ABSTRACT
The distributional profile of [3H] SCH 23390/dopamine D1-like
receptors in the hippocampal formation of the rat, monkey and human brains was
examined by quantitative receptor autoradiography. D1-like receptors were
found in the hippocampus of all three species. In the rat, the majority of
these receptors were located in the molecular layer of the dentate gyrus
whereas the Ammon's horn subfields exhibited lower levels of D1-like binding
sites. Interestingly, this distribution profile was reversed in the primate
hippocampus. In both the monkey and human hippocampus, the highest D1-like
receptor density was seen in the CA1 followed by the CA3 subfields, with the
dentate gyrus containing lower levels. These results demonstrate that, in
contrast to the situation seen in the basal ganglia, D1-like receptor
distribution in the hippocampal formation may not be evolutionary conserved.
INTRODUCTION
Dopamine (DA) neurons in the ventral tegmental area project to a number of
cortical and subcortical structures (Bjorklund and Lindvall, 1984; Lindvall
and Bjorklund, 1983). These dopaminergic projections which are referred to as
the mesocorticolimbic system have been implicated in a number of brain
functions such as cognition, motivation and reward (for reviews see Bozarth,
1989; Decker and McGaugh, 1991; Phillips et al., 1989; Wise and Hoffman,
1992). Mesocorticolimbic dysfunction is also thought to contribute to a number
of psychopathological conditions including schizophrenia and affective
disorders (Willner et al., 1989).
As a result, the characteristics of this dopaminergic innervation have been
studied in a variety of species including rat, monkey and human (Fallon and
Loughlin, 1987; Goldman-Rakic et al., 1992). For example, DA modulates long
term potentiation in the Schaffer collateral pathway of the rat hippocampus
via D1-like receptors (Huang and Kandel, 1995). Moreover, we recently reported
that D1-like receptors modulate hippocampal acetylcholine (ACh) release in the
rat and that this action may impact on cognitive abilities of the aged rat (Hersi
et al., 1995a and 1995b). An antisense approach revealed the likely
involvement of the D5 subtype of the D1-like receptor family in these effects
(Hersi et al., 1996). However, while it is true that basic commonalities of DA
mesocorticolimbic innervation exist across species, there are also important
differences. For example, the neocortical dopamine system appears to be more
extensive in the primate than in the rat brain (Roth and Elsworth, 1995; Roth
et al., 1987). A recent report also suggests the presence of differential
distribution of dopamine receptor subtypes in the primate cortex compared to
that of the rat (Huntley et al., 1992).
In order to establish if the data on the effects of D1-like receptors on
hippocampal ACh release in the rat may be applicable to other species
including man, we investigated the comparative distribution of [3H]
SCH 23390/D1-like receptors in the hippocampal formation of the rat, monkey
and man using quantitative receptor autoradiography.
MATERIALS AND METHODS
Materials
Male Long Evans rats (250-350g) obtained from Charles River Canada (St.
Constant, Quebec, Canada) were maintained on a 12 hr light-dark cycle (light
on at 7:00 a.m.) in temperature and humidity controlled rooms for at least 3-4
days prior to sacrifice. Animals were fed standard laboratory chow and had
access to tap water ad libitum. Animal care was according to protocols and
guidelines approved by the McGill University Animal Care Committee and the
Canadian Council for Animal Care (CCAC). Brains of monkeys (Callithrix Jacchus)
were kindly provided by Sanofi (France). Blocks of normal control human brains
from four individuals (3 males, 1 female; 74±3 years; neuropathological
examination revealed no evidence of neurological disorders, e.g. cell loss,
plaques, tangles, excessive gliosis) of approximately 3cm3
containing various regions were obtained from the Douglas Hospital Research
Centre Brain Bank. Post mortem intervals ranged from 6 to 26 hrs. These brain
blocks were frozen in isopentane, cooled on dry-ice and stored at -80oC
as previously described (Quirion et al, 1987).
[3H] SCH23390 (80.7Ci/mmol), 3H-Hyperfilms and microscale
standards were purchased from Amersham Canada (Oakville, Ontario, Canada). SCH
23390 HCl was obtained from RBI (Watick, MA, U.S.A.). Films, developer (D-19)
and fixer (Rapid Fix) were obtained from Kodak Chemical Inc. (Montreal Quebec,
Canada). All other reagents and chemicals were of HPLC or GC-MS grade and
purchased from either Fisher Scientific Co. (Montreal, Quebec, Canada) or
Aldrich Chemicals (Chicago, IL, U.S.A.).
Dopamine D1 Receptor Autoradiography The distribution of
dopaminergic D1-like receptors in the hippocampal formation and striatum of
rat, monkey and human brains was assessed as described in detail elsewhere (Debonnel
et al., 1990). In brief, following slicing at -17oC, 20mm sections
mounted on gelatin coated slides were washed twice (15min each time) at room
temperature in 50mM Tris HCl buffer (pH 7.4) containing 120mM NaCl, 5mM KCl,
2mM CaCl2, 1mM MgCl2. These
sections were then incubated for 60min at room temperature in the buffer in
the presence of 1.0nM [3H] SCH 23390. Adjacent sections were
incubated in the presence of the radioligand and 1
M
SCH 23390 to determine the level of specific binding. The sections were then
rinsed five times (2 min each) in fresh ice cold buffer. Buffer salts were
removed by a rapid dip in ice cold distilled water and the sections rapidly
air dried. Autoradiograms were generated by apposing the sections alongside
with tritium standards to tritium sensitive films for 6-8 weeks. The films
were then developed as described before (Quirion et al., 1981) and [3H]
SCH 23390 binding quantified (fmol/mg tissue, wet weight) using computer
assisted microdensitometric image analysis system (MCID System, Imaging
Research Inc., St-Catherines, Ontario, Canada). Anatomical areas were
identified according to the Paxinos and Watson's (1982), DeArmond et al.
(1989) and Gergen and MaClean (1962) atlases.
RESULTS AND DISCUSSION
The distribution of [3H] SCH 23390/D1-like receptors in the
hippocampal formation of the rat, monkey and human brains was examined by
quantitative receptor autoradiography. Our results demonstrate that D1-like
receptors are expressed in the hippocampal formation of all three species.
However, differences in the distribution profile of these receptors are seen
between the species.
The most extensive investigations examining the discrete localization of DA
receptors have been carried out in the basal ganglia (for example, Richfield
et al., 1987; Camps et al., 1990). Hence, in the present study, we examined [3H]
SCH 23390/D1-like binding sites in the caudate/dorsal striatum and used it as
control. As reported in these earlier studies, the highest density of D1-like
receptors in mammalian brain is found in basal ganglia structures such as the
caudate and putamen (Table 1). Moreover, the distributional profile of D1-like
receptors in these structures appears similar across species (Fig
1).
|
|
| Figure 1. Distribution profile of
D1-like/[3H] SCH 23390 binding sites. Rat, monkey and human brain
sections were incubated with 1.0nM [3H] SCH 23390 as described in the
Materials and Methods. Autoradiograms were generated by apposing the
sections against tritium sensitive films for 6-8 weeks (A-C). The bar
equals 1cm. Abbreviations: CA, Ammon's horn; Cd, Caudate; DG, Dentate
gyrus; Str, Dorsal striatum. |
________________________________________________________________________________
TABLE 1: Quantitative distribution of SCH 23390/D1-like receptor binding sites
--------------------------------------------------------------------------------
Area Rat Monkey Human
--------------------------------------------------------------------------------
Dorsal striatum / Caudate 143+28 126.2+10.4 71.0+11.8
Hippocampal formation
Dentate gyrus 19+1.7 14.4+0.6 13.2+0.5
CA1 subfield 9.0+0.5 39.7+5.8 22.1+2.5
CA3 subfield 7.0+0.4 16.2+2.2 14.7+1.5
_________________________________________________________________________________
The autoradiograms generated as described in the legend
to figure
1 were subsequently quantified using computer assisted image analysis
system. The data are expressed as fmol/mg tissue wet weight and represent
mean specific labelling + S.E.M. Rat, n=5; Human, n=4; Monkey, n=2.
The hippocampal formation of the three species studied here also expressed
D1-like receptors (Fig
1). Unlike in the basal ganglia structures, however, their pattern of
distribution was species-dependent (Table 1). In the rat, the highest
densities of D1-like receptors were seen in the molecular layer of the dentate
gyrus, followed by the pyramidal cell layer of the CA3 and CA1
subfields. In contrast, in the monkey and human hippocampi, [3H]
SCH 23390/D1-like receptor levels were highest in the CA1 area
(stratum oriens, pyramidale and radiatum) followed by the CA3
subfield (Fig 1). Lower amounts were seen in the molecular layer of the dentate gyrus.
Therefore, it appears that the distributional profile of D1-like receptors is
evolutionarily conserved in the basal ganglia but not in the hippocampal
formation (Camps et al., 1990).
The D1-like family of DA receptors consists of two members, namely D1 and
D5 (Jackson and Westlind-Danielsson, 1994). The mRNA of both these receptors
were shown to be expressed in the hippocampal formation (for recent reviews
see Mansour and Watson, 1995; Meodor-Woodruff et al., 1994a and 1994b). In the
rat, the D1 receptor mRNA is found primarily in the granular cell layer of the
dentate gyrus with no detectable levels in other areas of the hippocampal
formation. In contrast, the D5 receptor mRNA is seen throughout the rat
hippocampus, both in the granular cell layer of the dentate gyrus and the
pyramidal cell layer of the Ammon's horn. In the human hippocampus, D1
receptor mRNA is expressed in the subiculum and the pyramidal cell layer of
the CA1 subfield (Meador-Woodruff et al., 1994a). Similar to the
rat, D5 receptor messages in the human hippocampus are reportedly seen both in
the dentate gyrus and in the pyramidal cell layers (CA1-CA4).
However, due to translational efficiencies, turnover rates and possible
receptor protein transport, mRNA levels and distribution do not necessarily
reflect that of their respective receptor proteins. Selective ligands/antibodies
targeted against the individual receptor proteins are necessary to map the
precise location of these proteins. Presently, there are no selective
pharmacological ligands available that can distinguish between the two members
of the DA D1-like receptor family. Instead, the use of receptor
subtype-specific antibodies to determine receptor distribution has started
recently (Bergson et al., 1995; Ciliax et al., 1994). Preliminary results from
this immunocytochemical approach suggest that in the rat D1 receptors are
found in the CA1 subfield and not in the dentate gyrus (Ciliax et
al., 1994). This may be taken as an indication that [3H] SCH
23390/D1-like binding found in the dentate gyrus of the rat brain (Dawson et
al., 1986; This study) belongs to the D5 receptor subtype. Currently, no
immunocytochemical information is available regarding either the distribution
of the D5 receptor in the rat or the D1 and D5 receptor proteins in the human
hippocampus. In the monkey hippocampus, both D1 and D5 receptor proteins are
reportedly present in the pyramidal layers (CA1-CA4) as
well as in the dentate gyrus (Bergson et al., 1995). It is not possible, at
this time, to ascertain the respective contribution(s) of the two D1-like
receptor subtypes in the species-dependent distributional profile of [3H]
SCH 23390/D1-like binding sites reported in the present study.
The functional significance of the species-dependent differential
distribution of [3H] SCH 23390/D1-like receptors in the hippocampal
formation is unclear. We have recently shown that D1-like hippocampal
receptors are involved in modulating ACh release in the rat as monitored by in
vivo dialysis employing transverse probes (Hersi et al., 1995a). These probes
are implanted in such a way that they traverse both the dentate gyrus and the
various Ammon's horn subfields of the hippocampus (Damsma et al., 1987). As a
result, the effect on ACh release elicited by the stimulation of D1-like
receptors could be due to action in any subregion/laminae of the hippocampal
formation. Therefore, the presence of [3H] SCH 23390/D1-like
receptors in the hippocampus of monkey and human brains suggests that DA
acting via these receptors might also stimulate hippocampal ACh release in
primates. Given the differential distribution pattern of D1-like receptors
across species, however, the validation of this hypothesis must await the
conductance of functional investigations in primates. Fortunately, this is now
technically feasible with the development of in vivo dialysis techniques in
the monkey brain (Kolachana et al., 1994).
In summary, [3H] SCH 23390/D1-like receptors are expressed in
the rat, monkey and human hippocampi. However, a differential distribution
profile of these receptors was observed between the primate and rodent species
studied.
ACKNOWLEDGMENTS
This work was supported by a grant from the Medical Research Council of
Canada (MRCC) to Remi Quirion, a "Chercheur Boursier Senior" of the
Fonds de la Recherche en Sante du Quebec (FRSQ). Ali Hersi is a holder of a
Studentship from FRSQ while P. Gaudreau holds a Chercheur-Boursier award from
the FRSQ.
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