medial longitudinal fascicle; DLG= dorsal lateral geniculate nucleus; cp= cerebral peduncle; fr=fasciculus retroflexus; IP=interpeduncular nucleus; lfp=longitudinal fasciculus of pons; ll=lateral lemniscus; m5=motor root of the trigeminal nerve; MB= mammillary body; MGB= medial geniculate body; ml= medial lemniscus; Mn=median raphe nucleus; MT=medial terminal nucleus; PAG=periaqueductal gray; PaR=pararubral nucleus; pc=posterior commissure; PMR=paramedian raphe nucleus; Pn=pontine nuclei; PPT=pedunculopontine tegmental nucleus; R=red nucleus; rs=rubrospinal tract; RtTg=reticulotegmental nucleus of the pons; SNC=substantia nigra, compact part; SNR=substantia nigra, reticular part; ts=tectospinal tract; VL= ventral nucleus of the lateral lemniscus; xscp=decussation superior of the cerebellar peduncle.
Strain-dependent variation in mean
dopamine neuron number. Significant differences were found between C4A6 and
CXBI in the cell groups A9 (p<0.05)
and A10 (p<0.01; preplanned comparisons, independent samples T-test).
Error bars show standard error of the mean (SE).
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