Distribution of α1a-, α1b- and α1d-adrenergic receptor mRNA in the rat brain and spinal cord
Introduction
Since the first classical division of adrenergic receptors into alpha and beta subtypes (Ahlquist, 1948), further subdivisions of these broad receptor classes have been determined, based on criteria such as pre- versus post-synaptic location, affinity for different ligands and second messenger systems. In general, β-adrenergic receptors are positively coupled, and α2-adrenergic receptors negatively coupled, to adenylate cyclase. The phosphatidylinositol pathway is involved in α1-adrenergic receptor mediated responses. Although the adrenergic system has been studied extensively in the peripheral nervous system, its functions have not been explored as thoroughly in the central nervous system (CNS), and progress is hindered by the lack of highly specific ligands to differentiate between the different adrenergic receptor subtypes.
Thus far, nine distinct adrenergic receptor subtypes have been cloned, all of which are members of the seven transmembrane domain, G protein-coupled receptor superfamily (Gocayne et al., 1987; Buckland et al., 1990, Machida et al., 1990, Schwinn et al., 1990, Shimomura and Terada, 1990, Voigt et al., 1990, Zeng et al., 1990, Granneman et al., 1991, Lanier et al., 1991, Lomasney et al., 1991, Perez et al., 1991, Perez et al., 1994). Based on the type of criteria cited above, these receptors have been termed α1a (previously α1a/c), α1b, α1d (previously α1a or α1a/d), α2a, α2b, α2c, β1, β2 and β3. The cloning of these receptors has provided additional tools with which to explore the central adrenergic receptor system, and with the exception of the β3-AR, the presence of all of the cloned adrenergic receptors has been demonstrated in the rat CNS. Although the majority of the receptors have been mapped by in situ hybridization (McCune et al., 1993, Nicholas et al., 1993a, Nicholas et al., 1993b, Pieribone et al., 1994, Price et al., 1994, Rokosh et al., 1994, Scheinin et al., 1994), the presence of α1a-AR mRNA in rat brain has been demonstrated only by RNase protection assays (Price et al., 1994, Rokosh et al., 1994). Here we describe for the first time, the distribution of α1a-AR mRNA, by in situ hybridization using ribonucleotide probes. For comparison, the distributions of mRNA encoding the α1b- and α1d-AR were also examined. With a few exceptions, the results for the α1b- and α1d-AR were broadly similar to those obtained in previous studies using oligonucleotide probes (McCune et al., 1993, Pieribone et al., 1994).
At this point, it should be noted that the original nomenclature for the α1-AR differs from that currently used. Recent reviews summarize the literature supporting the current terminology, and distinctions between the cloned and pharmacologically characterized receptors (Ruffolo et al., 1994, Graham et al., 1996, Guarino et al., 1996). Briefly, according to the International Union of Pharmacology (IUPHAR), lower case subscripts refer to the cloned receptors α1a, α1b and α1d, whilst upper case subscripts refer to their pharmacological homologues α1A, α1B and α1D. The published in situ hybridization studies describing the distribution of the `α1A' or `α1A/D' adrenergic receptor refer to mRNA encoding the α1d-AR, according to the most recent classification. This receptor was originally designated α1a as it exhibited some features in common with the pharmacological α1A-AR, such as relatively high affinity for some α1A-AR-selective ligands and resistance to inactivation by chlorethylclonidine (CEC) (Lomasney et al., 1991). However, a more extensive pharmacological analysis of the 98% homologous receptor cloned by Perez et al. (1991), and the demonstration that this receptor was sensitive to inactivation to higher concentrations of CEC, suggested that this receptor differed from the pharmacological α1A-AR, and was consequently designated the α1d-AR. The initial cloning of the bovine adrenergic receptor, now termed α1a-AR, designated this receptor α1c-AR, because the pharmacological and distribution profiles of this receptor were not in total agreement with the pharmacological α1A-AR. More extensive analysis by more than one laboratory of the rat and human homologues of the bovine α1c-AR, provided evidence that the characteristics of this receptor were most similar to the pharmacological α1A-AR. At present, an α1c-AR has not been cloned.
The data presented here describe the mRNA distributions for the three α1-adrenergic receptors, α1a-AR, α1b-AR and α1d-AR, and complete the CNS mRNA distributions for the adrenergic receptors cloned thus far. It is clear that each receptor subtype has a distinct pattern of distribution, suggestive of unique roles for each receptor. For a given brain area, there may be a single receptor expressed, or considerable overlap of receptors. Although colocalization in the same neuron has yet to be demonstrated, these data suggest potential for significant complexity of cellular responses.
Section snippets
Animals
Six male Sprague–Dawley rats (Charles River Laboratories), weighing 300–350 g were used. Rats were housed three per cage, under conditions of constant temperature and humidity, on a 12 h light/dark cycle (lights on 06:00 h) with access to food and water ad libitum. Animals were killed by rapid decapitation 2 h after lights on. Brains and spinal cords were removed, frozen in isopentane cooled to −40 to −50°C and stored at −80°C until further processing.
In situ hybridization histochemistry
Serial 10 μm sections from brain and spinal
Results
The ribonucleotide probes used in the present study appeared to be specific for the three different α1-AR subtype mRNA examined. There was no specific labeling with sense probes for any of the α1-AR (Fig. 1A, 1B and 1C). In the hippocampus and cerebellar granule layer, some additional background labeling was apparent on autoradiographic films for the α1a-AR and α1b-AR probes, but this was clearly non-specific when examined on emulsion dipped slides (a diffuse labeling, rather than distinct
Discussion
The mRNA distribution of the three α1-adrenergic receptors cloned to date has been determined in the rat brain and spinal cord, using ribonucleotide probes for in situ hybridization. This study is the first to describe the distribution of the α1a-AR mRNA, which has a diverse and unique pattern of expression. Two other studies have used oligonucleotide probes to examine the distribution of α1b- and α1d-AR mRNA (McCune et al., 1993, Pieribone et al., 1994). The present study provides broadly
Acknowledgements
The authors are grateful to Sharon Burke for subcloning the α1- adrenergic receptors. This study was supported by NIMH #MH42251, NIDA #DA02265-18 and The Pritzker Network for the Study of Depression.
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