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<tr><td valign="top" align=left>© Neuroscience-Net<br> Volume 1, Article #10005
<td width=500 align=right>Received June 6, 1996<br>
Accepted for Publication July 8, 1996<br>
Published July 30, 1996<br>
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<font size=+1><b>COMPARATIVE DISTRIBUTION OF D1-LIKE RECEPTORS IN THE HIPPOCAMPAL FORMATION OF RAT, MONKEY AND HUMAN BRAINS</b></font><p>
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Ali I. Hersi<SUP>1,3,4</SUP>, Danielle Jacques<SUP>3</SUP>, Pierrette Gaudreau<SUP>4</SUP> and Remi Quirion<SUP>1,2,3</SUP>, Depts. of <SUP>1</SUP>Neurology/Neurosurgery, and <SUP>2</SUP>Psychiatry McGill University, Montreal, Quebec, Canada.; <SUP>3</SUP>Douglas Hospital Research Center, Verdun, Quebec, <STRONG>Canada</STRONG>. and <SUP>4</SUP>Neuroendocrinology Lab., Notre-Dame Hospital Research Center and Dept. of Medicine, Université‚ de Montreal, Montreal, Quebec, <STRONG>Canada</STRONG>.<p>
Send correspondence to:<p>
Professor Remi Quirion<br>
Dept. of Psychiatry<br>
Douglas Hospital Res Ctr<br>
6875 Blvd lasalle Verdun<BR>
Quebec, Canada H4H 1R3<BR>
E-mail:<A HREF="mailto:MCOU@MusicA.McGill.ca">MCOU@MusicA.McGill.ca</A><p>
<ul><ul>
<b>KEY WORDS</b>: Receptor Autoradiography; Hippocampus; D1 receptors; Monkey; Human; Rat.<p>
</ul></ul>
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<ul>
<li><a href="index.html" TARGET="_parent">Abstract</a><br>
<li><a href="#intro">Introduction</a><br>
<li><a href="#materials">Materials and Methods</a><br>
<li><a href="#legends">Legends</a><br>
<li><a href="#results">Results</a><br>
<li><a href="#discuss">Discussion</a><br>
<li><a href="#acknow">Acknowledgments</a><br>
<li><a href="#references">References</a><br>
</ul>
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<a name="intro">
<font size=+2><b>INTRODUCTION</b></font><p>
<FONT SIZE=+1></FONT>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).<P>
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).<P>
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 [<sup>3</sup>H] SCH 23390/D1-like receptors in the hippocampal formation of the rat, monkey and man using quantitative receptor autoradiography. <P>
<FONT SIZE=+1></FONT><p>
<hr>
<a name="materials">
<font size=+2><b>MATERIALS AND METHODS</b></font><p>
<FONT SIZE=+1></FONT>Materials<P>
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 3cm<SUP>3</SUP> 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 -80<SUP>o</SUP>C as previously described (Quirion et al, 1987).<P>
[<sup>3</sup>H] 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.).<P>
<STRONG>Dopamine D1 Receptor Autoradiography</STRONG>
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 -17<SUP>o</SUP>C, 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 C<SUB>a</SUB>Cl<SUB>2</SUB>, 1mM M<SUB>g</SUB>Cl<SUB>2</SUB>. These sections were then incubated for 60min at room temperature in the buffer in the presence of 1.0nM [<sup>3</sup>H] SCH 23390. Adjacent sections were incubated in the presence of the radioligand and 1<SUB><img src="../images/micro.gif" ALT="Greek Symbol for Micro"></SUB>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 [<sup>3</sup>H] 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.<P>
<hr>
<a name="results">
<font size=+2><b>RESULTS AND DISCUSSION </b></font><p>
<FONT SIZE=+1></FONT>The distribution of [<sup>3</sup>H] 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.<P>
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 [<sup>3</sup>H] 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 (<A HREF="#legends">Fig 1</A>).<P>
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<PRE><b>TABLE 1: Quantitative distribution of SCH 23390/D1-like receptor binding sites</B>
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Area Rat Monkey Human
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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
_________________________________________________________________________________
</pre><P>
<center><BLOCKQUOTE>
<font size=-1>The autoradiograms generated as described in the legend to <A HREF="#legends">figure 1</A> 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.
</font></BLOCKQUOTE></CENTER><P>
The hippocampal formation of the three species studied here also expressed D1-like receptors (<A HREF="#legends">Fig 1</A>). 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 CA<SUB>3</SUB> and CA<SUB>1</SUB> subfields. In contrast, in the monkey and human hippocampi, [<sup>3</sup>H] SCH 23390/D1-like receptor levels were highest in the CA<SUB>1</SUB> area (stratum oriens, pyramidale and radiatum) followed by the CA<SUB>3</SUB> subfield (<A HREF="#legends">Fig 1</A>). 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).<P>
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 CA<SUB>1</SUB> 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 (CA<SUB>1</SUB>-CA<SUB>4</SUB>).<P>
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 CA<SUB>1</SUB> subfield and not in the dentate gyrus (Ciliax et al., 1994). This may be taken as an indication that [<sup>3</sup>H] 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 (CA<SUB>1</SUB>-CA<SUB>4</SUB>) 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 [<sup>3</sup>H] SCH 23390/D1-like binding sites reported in the present study.<P>
The functional significance of the species-dependent differential distribution of [<sup>3</sup>H] 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 [<sup>3</sup>H] 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).<P>
In summary, [<sup>3</sup>H] 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.<P>
<hr>
<A NAME="legends"></A>
<FONT SIZE=+2><b>LEGENDS</b></FONT><p>
<A HREF="ano4f01l.html"> <IMG SRC="../images/an04f01s.jpg" WIDTH=117 HEIGHT=144 align=left>Figure 1.</A> Distribution profile of D1-like/[<sup>3</sup>H] SCH 23390 binding sites. Rat, monkey and human brain sections were incubated with 1.0nM [<sup>3</sup>H] 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.<br clear=both><P>
<hr>
<a name="acknow">
<font size=+2><b>ACKNOWLEDGMENTS</b></font><p>
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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<a name="references">
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