Distribution of alpha 1 adrenoceptors in rat brain revealed by in situ hybridization experiments utilizing subtype-specific probes

Abstract

The distribution of neurons in the rat CNS that synthesize mRNA for the alpha 1A/D and alpha 1B adrenoceptors was revealed by the in situ hybridization method. Forty-eight-mer DNA probes were synthesized to two different and unique regions of both the alpha 1A/D and alpha 1B mRNAs. Tissue sections from all levels of the CNS and some peripheral ganglia were incubated in a hybridization cocktail containing one of these four probes. The two mRNAs were expressed in a discrete and often complementary manner to each other, and identical hybridization patterns were seen for the probes directed against the same mRNA. The alpha 1A/D probes hybridized heavily with neurons in the internal granular and internal plexiform layers of the olfactory bulb, in layers II-V of most areas of the cerebral cortex, and in the lateral aspect of the lateral amygdaloid nucleus, with pyramidal neurons of CA1-CA4 regions, hilar and granular neurons of the dentate gyrus, and neurons in the reticular thalamic nucleus, cranial and spinal motor nuclei, and the inferior olivary nucleus. Light labeling was seen in a variety of other regions in the brain and spinal cord. The alpha 1B probes hybridized heavily with neurons in the mid layers of cerebral cortex and with virtually all neurons in the thalamus, except the reticular and habenular nuclei. In addition, labeling was seen in the lateral and central amygdaloid nuclei, in brainstem and spinal motor nuclei, over most neurons of the dorsal and medullary raphe nuclei and neurons of the intermediolateral cell column in the spinal cord. Light labeling was seen in the septal nucleus, the horizontal limb of the diagonal band, the paraventricular and lateral hypothalamic nuclei, the pontine and medullary reticular formation, and in most laminae in the spinal cord. The patterns of labeling obtained with the alpha 1B probes resemble the labeling seen in previous autoradiographic ligand binding studies utilizing "general" alpha 1 ligands, while the labeling patterns seen with the alpha 1A/D probes do not correspond to any published alpha 1 receptor distribution pattern, indicating that this mRNA likely encodes for a novel adrenoceptor. The present findings further expand the heterogeneity of adrenoceptor mRNAs presented in two accompanying studies (Nicholas et al., 1993a,b). This differential distribution of adrenoceptors subtypes provides a framework for the functional diversity to the apparently widespread, diffuse, and rather homogeneous noradrenergic innervation of the CNS.

There and Back Again: A Tale of Norepinephrine and Drug Addiction

“…Although much of the work has focused on a1AR modulation of VTA DA neurons' firing properties, neither radioligand binding nor mRNA in situ hybridization techniques has reliably detected a1ARs in the VTA (Jones et al, 1985;Palacios et al, 1987;Pieribone et al, 1994). Thus, it is important to bear in mind that, until more sensitive techniques (eg, single-cell RT-PCR, electron microscopy) are employed to identify the exact anatomical localization of a1ARs in the VTA, many of the following conclusions remain speculative.…”

Expression of α1 adrenoceptor subtypes in rat corpus cavernosum

“…These results were in complete accordance with previous studies. 33,34 B1 and B2 and D1 and D2 probes displayed a higher signal-to-noise ratio than the B3 and D3 probes. We therefore selected B1, B2, D1 and D2 probes for further ISH using corpus cavernosum sections.…”

“…Speci®c a1B (B1, B2, B3) and a1D (D1, D2, D3) oligonucleotide antisense probes were previously used in ISH experiments to establish the pattern of expression of a1B and a1D subtypes in the rat brain. 32,33 B1 (bases 247±292) and B3 (bases 1568±1612); D2 (bases 480±521) and D3 (bases 2076±2120) oligonucleotide antisense probes were constructed from the 5 H extracellular region and the 3 H intracellular region of a1B and a1D subtypes, respectively. B2 (bases 1028±1072) oligonucleotide antisense probe was derived from the third intracellular loop of a1B subtype and D1 (bases 241±288) oligonucleotide antisense probe was derived from the 5 H untranslated-region of a1D subtype.…”

“…Prior to use, labeled probes were puri®ed by centrifugation through Chroma Spin-100 columns (Clontech, Palo Alto, CA, USA). We used 33 P radioisotope instead of 35 S, as 33 P provides a shorter autoradiographic ®lm exposure time. 34 Firstly we studied a1A, a1B and a1D, a1b and a1d probe speci®city on rat brain sections then performed ISH on corpus cavernosum sections.…”

Regulation of hippocampal α1d adrenergic receptor mRNA by corticosterone in adrenalectomized rats

“…Levels of α 1a and α 1b ADR mRNA expression are relatively low in the hippocampus (McCune et al, 1993;Pieribone et al, 1994;Day et al, 1997). αIn contrast, α 1d ADR mRNA is expressed at high levels in all subfields of the rat hippocampus and dentate gyrus (McCune et al, 1993;Pieribone et al, 1994;Day et al, 1997), and this receptor is expressed in the human hippocampus also (Szot et al, 2005). There is extensive colocalization of α 1d ADR mRNA with both MR and GR within the rat hippocampal formation (Williams et al, 1997), and thus α 1D ADRs are in an excellent position to both modulate glucocorticoid sensitive hippocampal neurons, and in turn be modulated by glucocorticoids.…”