- Email: [email protected]
Distribution of angiotensin type-1 receptor messenger RNA expression in the adult rat brain
Pergamon
PII:
Neuroscience Vol. 82, No. 3, pp. 827–841, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00328-X
DISTRIBUTION OF ANGIOTENSIN TYPE-1 RECEPTOR MESSENGER RNA EXPRESSION IN THE ADULT RAT BRAIN Z. LENKEI,*† M. PALKOVITS,† P. CORVOL* and C. LLORENS-CORTES*‡ *INSERM U36, Chaire de Me´decine Expe´rimentale, Colle`ge de France, 3, rue d’Ulm, 75005 Paris, France †Laboratory of Neuromorphology, Semmelweis University Medical School, Budapest, Hungary Abstract––Angiotensin II and angiotensin III in the brain exert their various effects by acting on two pharmacologically well-defined receptors, the type-1 (AT1) and the type-2 (AT2) receptors. Receptor binding autoradiography has revealed the dominant presence of AT1 in brain nuclei involved in cardiovascular, body fluid and neuroendocrine control. The cloning of the AT1 complementary DNA has revealed the existence of two receptor subtypes in rodents, AT1A and AT1B. Using specific riboprobes for in situ hybridization, we have previously shown that the AT1A messenger RNA is predominantly expressed in the rat forebrain; in contrast the AT1B subtype predominates in the anterior pituitary. Using a similar technical approach, the aim of the present study was to establish the precise anatomical localization of cells synthetising the AT1A receptor in the adult rat brain. High AT1A messenger RNA expression was found in the vascular organ of the lamina terminalis, the median preoptic nucleus, the subfornical organ, the hypothalamic periventricular nucleus, the parvocellular parts of the paraventricular nucleus, the nucleus of the solitary tract and the area postrema, in agreement with previous autoradiographic studies, describing a high density of AT1 binding sites in these nuclei. In addition, AT1A messenger RNA expression was detected in several brain areas, where no AT1 binding was reported previously. Thus, we identify strong expression of AT1A messenger RNA expression in scattered cells of the lateral parts of the preoptic region, the lateral hypothalamus and several brainstem nuclei. In none of these structures was the AT1B messenger RNA detectable at the microscopic level. In conclusion, it is suggested that angiotensins may exert their central effects on body fluid and cardiovascular homeostasis mainly via the AT1A receptor subtype. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: angiotensin receptor, AT1, in situ hybridization, brain mapping, choroid plexus, glia.
Brain angiotensin II and angiotensin III play a major role in various physiological functions including regulation of blood pressure and body fluid homeostasis by enhancing vasopressin release, water intake, sodium appetite and sympathetic activity (for review see Refs 29 and 41). In addition, they influence anterior pituitary hormone release, learning, memory and sensory functions.6,7,41 Angiotensin effects are mediated through their interaction with specific angiotensin receptors. Two, pharmacologically different angiotensin II receptor types, type-1 (AT1) and type-2 (AT2) have been identified using selective non-peptidic and pseudopeptidic antagonists. In vivo studies using these ‡To whom correspondence should be addressed. Abbreviations: AP, area postrema; AT1, angiotensin type-1 receptor; AT2, angiotensin type-2 receptor; GFAP, glial fibrillary acidic protein; MnPO, median preoptic nucleus; NFR, Nuclear Fast Red; NTS, nucleus of the solitary tract; OVLT, vascular organ of the lamina terminalis; PBS, phosphate-buffered saline; Pe, periventricular nucleus; PVN, paraventricular nucleus hypothalamus; SFO, subfornical organ. 827
antagonists have indicated that the major central actions of angiotensin are mediated by the AT1 receptor. Accordingly, autoradiographic binding studies have established the predominant expression of the AT1 receptor in brain areas controlling body fluid and cardiovascular homeostasis as well as pituitary hormone release.2,8,30 AT2 binding sites, conversely, were found in brain regions participating in sensory, limbic and other functions, where the role of the AT2 receptor remains to be determined. Although, mice lacking the gene encoding for the AT2 receptor have an impaired drinking response to water deprivation,9,10 indicating a possible role also for AT2 in the regulation of body fluid control. The cloning of the AT1 receptor cDNA revealed that it belongs to the seven-transmembrane domain, G protein-coupled receptor family. Moreover, two AT1 receptor subtypes have been identified in rodents5,24,32,33 which have been designated AT1A and AT1B. These receptors are more than 95% identical at the amino acid level. In the rat, AT1A and AT1B cDNAs share 95% identity of the nucleotide sequence within their coding region, whereas the homology is
828
Z. Lenkei et al.
reduced to 35% within the 5*- and 3*-untranslated regions.31 Expression of AT1A and AT1B in COS-7 cells shows quite comparable binding and coupling properties for both subtypes.4,31 The intracellular signalling pathways of both receptors include coupling to a GTP-binding protein, activation of a phospholipase C resulting in inositol triphosphate generation, mobilization of intracellular Ca2+ stores and diacylglycerol formation leading to protein kinase C activation.24,32 However, AT1A and AT1B mRNAs are differentially expressed and regulated in various peripheral tissues suggesting that both receptor subtypes may have different roles in mediating biological effects of angiotensin II. In a previous study we have shown by in situ hybridization that the AT1A mRNA is predominantly expressed in the rat forebrain, in contrast the AT1B subtype dominates in the anterior pituitary.13 The neuronal expression of AT1A receptors was demonstrated in the subfornical organ and the hypothalamus.14,18 By using double label in situ hybridization, AT1A receptor expression was localized in corticotropin-releasinghormone, but not in vasopressin-containing neurons in the hypothalamus.1,14 In the present study, we attempted to give a detailed mapping of the AT1A mRNA expression in the adult rat brain, in order to identify the neuronal structures underlying AT1mediated central angiotensin II actions. EXPERIMENTAL PROCEDURES
Synthesis of complementary RNA probes The AT1A and AT1B antisense and sense cRNA probes were synthesized by in vitro transcription as previously described in detail.34 Briefly, two 2.2 kb cDNA fragments (clone pCa18b for AT1A, a gift from Dr K. Bernstein and clone RAG6D4.60 for AT1B, a gift from Dr K. Sandberg) were subcloned into Bluescript KS plasmid (Stratagene, La Jolla, California) and pCDNA II plasmid (Invitrogen, Oxon, U.K.), respectively. After linearization, in vitro transcription was performed using T3, T7 and SP6 RNA polymerases (Boehringer, Mannheim, Germany) in the presence of [35S]UTP (Amersham, Les Ullis, France). The yield of the transcription was checked by agarose gel electrophoresis.
Tissue preparation All animal experiments were carried out in accordance with current institutional guidelines for the care and use of experimental animals. Adult (250–350 g) Sprague–Dawley rats (Iffa–Credo, Les Oncins, France, n=6) were kept on 12 h light/dark cycle with free access to food and water. The anaesthetized rats were perfused transcardially after a brief saline rinse with 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS). Brains were removed and postfixed overnight in the same fixative, then embedded in paraffin using standard procedures. Coronal sections (5 µm thick) were made and collected on 3-aminopropyltriethoxysilane (Sigma–Aldrich S.a.r.l., L’Isle D’Abeau Chesnes, France) -coated slides. One series of sections was stained with levanol and Nuclear Fast Red (NFR) (Chroma Gesellschaft, Koengen, Germany) for histological orientation, while adjacent sections were processed for in situ hybridization. In situ hybridization After treatment by proteinase K, sections were hybridized with the AT1A antisense and sense cRNA probes (see technical details in Ref. 35). After washes of different temperature and stringency which included an RNase treatment, the sections were dehydrated and exposed to Amersham Hyperfilm â-max for four weeks. After developing the films selected sections were dipped in Kodak NTB2 liquid emulsion and exposed for six to eight weeks, then developed and counterstained with Toluidine Blue. Glial fibrillary acidic protein immunohistochemistry after in situ hybridization For the detection of glial fibrillary acidic protein (GFAP), sections after in situ hybridization were rinsed twice in PBS, incubated in 20% normal goat serum for 20 min then with anti-GFAP antibody (DAKO, Trappe, France, diluted to 1:1000 in PBS) for 90 min. After rinsing twice in PBS, sections were incubated in a 1:200 dilution of biotinylated anti-rabbit antibody (Vector, Compiegne, France) for 30 min then rinsed again twice in PBS. Colour reaction was developed with the Elite Vectestain Kit (Vector). The sections were washed in Tris–HCl buffer (50 mM, pH 7.6) overnight, then dehydrated, dipped in Ilford K5 liquid emulsion. RESULTS
The control sections hybridized with the AT1A sense cRNA probe showed no hybridization signal,
Abbreviations used in the figures 7 A1/C1 acp Amb AOD AOL AOV AP AVPO csf GFAP HDB LH lo LPO MFB MM MnPO
motor facial nucleus A1/C1 catecholaminergic cells anterior commissure, posterior nucleus ambiguus anterior olfactory nucleus, dorsal part anterior olfactory nucleus, lateral part anterior olfactory nucleus, ventral part area postrema anteroventral preoptic nucleus cerebrospinal fluid glial fibrillary acidic protein nucleus of the diagonal tract, horizontal limb lateral hypothalamic area lateral olfactory tract lateral preoptic area median forebrain bundle medial mammillary nucleus median preoptic nucleus
Mo5 MPB MS NTS opt OVLT ox Pe Pir POM PVN PVpo RCh RPa SFO SO VDB
motor trigeminal nucleus medial parabrachial nucleus medial septal nucleus nucleus of the solitary tract optic tract vascular organ of the lamina terminalis optic chiasm periventricular nucleus piriform cortex medial preoptic nucleus paraventricular nucleus hypothalamus preoptic periventricular nucleus retrochiasmatic area nucleus raphe pallidus subfornical organ supraoptic nucleus nucleus of the diagonal tract, vertical limb
AT1 receptor mRNA expression in the adult rat brain
829
Fig. 1. Comparison of labelling with the AT1A antisense (A) and the AT1B antisense (B) probes at the level of the subfornical organ (SFO). Note the complete lack of specific labelling on B. This labelling is equivalent to the labelling obtained with the AT1A sense probe. Scale bar=500 µm. All photographs were scanned, brightness/contrast adjusted and labelled using Adobe Photoshop 3.0 for Macintosh (Adobe Systems Inc., Mountain View, CA), then printed on a Fuji Pictro 3000 printer. The data content of the images was not altered in any way.
with the exception of the hippocampus and the Purkinje cells of the cerebellum, where a constant, weak nonspecific labelling was observed. Hybridization with the AT1B antisense cRNA probe (Fig. 1) gave similar results to that of the AT1A sense cRNA probe, with the exception of the subfornical organ (SFO) and the parvocellular hypothalamic paraventricular nucleus, where a very weak AT1B specific labelling was detected. However, this very weak labelling was detectable only on film autoradiograms but not at the cellular level using emulsion autoradiography (Fig. 1). Hybridization with the AT1A antisense cRNA probe resulted in a well-localized cellular labelling in many brain nuclei. The results are summarized in Table 1 and some representative sections with high or very high level of AT1A mRNA expression are shown in Figs 2–15. As a guide to orientation, each photographic plate includes a low power microphotograph of a levanol–NFR stained coronal section of the rat brain, adjacent to the sections processed for in situ hybridization. Telencephalic structures Very high expression of AT1A mRNA was found in the anterior olfactory nucleus (Fig. 2) and the piriform cortex (Fig. 3). Low levels of AT1A mRNA expression were found in the cingulate, frontal and parietal cortex (Table 1). The vertical and horizontal limb of the nucleus of the diagonal band (Fig. 3), all parts of the hippocampus with the exception of the dentate gyrus displayed low level of AT1A mRNA expression (Table 1). Low density of AT1A mRNA expression was also found in the medial septal
nucleus (Fig. 3), the claustrum, the nucleus of the lateral olfactory tract, the anterior amygdaloid area, the central and lateral amygdaloid nuclei (Table 1). Basal ganglia did not contain detectable amounts of AT1A mRNA. Diencephalic structures In the hypothalamus, high to very high level of AT1A mRNA expression was detected in the preoptic area, especially in the vascular organ of the lamina terminalis (OVLT, Figs 4, 5), the median preoptic nucleus (MnPO, Figs 5–7), the preoptic part of the medial forebrain bundle (Fig. 6), and in scattered cells of the preoptic periventricular nucleus, the medial preoptic and the lateral preoptic area and anteroventral preoptic nucleus (Fig. 8). In the anterior hypothalamus, the periventricular nucleus (Pe, Fig. 9), all parvicellular parts of the paraventricular nucleus (PVN, Fig. 10) and the lateral retrochiasmatic area (Figs 10, 11) was established as high density of AT1A mRNA-expressing cells. High AT1A mRNA expression was also detected in cells in the rostral part of the lateral hypothalamic area (Figs 9–11). A number of the hypothalamic nuclei did not contain labelled cells (Table 1) except the perifornical nucleus (Fig. 11) and the median part of the medial mamillary nucleus (Fig. 12) where moderate– high, and the lateral preoptic area (Fig. 8), the caudal part of the lateral hypothalamic area (Table 1), the medial retrochiasmatic area, the dorsomedial nucleus (Fig. 11), the supramammilary nucleus where low AT1A mRNA expression was found (Table 1). In the thalamus, low AT1A mRNA expression was detected in the anterior ventral thalamic nucleus, the
830
Z. Lenkei et al. Table 1. Relative densities of AT1A messenger RNA expression in various brain nuclei 1. Telencephalon 1.1. Rhinencephalon Anterior olfactory nucleus Olfactory tubercle Nucl. of the diagonal tract Hippocampus
Nucleus of the lateral olfactory tract 1.2. Cerebral cortex Cingulate cortex* Piriform cortex Entorhinal cortex Frontal cortex* Parietal cortex* Insular cortex Temporal cortex Occipital cortex 1.3. Basal ganglia Accumbens nucleus Caudate nucleus Caudoputamen Substantia innominata Globus pallidus Endopiriform nucleus Claustrum** 1.4. Septum Medial septal nucleus Lateral septal nucleus Dorsal septal nucleus Fimbrial septal nucleus 1.5. Amygdala Anterior amygdaloid area Nucl. of the lateral olfactory tract Central nucleus amygdala Lateral nucleus amygdala Medial nucleus amygdala Basal nucleus amygdala Cortical nucleus amygdala Posterior nucleus amygdala Bed nucleus stria terminalis 2. Diencephalon 2.1. Thalamus Paratenial nucleus thalamus Paraventricular nucleus thalamus Anterodorsal nucleus thalamus Anteroventral nucleus thalamus Anteromedial nucleus thalamus Mediodorsal nucleus thalamus Central medial nucleus thalamus Central lateral nucleus thalamus Paracentral thalamic nuclei Midline thalamic nuclei Reuniens nucleus thalamus Rhomboid nucleus thalamus Gelatinose nucleus thalamus Reticular nucleus thalamus Lateral nucleus thalamus Lateral posterior nucleus thalamus Ventrolateral nucleus thalamus Ventromedial nucleus thalamus Ventral posterolateral nucleus thalamus Ventral posteromedial nucleus thalamus Suprageniculate nucleus Posterior nucleus thalamus 2.2. Epithalamus Medial habenula Lateral habenula
vertical limb horizontal limb anterior hilus (CA1–CA2) CA1 CA2 CA3 dentate gyrus subiculum
+++ – + + + + + + + – + ++ + +++ – + + – – – – – – – – – + + – – – + + + + – – – – – – – – + – + – – – – – – – – – – + – + + – – – –
AT1 receptor mRNA expression in the adult rat brain
831
Table 1. Continued 2.3. Metathalamus Lateral geniculate complex Medial geniculate complex Pretectal area Nucleus of the optic tract Nucleus of the posterior commissure 2.4. Subthalamus Zona incerta Subthalamic nucleus Fields of Forel Entopeduncular nucleus Bed nucleus of the supraoptic decussation 2.5. Preoptic region Median preoptic nucleus Medial preoptic nucleus Preoptic periventricular nucleus Anteroventral preoptic nucleus Preoptic suprachiasmatic nucleus Lateral preoptic area Medial forebrain bundle, preoptic part 2.6. Hypothalamus Lateral hypothalamic area Suprachiasmatic nucleus Periventricular nucleus Supraoptic nucleus Paraventricular nucleus
Anterior hypothalamic nucleus Retrochiasmatic area Median eminence Arcuate nucleus Ventromedial nucleus Dorsomedial nucleus Perifornical nucleus Dorsal premamillary nucleus Ventral premamillary nucleus Posterior hypothalamic nucleus Supramamillary nucleus Tuberomamillary nucleus A11 catecholaminergic cells 2.7. Mamillary body Medial mamillary nucleus
Lateral mamillary nucleus 3. Mesencephalon Medial forebrain bundle, mesencephalic part Ventral tegmental area Periaqueductal central gray matter Dorsal raphe nucleus Motor oculomotor nucleus Trochlear nucleus Interstitial nucleus of Cajal Linear raphe nuclei Midline raphe nucleus Colliculus superior Substantia nigra Peripeduncular nucleus Interpeduncular nucleus Red nucleus Cuneiform nucleus Pedunculopontine tegmental nucleus Inferior colliculus
dorsal part ventral part intergeniculate leaflet
– – + – – – – – – + – – +++ + s ++ – + ++
anterior part posterior part
periventricular medial-dorsal post. parvicell. magnocellular medial part lateral part
median part medial part lateral part posterior part
reticular zone compact zone lateral part
++ + – ++ – +++ +++ ++ – s + – – – – + ++ – – s + – + ++ + + – – + s + s/+ – – – – – – – – – – – – – + –
Table 1. Continued 4. Pons Pontine nuclei Nuclei of the lateral lemniscus Pontine reticular nucleus Reticular tegmental pontine nucleus Pontine raphe nucleus Dorsal tegmental nucleus Dorsolateral tegmental nucleus Locus coeruleus Subcoeruleus nucleus Nucleus of Barrington Paragenual nucleus Mesenchephalic nucleus of the trigeminal Medial parabrachial nucleus Lateral parabrachial nucleus Kolliker–Fuse nucleus Ventral tegmental nucleus Trigeminal motor nucleus Principal sensory nucleus of the trigeminal Superior olive Abducens nucleus 5. Cerebellum 5.1. Cerebellar cortex*** 5.2. Cerebellar nuclei Medial (fastigial) nucleus Interpositus nuclei Lateral nucleus 6. Medulla oblongata Cochlear nuclei Superior vestibular nucleus Medial vestibular nucleus Lateral vestibular nucleus Spinal vestibular nucleus Nucleus raphe magnus Nucleus raphe obscurus Nucleus raphe pallidus Gigantocellular reticular nucleus Paragigantocellular reticular nucleus Parvocellular reticular nucleus Medullary reticular nucleus Paramedian reticular nucleus Lateral reticular nucleus Spinal nucleus of the trigeminal, oral part Paratrigeminal nucleus Facial motor nucleus Inferior olivary complex Prepositus hypoglossal nucleus Nucleus of the solitary tract
Intercalate nucleus Dorsal motor nucleus of the vagus Ambiguus nucleus Linear nucleus of Rolando Motor hypoglossal nucleus Peritrigeminal nucleus External cuneate nucleus Cuneate nucleus of Burdach Gracile nucleus of Goll 7. Circumventricular organs Organum vasculosum laminae terminalis (OVLT) Subfornical organ (SFO) Median eminence Subcomissural organ Area postrema Choroid plexus
– – + + + + ++ + + – – + ++ + + – ++ ++ + – + + s +
interpositus part gelatinose part principal part dorsal accessory medial accesory rostral medial part caudal medial part lateral part commissural part
+ – – – – s++ – +++ + + – + – s++ + + – – ++ – – – + +++ +++ + +++ – s/+ +++ + + – – – – +++ +++ – – ++ ++
‘‘+++’’ very high, ‘‘++’’ high and ‘‘+’’ low level of expression. *V. layer; **Superficial layer; ***Purkinje cells. Label ‘‘s’’ indicates scattered labelled cells through the region. The results were consistent from animal to animal specially at the higher values. At lower values sometimes variation was detected +/-, partly due to the inhomogeneity of AT1A mRNA expression within the nucleus.
AT1 receptor mRNA expression in the adult rat brain
833
Fig. 2. Expression of AT1A receptor mRNA in the anterior olfactory nucleus. (A) Levanol–NFR-stained coronal section, approximately 4.20 mm rostral to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the ventral (AOV) and in the lateral (AOL) parts of the anterior olfactory nucleus. lo, lateral olfactory tract. Scale bar=1 mm.
Fig. 3. Expression of AT1A receptor mRNA in the septum and the piriform cortex. (A) Levanol–NFRstained coronal section, approximately 0.70 mm rostral to the level of the bregma. (B,C) Dark-field photomicrographs of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the piriform cortex (Pir) and low in the medial septal nucleus (MS) the vertical (VDB) and horizontal (HDB) limbs of the nucleus of the diagonal band. lo, lateral olfactory tract. Scale bar=1 mm.
Fig. 4. Expression of AT1A receptor mRNA in the preoptic region. (A) Levanol–NFR-stained coronal section, approximately 0.40 mm rostral to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the vascular organ of the lamina terminalis (OVLT). ox, optic chiasm and the asterisk labels the optic recess of the third ventricle. Scale bar=1 mm.
mediodorsal thalamic nucleus, in the ventrolateral, ventromedial and ventroposterior lateral thalamic nuclei (Table 1). The metathalamus (the intergenicu-
late leaflet of the lateral geniculate body) and the subthalamus (the fields of Forel) were poor in AT1A mRNA expressing cells (Table 1).
834
Z. Lenkei et al.
Fig. 5. Expression of AT1A receptor mRNA in the preoptic region. (A) Levanol–NFR-stained coronal section, approximately 0.20 mm rostral to the level of the bregma. (B). Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the caudal end of the vascular organ of the lamina terminalis (OVLT) and in the rostroventral part of the median preoptic nucleus (MnPO) ox, optic chiasm, acp, anterior commissure, posterior part. Scale bar=1 mm.
Fig. 6. Expression of AT1A receptor mRNA in the preoptic region. (A) Levanol–NFR-stained coronal section, approximately 0.10 mm rostral to the level of the bregma. (B,C). Darkfield photomicrographs of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the median preoptic nucleus (MnPO) and high in the medial forebrain bundle (MFB). ox = optic chiasm, acp = anterior commissure, posterior part and the asterisk labels the third ventricle. Scale bar =1 mm.
Mesencephalic structures High AT1A mRNA expression was detected in the rostral part of the midbrain periaqueductal gray matter (Table 1). Low level of labelling was present in the mesencephalic part of the medial forebrain bundle, in the dorsal raphe nucleus and in the pedunculopontine tegmental nucleus, while neurons in other midbrain structures were unlabelled (Table 1).
nucleus, the lateral parabrachial nucleus, the Kolliker–Fuse nucleus, the superior olive and the trapezoid body (Table 1). Cerebellum Weak expression of AT1A mRNA was detected in the Purkinje cell layer of the cerebellar cortex and in the medial, interpositus and lateral cerebellar nuclei (Table 1).
Pons High AT1A mRNA expression was detected in the medial parabrachial nucleus (Table 1), in the dorsolateral tegmental nucleus (Table 1), the motor and principal sensory trigeminal nuclei (Table 1). Weak labelling was also found in the pontine reticular nucleus, the reticular tegmental pontine nucleus, the pontine raphe nucleus, the dorsal tegmental nucleus, the locus coeruleus, the subcoeruleus area, the nucleus of Barrington, the mesencephalic trigeminal
Medulla oblongata Very high AT1A mRNA expression was detected in the nucleus raphe pallidus (Fig. 13), the retrofacial (Table 1), the ambiguus (Figs 14, 15) and linear nuclei (Table 1), the medial and commisural parts of the nucleus of the solitary tract as well as in the area postrema (Fig. 15). High expression of AT1A mRNA was detected in the nucleus raphe magnus (Table 1) and the facial motor nucleus (Fig. 13). Weak AT1A
AT1 receptor mRNA expression in the adult rat brain
835
Fig. 7. Expression of AT1A receptor mRNA in the preoptic region. (A) Levanol–NFR-stained coronal section, approximately at the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the median preoptic nucleus (MnPO). acp, anterior commissure, posterior part and the asterisk labels the third ventricle. Scale bar=1 mm.
Fig. 8. Expression of AT1A receptor mRNA in the anterior hypothalamus. (A) Levanol–NFR-stained coronal section, approximately 0.85 mm caudal to the level of the bregma. (B,C) Dark-field photomicrographs of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the subfornical organ (SFO), high in scattered cells of the medial preoptic nucleus (POM) the preoptic periventricular nucleus (PVpo) and the lateral preoptic area (LPO). The lateral part of the POM corresponds also to the anteroventral preoptic nucleus (AVPO) of Paxinos and Watson.27 ox, optic chiasm and the asterisk labels the third ventricle. Scale bar=1 mm.
mRNA expression was detected in the cochlear nuclei, the lateral vestibular nucleus, the giganto-cellular, paragigantocellular, lateral and medullary reticular nuclei, in the motor hypoglossal nucleus, in the oral and interpositus part of the spinal trigeminal nucleus and the prepositus hypoglossal nucleus (Table 1).
the anterior commissure (Figs 4–6) and the optic tract (Figs 8–10), but not in other white matter tracts as the corpus callosum or the pyramidal tract, etc. This labelling was not detected either with the AT1B antisense nor with the AT1A sense probes. However, we were not able to clearly associate this labelling with cells.
Choroid plexus Expression of AT1A mRNA was detected in connective tissue cells of the choroid plexus (Fig. 16). Myelinated structures Expression of AT1A mRNA is indicated by small clusters of silver grains in several fibre tracts such as
Identification of the cell types expressing AT1A messenger RNA GFAP immunohistochemistry performed after in situ hybridization in the hypothalamus shows that AT1A mRNA is expressed exclusively by large GFAP
836
Z. Lenkei et al.
Fig. 9. Expression of AT1A receptor mRNA in the anterior hypothalamus. (A) Levanol–NFR-stained coronal section, approximately 1.40 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is high in the periventricular nucleus (Pe) and in scattered cells of the lateral hypothalamic area (LH). ox, optic chiasm and the asterisk labels the third ventricle. Scale bar=1 mm.
Fig. 10. Expression of AT1A receptor mRNA in the anterior hypothalamus. (A) Levanol–NFR-stained coronal section, approximately 1.80 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the parvocellular parts of the paraventricular nucleus (PVN) high in scattered cells of the lateral hypothalamic area (LH) and retrochiasmatic area (RCh). opt, optic tract and the asterisk labels the third ventricle. Scale bar=1 mm.
negative cells (Fig. 17), indicating a neuronal expression of AT1A mRNA. DISCUSSION
Using radiolabelled riboprobes for in situ hybridization histochemistry, we established the detailed distribution of the AT1 receptor subtype mRNAs in the adult rat brain at the cellular level. The specificity of the probes used was previously established in transfected cell lines overexpressing either the AT1A or the AT1B receptor.13 In the hypothalamus, where we identified the nature of AT1A expressing cells by using immunohistochemical detection of GFAP after the in situ hybridization, the expression of AT1A mRNA was found exclusively in neurons. High level of AT1A mRNA expression was found in brain areas involved in mediating angiotensin effects on drinking, blood pressure and endocrine status (Fig. 18), such as the OVLT, median preoptic nucleus (MnPO), subfornical organ (SFO), paraventricular nucleus hypotha-
lamus (PVN), nucleus of the solitary tract (NTS) and area postrema (AP), but also in brain regions where the role of angiotensin is less well defined as the piriform cortex, the trigeminal nuclei, the facial motor nucleus, the nucleus raphe pallidus and the ambiguus nucleus. In accordance with our previous report,13 labelling with the AT1B receptor cRNA probe was very weak and restricted to the brain nuclei which displayed the highest expression of the AT1A mRNA. In addition, this very weak labelling was detectable only on film autoradiograms but not at the cellular level using emulsion autoradiography. It could not be exluded, however, that AT1B mRNA is expressed in the hippocampus and the Purkinje cells of the cerebellum, where the observed constant, weak non-specific labelling may mask the presence of a weak specific signal. In conclusion, if AT1B mRNA is expressed in the brain, in our hands it is below the detection limit of the currently known most sensitive method for in situ hybridization, using radioactivelylabelled long riboprobes.
AT1 receptor mRNA expression in the adult rat brain
837
Fig. 11. Expression of AT1A receptor mRNA in the middle part of the hypothalamus. (A) Levanol– NFR-stained coronal section, approximately 2.30 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is high in scattered cells of the lateral hypothalamic area (LH) and lateral retrochiasmatic area (RCh). opt, optic tract and the asterisk labels the third ventricle. Scale bar=1 mm.
Fig. 12. Expression of AT1A receptor mRNA in the mamillary body. (A) Levanol–NFR-stained coronal section, approximately 4.65 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is high in the median part of the medial mammillary nucleus (MM). Scale bar=1 mm.
Fig. 13. Expression of AT1A receptor mRNA in the rostral medulla oblongata. (A) Levanol–NFR-stained coronal section, approximately 11.0 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the nucleus raphe pallidus (RPa) and high in the motor facial nucleus (7). Scale bar=1 mm.
In general, the distribution of AT1A mRNA presented here is in agreement with AT1 mRNA mapping of Bunneman et al.,3 which was performed at the macroscopic level. Our results confirm and expand on a recent report12 which studied the expres-
sion of AT1 receptor subtype mRNAs in the forebrain of two-week-old rats and found AT1A mRNA expression in the OVLT, MnPO, SFO, PVN, Pe and lateral hypothalamus. However, in contrast with this latter study which reported expression
838
Z. Lenkei et al.
Fig. 14. Expression of AT1A receptor mRNA in the medulla oblongata. (A) Levanol–NFR-stained coronal section, approximately 12.60 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the ambiguus nucleus (Amb). Scale bar=1 mm.
Fig. 15. Expression of AT1A receptor mRNA in the caudal medulla oblongata. (A) Levanol–NFR-stained coronal section, approximately 13.60 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the medial part of the nucleus of the solitary tract (NTS) and in the area postrema (AP) and low but visible in the lateral reticular nucleus (A1/C1). Scale bar=1 mm.
of AT1B mRNA in the hippocampus, we detected only AT1A mRNA expression, as discussed above. The distribution of the AT1A mRNA shows a strong correlation with the distribution of AT1 binding sites previously established using non-peptidic AT1 ligands for receptor binding autoradiography.2,8 These studies showed a high density of AT1 binding sites in the OVLT, the MnPO, the SFO, the hypothalamic periventricular nucleus, the parvocellular parts of the paraventricular nucleus, the NTS and the AP. Our results further document these findings at the microscopic level and show that the AT1 receptor in these nuclei is of the AT1A subtype. In these nuclei the co-localization of AT1A mRNA with angiotensin II binding sites and angiotensin-immunoreactive terminal fields15 suggest that AT1 receptors are likely present on the cell bodies or dendrites, serving as postsynaptic receptors for neuronal angiotensins. Angiotensin-containing nerve terminals have also been visualized in the OVLT and SFO.15,25 In the central, heavily vascularized regions of these circumventricular organs, they do not form synaptic specializations and are closely opposed to fenestrated
capillaries, whereas they are in synaptic contact with neurons in peripheral parts of these structures.25 This indicates that a part of the neuronally released angiotensins is secreted directly into the circulation, and suggests that in these organs, some neuronal AT1A receptors may not constitute postsynaptic receptors for neuronal angiotensins. In fact, these circumventricular organs are targets for circulating angiotensin II.23,26 The respective roles of the circulating angiotensin II originating from the periphery versus that from the neighbouring angiotensinergic neurons, on activation of AT1A receptors, is unknown. On the other hand, AT1 mRNA expression was detected in several brain areas, where no AT1 binding was previously reported (Table 1). Thus, taking advantage of the increased resolution of the in situ hybridization technique, we identified strong expression of AT1A mRNA expression in scattered cells of the lateral parts of the preoptic region, the lateral hypothalamus and several brainstem nuclei. By contrast, we did not detect AT1A mRNA expression in the suprachiasmatic nucleus (visible but not marked on Fig. 7B), the median eminence and the ventral
AT1 receptor mRNA expression in the adult rat brain
839
Fig. 16. Expression of AT1A receptor mRNA in the choroid plexus of the lateral ventricle. A cell in the connective tissue shows accumulation of silver grains over its cytoplasm. csf, cerebrospinal fluid. Scale bar=5 µm.
Fig. 17. Neuronal expression of AT1A mRNA in the lateral hypothalamic area. The in situ hybridization with a radioactive AT1A receptor riboprobe was followed by immunohistochemistry on the same brain slice using an anti-glial fibrillary acidic protein (GFAP) antibody. Arrowheads indicate GFAP-immunoreactive astrocytes and the arrow shows a GFAP-negative large cell labelled by accumulation of silver grains over its cytoplasm, indicating the expression AT1A mRNA. Scale bar=10 µm.
part of the bed nucleus of the stria terminalis, structures where high density of AT1 binding sites were reported.2,8 Possibly, the AT1 receptors are synthesized in perikaryons located elsewhere and transported axonally to these structures to mediate angiotensin II or angiotensin III action on the release of neurotransmitters at the presynaptic level. Distribution of AT1 receptors was also studied in the brain with immunohistochemical staining, using an antibody to a portion of the 3rd cytoplasmic loop of the AT1 receptor.28 The results of this report show an overlap with the results of the receptor binding studies2,8 as well as with our present data by showing AT1 receptor expression in the OVLT, the MnPO, the SFO, the locus coeruleus, the mesencephalic and motor nuclei of the trigeminal nerve, the cochlear nucleus and superior olivary nuclei, the nucleus of the
solitary tract, the nucleus hypoglossus, the rostroventrolateral medullary area and the nucleus ambiguus. However, very strong immunohistochemical staining has also been reported in the magnocellular neurons of the paraventricular and supraoptic nuclei, devoid of angiotensin II binding sites2,8 or AT1A or AT1B mRNA expression.1,12–14 One possible explanation is that this antibody also detects a non-AT1A non-AT1B receptor which does not bind radiolabelled angiotensin II analogues but shows an identity in its amino acid sequence to the portion of the 3rd cytoplasmic loop of the AT1 receptor. Structures associated with the lamina terminalis include the MnPO and the circumventricular organs SFO and OVLT. Current evidence indicates that blood-borne signals, such as angiotensin II, reach the SFO and OVLT where they are transduced. This information is then carried via neural pathways to brain nuclei, such as MnPO, PVN, supraoptic nucleus, NTS, where it is integrated with other inputs derived from systemic arterial blood pressure and volume receptors. Because of their receptive and integrative functions, lamina terminalis structures are essential for the normal control of hormone release (e.g., vasopressin), sympathetic activation and behaviours (thirst and salt appetite), which collectively contribute to maintenance of cardiovascular and body fluid homeostasis (for review see Ref. 11). In the choroid plexus, angiotensin II was shown to decrease the blood flow19,20 and the production rate of cerebrospinal fluid.21,40 This organ is rich in angiotensin converting enzyme23 and contains AT1 binding sites.36 The angiotensin II receptors can be synthetized locally as cells in the connective tissue of this organ show a moderate expression of AT1A mRNA. Alternatively, angiotensin II receptors synthesized elsewhere could be transported to this structure as cholinergic fibres from the vagus and adrenergic fibres from the superior cervical ganglion
840
Z. Lenkei et al.
Fig. 18. Distribution of AT1A receptor mRNA in the adult rat brain, sagittal section. Nuclei where the labelling was ‘‘+++’’ or ‘‘++’’ (Table 1) are marked with dark grey, nuclei with high but scattered labelling are marked with light grey. For abbreviations see list.
innervate the blood vessels and epithelia of the choroid plexus.16,17 The possible presence of AT1A mRNA in small cells of several fibre tracts (anterior commisure, optic tract) is in line with the observation that AT1 receptor expression was found in cultured glial cell lines, derived from embryonic tissues37,38,39 or from the adult corpus callosum.22 However, immunohistochemical detection of the glial marker GFAP after in situ hybridization for the AT1A mRNA in the hypothalamus, shows that the AT1A mRNA is expressed only in neurons. In fibre tracts, the small clusters of silver grains could not be associated either to GFAPpositive cells, or to other non-labelled cells. Thus, this AT1A specific labelling might be expressed by small GFAP-negative glial cells, whose small size makes the cellular localization of the labelling difficult, but further studies using glial-specific markers for double labelling studies might elucidate this question. Alternatively, we are detecting a non-specific phenomena
characteristic of the AT1A antisense riboprobe, since this labelling was detected neither with the AT1B antisense nor with the AT1A sense probe. CONCLUSION
In conclusion, by the demonstration of a relatively wide distribution pattern of AT1A mRNA in the adult rat brain we provide a basis for further investigations concerning the role of the brain angiotensin system in the control of the salt water homeostasis and blood pressure, and probably other regulatory mechanisms. Acknowledgements—The authors thank K. Bernstein for the AT1A receptor cDNA clone pCa18b, K. Sandberg for AT1B receptor cDNA clone RAG6D4.60, J. Helfferich for skilfull technical assistence. This work received the financial support of Bristol–Myers–Squibb Laboratories and of a French–Hungarian cooperation (Balaton) managed by the french ‘‘Ministere des Affaires Etrange`res’’ and OMFB.
REFERENCES
1. 2. 3. 4. 5. 6. 7.
Aguilera G., Young W. S., Kiss A. and Bathia A. (1995) Direct regulation of hypothalamic corticotropin-releasing hormone neurons by angiotensin II. Neuroendocrinology 61, 437–444. Allen A. M., Paxinos G., Song K. F. and Mendelsohn F. A. O. (1992) Localization of angiotensin receptor binding sites in the rat brain. In Handbook of Chemical Neuroanatomy Vol. 11: Neuropeptide Receptors in the CNS (eds A. Bjo¨rklund, T. Ho¨kfelt and M. J. Kuhar), Vol. 11, pp. 1–37. Elsevier, Amsterdam. Bunnemann B., Iwai N., Metzger R., Fuxe K., Inagami T. and Ganten D. (1992) The distribution of angiotensin II AT1 receptor subtype mRNA in the rat brain. Neurosci. Lett. 142, 155–158. Clauser E., Curnow K. M., Conchon S., Davies E., Teutsch B., Vianello B., Monnot C. and Corvol P. (1995) Molecular structure and mechanisms of action of mammalian angiotensin receptors. Curr. Opin. Endocr. Diab. 2, 404–411. Elton T. S., Stephan C. C., Taylor G. R., Kimball M. G., Martin M. M., Durand J. N. and Oparil S. (1992) Isolation of two distinct type I angiotensin II receptor genes. Biochem. biophys. Res. Commun. 184, 1067–1073. Ganong W. F. (1993) Blood, pituitary, and brain renin–angiotensin systems and regulation of secretion of anterior pituitary gland. Front. Neuroendocrinol. 14, 233–249. Ganong W. F. (1995) Reproduction and the renin–angiotensin system. Neurosci. Biobehav. Rev. 19, 241–250.
AT1 receptor mRNA expression in the adult rat brain 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
841
Gehlert D. R., Gackenheimer S. L. and Schober D. A. (1991) Autoradiographic localization of subtypes of angiotensin II antagonist binding in the rat brain. Neuroscience 44, 501–514. Hein L., Barsh G., Pratt R. E., Dzau V. J. and Koblika B. K. (1995) Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor gene in mice. Nature 377, 744–747. Ichiki T., Labosky P. A., Shiota C., Okuyama S., Imagawa Y., Fogo A., Niimura F., Ichikawa I., Hogan B. L. and Inagami T. (1995) Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature 377, 748–750. Johnson A. K., Cunningham J. T. and Thunhorst R. L. (1996) Integrative role of the lamina terminalis in the regulation of cardiovascular and body fluid homeostasis. Clin. exp. Pharm. Physiol. 23, 183–191. Jo¨hren O. and Saavedra J. M. (1996) Expression of AT1A and AT1B angiotensin II receptor messenger RNA in forebrain of 2-wk-old rats. Am. J. Physiol. – Endocrinol. Met. 34, E104–E112. Lenkei Z., Corvol P. and Llorens-Cortes C. (1995) The angiotensin receptor subtype AT1A predominates in rat forebrain areas involved in blood pressure, body fluid homeostasis and neuroendocrine control. Molec. Brain Res. 30, 53–60. Lenkei Z., Corvol P. and Llorens-Cortes C. (1995) Comparative expression of vasopressin and angiotensin type-1 receptor mRNA in rat hypothalamic nuclei: a double in situ hybridization study. Molec. Brain Res. 34, 135–142. Lind R. W. and Ganten D. (1990) Angiotensin. In Handbook of Chemical Neuroanatomy (eds A. Bjo¨rklund, T. Ho¨kfelt and M. J. Kuhar), Vol. 9, Part II, pp. 165–286. Elsevier, Amsterdam. Lindvall M., Edvinsson L. and Owman C. (1978) Sympathetic nervous control of cerebrospinal fluid production from the choroid plexus. Science 201, 176–178. Lindvall M. and Owman C. (1981) Autonomic nerves in the mammalian choroid plexus and their influence on the formation of cerebrospinal fluid. J. cerebr. Blood Flow Metab. 1, 245–266. Lippoldt A., Bunnemann B., Iwai N., Metzger R., Inagami T., Fuxe K. and Ganten D. (1993) Cellular localization of angiotensin type 1 receptor and angiotensinogen mRNAs in the subfornical organ of the rat brain. Neurosci. Lett. 150, 153–158. Maktabi M. A., Heistad D. D. and Faraci F. M. (1990) Effects of angiotensin II on blood flow to choroid plexus. Am. J. Physiol. 258, H414–H418. Maktabi M. A., Heistad D. D. and Faraci F. M. (1991) Effects of central and intravascular angiotensin I and II on the choroid plexus. Am. J. Physiol. 261, R1126–R1132. Maktabi M. A., Stachovic G. C. and Faraci F. M. (1993) Angiotensin II decreases the rate of production of cerebrospinal fluid. Brain Res. 606, 44–49. Matute C., Pulakat L., Rı´o C., Varca´rcel C. and Miledi R. (1994) Properties of angiotensin II receptors in glial cells from the adult corpus callosum. Proc. natn. Acad. Sci. U.S.A. 91, 3774–3778. McKinley M. J., McAllen R. M., Mendelsohn F. A. O., Allen A. M., Chai S. Y. and Oldfield B. J. (1990) Circumventricular organs: neuroendocrine interfaces between the brain and the hemal milieu. Front. Neuroendocrinol. 11, 91–127. Murphy T. J., Alexander R. W., Griendling K. K., Runge M. S. and Bernstein K. E. (1991) Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature 351, 233–236. Oldfield B. J., Ganten D. and McKinley M. J. (1989) An ultrastructural analysis of the distribution of angiotensin II in the rat brain. J. Neuroendocrinol. 1, 121–128. Oldfield B. O. and McKinley M. J. (1994) Circumventricular organs. In The Rat Nervous System, 2nd edn (ed. G. Paxinos). Academic, London. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic, Sydney. Phillips M. I., Shen L., Richards E. M. and Raizada M. K. (1993) Immunohistochemical mapping of angiotensin AT1 receptors in the brain. Regul. Pept. 44, 95–107. Saavedra J. M. (1992) Brain and pituitary angiotensin. Endocr. Rev. 13, 329–380. Saavedra J. M. (1994) Brain angiotensin II receptor subtypes. In Angiotensin Receptors (eds J. M. Saavedra and P. B. M. W. M. Timmermans), pp. 151–175. Plenum, New York. Sandberg K. (1994) Structural analysis and regulation of angiotensin II receptors. Trends Endocrinol. Metab. 5, 28–35. Sandberg K., Ji H., Clark A. J., Shapira H. and Catt K. J. (1992) Cloning and expression of a novel angiotensin II receptor subtype. J. biol. Chem. 267, 9455–9458. Sasamura H., Hein L., Krieger J. E., Pratt R. E., Kobilka B. K. and Dzau V. J. (1992) Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome. Biochem. biophys. Res. Commun. 185, 253–259. Shanmugam S., Monnot C., Corvol P. and Gasc J.-M. (1994) Distribution of type 1 angiotensin II receptor subtype messenger RNAs in the rat fetus. Hypertension 23, 137–141. Sibony M., Commo F., Callard P. and Gasc J. M. (1995) Enhancement of mRNA in situ hybridization signal by microwave heating. Lab. Invest. 73, 586–591. Song K., Allen A. M., Paxinos G. and Mendelsohn F. A. (1992) Mapping of angiotensin II receptor subtype heterogeneity in rat brain. J. comp. Neurol. 316, 467–484. Sumners C., Myers L. M., Kalberg C. J. and Raizada M. K. (1990) Physiological and pharmacological comparisons of angiotensin II receptors in neuronal and astrocyte glial cultures. Prog. Neurobiol. 34, 355–385. Sumners C., Tang W., Paulding W. and Raizada M. K. (1994) Peptide receptors in astroglia: focus on angiotensin II and atrial natriuretic peptide. Glia 11, 110–116. Sumners C., Tang W., Zelezna B. and Raizada M. K. (1991) Angiotensin II receptor subtypes are coupled with distinct signal-transduction mechanisms in neurons and astrocytes from rat brain. Proc. natn. Acad. Sci. U.S.A. 88, 7567–7571. Vogh B. P. and Godman D. R. (1989) Effects of inhibition of angiotensin converting enzyme and carbonic anhydrase on fluid production by ciliary process, choroid plexus, and pancreas. J. ocul. Pharmac. 5, 303–311. Wright J. W. and Harding J. W. (1992) Regulatory role of brain angiotensins in the control of physiological and behavioral responses. Brain Res. Rev. 17, 227–262. (Accepted 10 June 1997)
PII:
Neuroscience Vol. 82, No. 3, pp. 827–841, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00328-X
DISTRIBUTION OF ANGIOTENSIN TYPE-1 RECEPTOR MESSENGER RNA EXPRESSION IN THE ADULT RAT BRAIN Z. LENKEI,*† M. PALKOVITS,† P. CORVOL* and C. LLORENS-CORTES*‡ *INSERM U36, Chaire de Me´decine Expe´rimentale, Colle`ge de France, 3, rue d’Ulm, 75005 Paris, France †Laboratory of Neuromorphology, Semmelweis University Medical School, Budapest, Hungary Abstract––Angiotensin II and angiotensin III in the brain exert their various effects by acting on two pharmacologically well-defined receptors, the type-1 (AT1) and the type-2 (AT2) receptors. Receptor binding autoradiography has revealed the dominant presence of AT1 in brain nuclei involved in cardiovascular, body fluid and neuroendocrine control. The cloning of the AT1 complementary DNA has revealed the existence of two receptor subtypes in rodents, AT1A and AT1B. Using specific riboprobes for in situ hybridization, we have previously shown that the AT1A messenger RNA is predominantly expressed in the rat forebrain; in contrast the AT1B subtype predominates in the anterior pituitary. Using a similar technical approach, the aim of the present study was to establish the precise anatomical localization of cells synthetising the AT1A receptor in the adult rat brain. High AT1A messenger RNA expression was found in the vascular organ of the lamina terminalis, the median preoptic nucleus, the subfornical organ, the hypothalamic periventricular nucleus, the parvocellular parts of the paraventricular nucleus, the nucleus of the solitary tract and the area postrema, in agreement with previous autoradiographic studies, describing a high density of AT1 binding sites in these nuclei. In addition, AT1A messenger RNA expression was detected in several brain areas, where no AT1 binding was reported previously. Thus, we identify strong expression of AT1A messenger RNA expression in scattered cells of the lateral parts of the preoptic region, the lateral hypothalamus and several brainstem nuclei. In none of these structures was the AT1B messenger RNA detectable at the microscopic level. In conclusion, it is suggested that angiotensins may exert their central effects on body fluid and cardiovascular homeostasis mainly via the AT1A receptor subtype. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: angiotensin receptor, AT1, in situ hybridization, brain mapping, choroid plexus, glia.
Brain angiotensin II and angiotensin III play a major role in various physiological functions including regulation of blood pressure and body fluid homeostasis by enhancing vasopressin release, water intake, sodium appetite and sympathetic activity (for review see Refs 29 and 41). In addition, they influence anterior pituitary hormone release, learning, memory and sensory functions.6,7,41 Angiotensin effects are mediated through their interaction with specific angiotensin receptors. Two, pharmacologically different angiotensin II receptor types, type-1 (AT1) and type-2 (AT2) have been identified using selective non-peptidic and pseudopeptidic antagonists. In vivo studies using these ‡To whom correspondence should be addressed. Abbreviations: AP, area postrema; AT1, angiotensin type-1 receptor; AT2, angiotensin type-2 receptor; GFAP, glial fibrillary acidic protein; MnPO, median preoptic nucleus; NFR, Nuclear Fast Red; NTS, nucleus of the solitary tract; OVLT, vascular organ of the lamina terminalis; PBS, phosphate-buffered saline; Pe, periventricular nucleus; PVN, paraventricular nucleus hypothalamus; SFO, subfornical organ. 827
antagonists have indicated that the major central actions of angiotensin are mediated by the AT1 receptor. Accordingly, autoradiographic binding studies have established the predominant expression of the AT1 receptor in brain areas controlling body fluid and cardiovascular homeostasis as well as pituitary hormone release.2,8,30 AT2 binding sites, conversely, were found in brain regions participating in sensory, limbic and other functions, where the role of the AT2 receptor remains to be determined. Although, mice lacking the gene encoding for the AT2 receptor have an impaired drinking response to water deprivation,9,10 indicating a possible role also for AT2 in the regulation of body fluid control. The cloning of the AT1 receptor cDNA revealed that it belongs to the seven-transmembrane domain, G protein-coupled receptor family. Moreover, two AT1 receptor subtypes have been identified in rodents5,24,32,33 which have been designated AT1A and AT1B. These receptors are more than 95% identical at the amino acid level. In the rat, AT1A and AT1B cDNAs share 95% identity of the nucleotide sequence within their coding region, whereas the homology is
828
Z. Lenkei et al.
reduced to 35% within the 5*- and 3*-untranslated regions.31 Expression of AT1A and AT1B in COS-7 cells shows quite comparable binding and coupling properties for both subtypes.4,31 The intracellular signalling pathways of both receptors include coupling to a GTP-binding protein, activation of a phospholipase C resulting in inositol triphosphate generation, mobilization of intracellular Ca2+ stores and diacylglycerol formation leading to protein kinase C activation.24,32 However, AT1A and AT1B mRNAs are differentially expressed and regulated in various peripheral tissues suggesting that both receptor subtypes may have different roles in mediating biological effects of angiotensin II. In a previous study we have shown by in situ hybridization that the AT1A mRNA is predominantly expressed in the rat forebrain, in contrast the AT1B subtype dominates in the anterior pituitary.13 The neuronal expression of AT1A receptors was demonstrated in the subfornical organ and the hypothalamus.14,18 By using double label in situ hybridization, AT1A receptor expression was localized in corticotropin-releasinghormone, but not in vasopressin-containing neurons in the hypothalamus.1,14 In the present study, we attempted to give a detailed mapping of the AT1A mRNA expression in the adult rat brain, in order to identify the neuronal structures underlying AT1mediated central angiotensin II actions. EXPERIMENTAL PROCEDURES
Synthesis of complementary RNA probes The AT1A and AT1B antisense and sense cRNA probes were synthesized by in vitro transcription as previously described in detail.34 Briefly, two 2.2 kb cDNA fragments (clone pCa18b for AT1A, a gift from Dr K. Bernstein and clone RAG6D4.60 for AT1B, a gift from Dr K. Sandberg) were subcloned into Bluescript KS plasmid (Stratagene, La Jolla, California) and pCDNA II plasmid (Invitrogen, Oxon, U.K.), respectively. After linearization, in vitro transcription was performed using T3, T7 and SP6 RNA polymerases (Boehringer, Mannheim, Germany) in the presence of [35S]UTP (Amersham, Les Ullis, France). The yield of the transcription was checked by agarose gel electrophoresis.
Tissue preparation All animal experiments were carried out in accordance with current institutional guidelines for the care and use of experimental animals. Adult (250–350 g) Sprague–Dawley rats (Iffa–Credo, Les Oncins, France, n=6) were kept on 12 h light/dark cycle with free access to food and water. The anaesthetized rats were perfused transcardially after a brief saline rinse with 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS). Brains were removed and postfixed overnight in the same fixative, then embedded in paraffin using standard procedures. Coronal sections (5 µm thick) were made and collected on 3-aminopropyltriethoxysilane (Sigma–Aldrich S.a.r.l., L’Isle D’Abeau Chesnes, France) -coated slides. One series of sections was stained with levanol and Nuclear Fast Red (NFR) (Chroma Gesellschaft, Koengen, Germany) for histological orientation, while adjacent sections were processed for in situ hybridization. In situ hybridization After treatment by proteinase K, sections were hybridized with the AT1A antisense and sense cRNA probes (see technical details in Ref. 35). After washes of different temperature and stringency which included an RNase treatment, the sections were dehydrated and exposed to Amersham Hyperfilm â-max for four weeks. After developing the films selected sections were dipped in Kodak NTB2 liquid emulsion and exposed for six to eight weeks, then developed and counterstained with Toluidine Blue. Glial fibrillary acidic protein immunohistochemistry after in situ hybridization For the detection of glial fibrillary acidic protein (GFAP), sections after in situ hybridization were rinsed twice in PBS, incubated in 20% normal goat serum for 20 min then with anti-GFAP antibody (DAKO, Trappe, France, diluted to 1:1000 in PBS) for 90 min. After rinsing twice in PBS, sections were incubated in a 1:200 dilution of biotinylated anti-rabbit antibody (Vector, Compiegne, France) for 30 min then rinsed again twice in PBS. Colour reaction was developed with the Elite Vectestain Kit (Vector). The sections were washed in Tris–HCl buffer (50 mM, pH 7.6) overnight, then dehydrated, dipped in Ilford K5 liquid emulsion. RESULTS
The control sections hybridized with the AT1A sense cRNA probe showed no hybridization signal,
Abbreviations used in the figures 7 A1/C1 acp Amb AOD AOL AOV AP AVPO csf GFAP HDB LH lo LPO MFB MM MnPO
motor facial nucleus A1/C1 catecholaminergic cells anterior commissure, posterior nucleus ambiguus anterior olfactory nucleus, dorsal part anterior olfactory nucleus, lateral part anterior olfactory nucleus, ventral part area postrema anteroventral preoptic nucleus cerebrospinal fluid glial fibrillary acidic protein nucleus of the diagonal tract, horizontal limb lateral hypothalamic area lateral olfactory tract lateral preoptic area median forebrain bundle medial mammillary nucleus median preoptic nucleus
Mo5 MPB MS NTS opt OVLT ox Pe Pir POM PVN PVpo RCh RPa SFO SO VDB
motor trigeminal nucleus medial parabrachial nucleus medial septal nucleus nucleus of the solitary tract optic tract vascular organ of the lamina terminalis optic chiasm periventricular nucleus piriform cortex medial preoptic nucleus paraventricular nucleus hypothalamus preoptic periventricular nucleus retrochiasmatic area nucleus raphe pallidus subfornical organ supraoptic nucleus nucleus of the diagonal tract, vertical limb
AT1 receptor mRNA expression in the adult rat brain
829
Fig. 1. Comparison of labelling with the AT1A antisense (A) and the AT1B antisense (B) probes at the level of the subfornical organ (SFO). Note the complete lack of specific labelling on B. This labelling is equivalent to the labelling obtained with the AT1A sense probe. Scale bar=500 µm. All photographs were scanned, brightness/contrast adjusted and labelled using Adobe Photoshop 3.0 for Macintosh (Adobe Systems Inc., Mountain View, CA), then printed on a Fuji Pictro 3000 printer. The data content of the images was not altered in any way.
with the exception of the hippocampus and the Purkinje cells of the cerebellum, where a constant, weak nonspecific labelling was observed. Hybridization with the AT1B antisense cRNA probe (Fig. 1) gave similar results to that of the AT1A sense cRNA probe, with the exception of the subfornical organ (SFO) and the parvocellular hypothalamic paraventricular nucleus, where a very weak AT1B specific labelling was detected. However, this very weak labelling was detectable only on film autoradiograms but not at the cellular level using emulsion autoradiography (Fig. 1). Hybridization with the AT1A antisense cRNA probe resulted in a well-localized cellular labelling in many brain nuclei. The results are summarized in Table 1 and some representative sections with high or very high level of AT1A mRNA expression are shown in Figs 2–15. As a guide to orientation, each photographic plate includes a low power microphotograph of a levanol–NFR stained coronal section of the rat brain, adjacent to the sections processed for in situ hybridization. Telencephalic structures Very high expression of AT1A mRNA was found in the anterior olfactory nucleus (Fig. 2) and the piriform cortex (Fig. 3). Low levels of AT1A mRNA expression were found in the cingulate, frontal and parietal cortex (Table 1). The vertical and horizontal limb of the nucleus of the diagonal band (Fig. 3), all parts of the hippocampus with the exception of the dentate gyrus displayed low level of AT1A mRNA expression (Table 1). Low density of AT1A mRNA expression was also found in the medial septal
nucleus (Fig. 3), the claustrum, the nucleus of the lateral olfactory tract, the anterior amygdaloid area, the central and lateral amygdaloid nuclei (Table 1). Basal ganglia did not contain detectable amounts of AT1A mRNA. Diencephalic structures In the hypothalamus, high to very high level of AT1A mRNA expression was detected in the preoptic area, especially in the vascular organ of the lamina terminalis (OVLT, Figs 4, 5), the median preoptic nucleus (MnPO, Figs 5–7), the preoptic part of the medial forebrain bundle (Fig. 6), and in scattered cells of the preoptic periventricular nucleus, the medial preoptic and the lateral preoptic area and anteroventral preoptic nucleus (Fig. 8). In the anterior hypothalamus, the periventricular nucleus (Pe, Fig. 9), all parvicellular parts of the paraventricular nucleus (PVN, Fig. 10) and the lateral retrochiasmatic area (Figs 10, 11) was established as high density of AT1A mRNA-expressing cells. High AT1A mRNA expression was also detected in cells in the rostral part of the lateral hypothalamic area (Figs 9–11). A number of the hypothalamic nuclei did not contain labelled cells (Table 1) except the perifornical nucleus (Fig. 11) and the median part of the medial mamillary nucleus (Fig. 12) where moderate– high, and the lateral preoptic area (Fig. 8), the caudal part of the lateral hypothalamic area (Table 1), the medial retrochiasmatic area, the dorsomedial nucleus (Fig. 11), the supramammilary nucleus where low AT1A mRNA expression was found (Table 1). In the thalamus, low AT1A mRNA expression was detected in the anterior ventral thalamic nucleus, the
830
Z. Lenkei et al. Table 1. Relative densities of AT1A messenger RNA expression in various brain nuclei 1. Telencephalon 1.1. Rhinencephalon Anterior olfactory nucleus Olfactory tubercle Nucl. of the diagonal tract Hippocampus
Nucleus of the lateral olfactory tract 1.2. Cerebral cortex Cingulate cortex* Piriform cortex Entorhinal cortex Frontal cortex* Parietal cortex* Insular cortex Temporal cortex Occipital cortex 1.3. Basal ganglia Accumbens nucleus Caudate nucleus Caudoputamen Substantia innominata Globus pallidus Endopiriform nucleus Claustrum** 1.4. Septum Medial septal nucleus Lateral septal nucleus Dorsal septal nucleus Fimbrial septal nucleus 1.5. Amygdala Anterior amygdaloid area Nucl. of the lateral olfactory tract Central nucleus amygdala Lateral nucleus amygdala Medial nucleus amygdala Basal nucleus amygdala Cortical nucleus amygdala Posterior nucleus amygdala Bed nucleus stria terminalis 2. Diencephalon 2.1. Thalamus Paratenial nucleus thalamus Paraventricular nucleus thalamus Anterodorsal nucleus thalamus Anteroventral nucleus thalamus Anteromedial nucleus thalamus Mediodorsal nucleus thalamus Central medial nucleus thalamus Central lateral nucleus thalamus Paracentral thalamic nuclei Midline thalamic nuclei Reuniens nucleus thalamus Rhomboid nucleus thalamus Gelatinose nucleus thalamus Reticular nucleus thalamus Lateral nucleus thalamus Lateral posterior nucleus thalamus Ventrolateral nucleus thalamus Ventromedial nucleus thalamus Ventral posterolateral nucleus thalamus Ventral posteromedial nucleus thalamus Suprageniculate nucleus Posterior nucleus thalamus 2.2. Epithalamus Medial habenula Lateral habenula
vertical limb horizontal limb anterior hilus (CA1–CA2) CA1 CA2 CA3 dentate gyrus subiculum
+++ – + + + + + + + – + ++ + +++ – + + – – – – – – – – – + + – – – + + + + – – – – – – – – + – + – – – – – – – – – – + – + + – – – –
AT1 receptor mRNA expression in the adult rat brain
831
Table 1. Continued 2.3. Metathalamus Lateral geniculate complex Medial geniculate complex Pretectal area Nucleus of the optic tract Nucleus of the posterior commissure 2.4. Subthalamus Zona incerta Subthalamic nucleus Fields of Forel Entopeduncular nucleus Bed nucleus of the supraoptic decussation 2.5. Preoptic region Median preoptic nucleus Medial preoptic nucleus Preoptic periventricular nucleus Anteroventral preoptic nucleus Preoptic suprachiasmatic nucleus Lateral preoptic area Medial forebrain bundle, preoptic part 2.6. Hypothalamus Lateral hypothalamic area Suprachiasmatic nucleus Periventricular nucleus Supraoptic nucleus Paraventricular nucleus
Anterior hypothalamic nucleus Retrochiasmatic area Median eminence Arcuate nucleus Ventromedial nucleus Dorsomedial nucleus Perifornical nucleus Dorsal premamillary nucleus Ventral premamillary nucleus Posterior hypothalamic nucleus Supramamillary nucleus Tuberomamillary nucleus A11 catecholaminergic cells 2.7. Mamillary body Medial mamillary nucleus
Lateral mamillary nucleus 3. Mesencephalon Medial forebrain bundle, mesencephalic part Ventral tegmental area Periaqueductal central gray matter Dorsal raphe nucleus Motor oculomotor nucleus Trochlear nucleus Interstitial nucleus of Cajal Linear raphe nuclei Midline raphe nucleus Colliculus superior Substantia nigra Peripeduncular nucleus Interpeduncular nucleus Red nucleus Cuneiform nucleus Pedunculopontine tegmental nucleus Inferior colliculus
dorsal part ventral part intergeniculate leaflet
– – + – – – – – – + – – +++ + s ++ – + ++
anterior part posterior part
periventricular medial-dorsal post. parvicell. magnocellular medial part lateral part
median part medial part lateral part posterior part
reticular zone compact zone lateral part
++ + – ++ – +++ +++ ++ – s + – – – – + ++ – – s + – + ++ + + – – + s + s/+ – – – – – – – – – – – – – + –
Table 1. Continued 4. Pons Pontine nuclei Nuclei of the lateral lemniscus Pontine reticular nucleus Reticular tegmental pontine nucleus Pontine raphe nucleus Dorsal tegmental nucleus Dorsolateral tegmental nucleus Locus coeruleus Subcoeruleus nucleus Nucleus of Barrington Paragenual nucleus Mesenchephalic nucleus of the trigeminal Medial parabrachial nucleus Lateral parabrachial nucleus Kolliker–Fuse nucleus Ventral tegmental nucleus Trigeminal motor nucleus Principal sensory nucleus of the trigeminal Superior olive Abducens nucleus 5. Cerebellum 5.1. Cerebellar cortex*** 5.2. Cerebellar nuclei Medial (fastigial) nucleus Interpositus nuclei Lateral nucleus 6. Medulla oblongata Cochlear nuclei Superior vestibular nucleus Medial vestibular nucleus Lateral vestibular nucleus Spinal vestibular nucleus Nucleus raphe magnus Nucleus raphe obscurus Nucleus raphe pallidus Gigantocellular reticular nucleus Paragigantocellular reticular nucleus Parvocellular reticular nucleus Medullary reticular nucleus Paramedian reticular nucleus Lateral reticular nucleus Spinal nucleus of the trigeminal, oral part Paratrigeminal nucleus Facial motor nucleus Inferior olivary complex Prepositus hypoglossal nucleus Nucleus of the solitary tract
Intercalate nucleus Dorsal motor nucleus of the vagus Ambiguus nucleus Linear nucleus of Rolando Motor hypoglossal nucleus Peritrigeminal nucleus External cuneate nucleus Cuneate nucleus of Burdach Gracile nucleus of Goll 7. Circumventricular organs Organum vasculosum laminae terminalis (OVLT) Subfornical organ (SFO) Median eminence Subcomissural organ Area postrema Choroid plexus
– – + + + + ++ + + – – + ++ + + – ++ ++ + – + + s +
interpositus part gelatinose part principal part dorsal accessory medial accesory rostral medial part caudal medial part lateral part commissural part
+ – – – – s++ – +++ + + – + – s++ + + – – ++ – – – + +++ +++ + +++ – s/+ +++ + + – – – – +++ +++ – – ++ ++
‘‘+++’’ very high, ‘‘++’’ high and ‘‘+’’ low level of expression. *V. layer; **Superficial layer; ***Purkinje cells. Label ‘‘s’’ indicates scattered labelled cells through the region. The results were consistent from animal to animal specially at the higher values. At lower values sometimes variation was detected +/-, partly due to the inhomogeneity of AT1A mRNA expression within the nucleus.
AT1 receptor mRNA expression in the adult rat brain
833
Fig. 2. Expression of AT1A receptor mRNA in the anterior olfactory nucleus. (A) Levanol–NFR-stained coronal section, approximately 4.20 mm rostral to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the ventral (AOV) and in the lateral (AOL) parts of the anterior olfactory nucleus. lo, lateral olfactory tract. Scale bar=1 mm.
Fig. 3. Expression of AT1A receptor mRNA in the septum and the piriform cortex. (A) Levanol–NFRstained coronal section, approximately 0.70 mm rostral to the level of the bregma. (B,C) Dark-field photomicrographs of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the piriform cortex (Pir) and low in the medial septal nucleus (MS) the vertical (VDB) and horizontal (HDB) limbs of the nucleus of the diagonal band. lo, lateral olfactory tract. Scale bar=1 mm.
Fig. 4. Expression of AT1A receptor mRNA in the preoptic region. (A) Levanol–NFR-stained coronal section, approximately 0.40 mm rostral to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the vascular organ of the lamina terminalis (OVLT). ox, optic chiasm and the asterisk labels the optic recess of the third ventricle. Scale bar=1 mm.
mediodorsal thalamic nucleus, in the ventrolateral, ventromedial and ventroposterior lateral thalamic nuclei (Table 1). The metathalamus (the intergenicu-
late leaflet of the lateral geniculate body) and the subthalamus (the fields of Forel) were poor in AT1A mRNA expressing cells (Table 1).
834
Z. Lenkei et al.
Fig. 5. Expression of AT1A receptor mRNA in the preoptic region. (A) Levanol–NFR-stained coronal section, approximately 0.20 mm rostral to the level of the bregma. (B). Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the caudal end of the vascular organ of the lamina terminalis (OVLT) and in the rostroventral part of the median preoptic nucleus (MnPO) ox, optic chiasm, acp, anterior commissure, posterior part. Scale bar=1 mm.
Fig. 6. Expression of AT1A receptor mRNA in the preoptic region. (A) Levanol–NFR-stained coronal section, approximately 0.10 mm rostral to the level of the bregma. (B,C). Darkfield photomicrographs of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the median preoptic nucleus (MnPO) and high in the medial forebrain bundle (MFB). ox = optic chiasm, acp = anterior commissure, posterior part and the asterisk labels the third ventricle. Scale bar =1 mm.
Mesencephalic structures High AT1A mRNA expression was detected in the rostral part of the midbrain periaqueductal gray matter (Table 1). Low level of labelling was present in the mesencephalic part of the medial forebrain bundle, in the dorsal raphe nucleus and in the pedunculopontine tegmental nucleus, while neurons in other midbrain structures were unlabelled (Table 1).
nucleus, the lateral parabrachial nucleus, the Kolliker–Fuse nucleus, the superior olive and the trapezoid body (Table 1). Cerebellum Weak expression of AT1A mRNA was detected in the Purkinje cell layer of the cerebellar cortex and in the medial, interpositus and lateral cerebellar nuclei (Table 1).
Pons High AT1A mRNA expression was detected in the medial parabrachial nucleus (Table 1), in the dorsolateral tegmental nucleus (Table 1), the motor and principal sensory trigeminal nuclei (Table 1). Weak labelling was also found in the pontine reticular nucleus, the reticular tegmental pontine nucleus, the pontine raphe nucleus, the dorsal tegmental nucleus, the locus coeruleus, the subcoeruleus area, the nucleus of Barrington, the mesencephalic trigeminal
Medulla oblongata Very high AT1A mRNA expression was detected in the nucleus raphe pallidus (Fig. 13), the retrofacial (Table 1), the ambiguus (Figs 14, 15) and linear nuclei (Table 1), the medial and commisural parts of the nucleus of the solitary tract as well as in the area postrema (Fig. 15). High expression of AT1A mRNA was detected in the nucleus raphe magnus (Table 1) and the facial motor nucleus (Fig. 13). Weak AT1A
AT1 receptor mRNA expression in the adult rat brain
835
Fig. 7. Expression of AT1A receptor mRNA in the preoptic region. (A) Levanol–NFR-stained coronal section, approximately at the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the median preoptic nucleus (MnPO). acp, anterior commissure, posterior part and the asterisk labels the third ventricle. Scale bar=1 mm.
Fig. 8. Expression of AT1A receptor mRNA in the anterior hypothalamus. (A) Levanol–NFR-stained coronal section, approximately 0.85 mm caudal to the level of the bregma. (B,C) Dark-field photomicrographs of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the subfornical organ (SFO), high in scattered cells of the medial preoptic nucleus (POM) the preoptic periventricular nucleus (PVpo) and the lateral preoptic area (LPO). The lateral part of the POM corresponds also to the anteroventral preoptic nucleus (AVPO) of Paxinos and Watson.27 ox, optic chiasm and the asterisk labels the third ventricle. Scale bar=1 mm.
mRNA expression was detected in the cochlear nuclei, the lateral vestibular nucleus, the giganto-cellular, paragigantocellular, lateral and medullary reticular nuclei, in the motor hypoglossal nucleus, in the oral and interpositus part of the spinal trigeminal nucleus and the prepositus hypoglossal nucleus (Table 1).
the anterior commissure (Figs 4–6) and the optic tract (Figs 8–10), but not in other white matter tracts as the corpus callosum or the pyramidal tract, etc. This labelling was not detected either with the AT1B antisense nor with the AT1A sense probes. However, we were not able to clearly associate this labelling with cells.
Choroid plexus Expression of AT1A mRNA was detected in connective tissue cells of the choroid plexus (Fig. 16). Myelinated structures Expression of AT1A mRNA is indicated by small clusters of silver grains in several fibre tracts such as
Identification of the cell types expressing AT1A messenger RNA GFAP immunohistochemistry performed after in situ hybridization in the hypothalamus shows that AT1A mRNA is expressed exclusively by large GFAP
836
Z. Lenkei et al.
Fig. 9. Expression of AT1A receptor mRNA in the anterior hypothalamus. (A) Levanol–NFR-stained coronal section, approximately 1.40 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is high in the periventricular nucleus (Pe) and in scattered cells of the lateral hypothalamic area (LH). ox, optic chiasm and the asterisk labels the third ventricle. Scale bar=1 mm.
Fig. 10. Expression of AT1A receptor mRNA in the anterior hypothalamus. (A) Levanol–NFR-stained coronal section, approximately 1.80 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the parvocellular parts of the paraventricular nucleus (PVN) high in scattered cells of the lateral hypothalamic area (LH) and retrochiasmatic area (RCh). opt, optic tract and the asterisk labels the third ventricle. Scale bar=1 mm.
negative cells (Fig. 17), indicating a neuronal expression of AT1A mRNA. DISCUSSION
Using radiolabelled riboprobes for in situ hybridization histochemistry, we established the detailed distribution of the AT1 receptor subtype mRNAs in the adult rat brain at the cellular level. The specificity of the probes used was previously established in transfected cell lines overexpressing either the AT1A or the AT1B receptor.13 In the hypothalamus, where we identified the nature of AT1A expressing cells by using immunohistochemical detection of GFAP after the in situ hybridization, the expression of AT1A mRNA was found exclusively in neurons. High level of AT1A mRNA expression was found in brain areas involved in mediating angiotensin effects on drinking, blood pressure and endocrine status (Fig. 18), such as the OVLT, median preoptic nucleus (MnPO), subfornical organ (SFO), paraventricular nucleus hypotha-
lamus (PVN), nucleus of the solitary tract (NTS) and area postrema (AP), but also in brain regions where the role of angiotensin is less well defined as the piriform cortex, the trigeminal nuclei, the facial motor nucleus, the nucleus raphe pallidus and the ambiguus nucleus. In accordance with our previous report,13 labelling with the AT1B receptor cRNA probe was very weak and restricted to the brain nuclei which displayed the highest expression of the AT1A mRNA. In addition, this very weak labelling was detectable only on film autoradiograms but not at the cellular level using emulsion autoradiography. It could not be exluded, however, that AT1B mRNA is expressed in the hippocampus and the Purkinje cells of the cerebellum, where the observed constant, weak non-specific labelling may mask the presence of a weak specific signal. In conclusion, if AT1B mRNA is expressed in the brain, in our hands it is below the detection limit of the currently known most sensitive method for in situ hybridization, using radioactivelylabelled long riboprobes.
AT1 receptor mRNA expression in the adult rat brain
837
Fig. 11. Expression of AT1A receptor mRNA in the middle part of the hypothalamus. (A) Levanol– NFR-stained coronal section, approximately 2.30 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is high in scattered cells of the lateral hypothalamic area (LH) and lateral retrochiasmatic area (RCh). opt, optic tract and the asterisk labels the third ventricle. Scale bar=1 mm.
Fig. 12. Expression of AT1A receptor mRNA in the mamillary body. (A) Levanol–NFR-stained coronal section, approximately 4.65 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is high in the median part of the medial mammillary nucleus (MM). Scale bar=1 mm.
Fig. 13. Expression of AT1A receptor mRNA in the rostral medulla oblongata. (A) Levanol–NFR-stained coronal section, approximately 11.0 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the nucleus raphe pallidus (RPa) and high in the motor facial nucleus (7). Scale bar=1 mm.
In general, the distribution of AT1A mRNA presented here is in agreement with AT1 mRNA mapping of Bunneman et al.,3 which was performed at the macroscopic level. Our results confirm and expand on a recent report12 which studied the expres-
sion of AT1 receptor subtype mRNAs in the forebrain of two-week-old rats and found AT1A mRNA expression in the OVLT, MnPO, SFO, PVN, Pe and lateral hypothalamus. However, in contrast with this latter study which reported expression
838
Z. Lenkei et al.
Fig. 14. Expression of AT1A receptor mRNA in the medulla oblongata. (A) Levanol–NFR-stained coronal section, approximately 12.60 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the ambiguus nucleus (Amb). Scale bar=1 mm.
Fig. 15. Expression of AT1A receptor mRNA in the caudal medulla oblongata. (A) Levanol–NFR-stained coronal section, approximately 13.60 mm caudal to the level of the bregma. (B) Dark-field photomicrograph of an adjacent section showing the distribution of AT1A receptor mRNA. The labelling is very high in the medial part of the nucleus of the solitary tract (NTS) and in the area postrema (AP) and low but visible in the lateral reticular nucleus (A1/C1). Scale bar=1 mm.
of AT1B mRNA in the hippocampus, we detected only AT1A mRNA expression, as discussed above. The distribution of the AT1A mRNA shows a strong correlation with the distribution of AT1 binding sites previously established using non-peptidic AT1 ligands for receptor binding autoradiography.2,8 These studies showed a high density of AT1 binding sites in the OVLT, the MnPO, the SFO, the hypothalamic periventricular nucleus, the parvocellular parts of the paraventricular nucleus, the NTS and the AP. Our results further document these findings at the microscopic level and show that the AT1 receptor in these nuclei is of the AT1A subtype. In these nuclei the co-localization of AT1A mRNA with angiotensin II binding sites and angiotensin-immunoreactive terminal fields15 suggest that AT1 receptors are likely present on the cell bodies or dendrites, serving as postsynaptic receptors for neuronal angiotensins. Angiotensin-containing nerve terminals have also been visualized in the OVLT and SFO.15,25 In the central, heavily vascularized regions of these circumventricular organs, they do not form synaptic specializations and are closely opposed to fenestrated
capillaries, whereas they are in synaptic contact with neurons in peripheral parts of these structures.25 This indicates that a part of the neuronally released angiotensins is secreted directly into the circulation, and suggests that in these organs, some neuronal AT1A receptors may not constitute postsynaptic receptors for neuronal angiotensins. In fact, these circumventricular organs are targets for circulating angiotensin II.23,26 The respective roles of the circulating angiotensin II originating from the periphery versus that from the neighbouring angiotensinergic neurons, on activation of AT1A receptors, is unknown. On the other hand, AT1 mRNA expression was detected in several brain areas, where no AT1 binding was previously reported (Table 1). Thus, taking advantage of the increased resolution of the in situ hybridization technique, we identified strong expression of AT1A mRNA expression in scattered cells of the lateral parts of the preoptic region, the lateral hypothalamus and several brainstem nuclei. By contrast, we did not detect AT1A mRNA expression in the suprachiasmatic nucleus (visible but not marked on Fig. 7B), the median eminence and the ventral
AT1 receptor mRNA expression in the adult rat brain
839
Fig. 16. Expression of AT1A receptor mRNA in the choroid plexus of the lateral ventricle. A cell in the connective tissue shows accumulation of silver grains over its cytoplasm. csf, cerebrospinal fluid. Scale bar=5 µm.
Fig. 17. Neuronal expression of AT1A mRNA in the lateral hypothalamic area. The in situ hybridization with a radioactive AT1A receptor riboprobe was followed by immunohistochemistry on the same brain slice using an anti-glial fibrillary acidic protein (GFAP) antibody. Arrowheads indicate GFAP-immunoreactive astrocytes and the arrow shows a GFAP-negative large cell labelled by accumulation of silver grains over its cytoplasm, indicating the expression AT1A mRNA. Scale bar=10 µm.
part of the bed nucleus of the stria terminalis, structures where high density of AT1 binding sites were reported.2,8 Possibly, the AT1 receptors are synthesized in perikaryons located elsewhere and transported axonally to these structures to mediate angiotensin II or angiotensin III action on the release of neurotransmitters at the presynaptic level. Distribution of AT1 receptors was also studied in the brain with immunohistochemical staining, using an antibody to a portion of the 3rd cytoplasmic loop of the AT1 receptor.28 The results of this report show an overlap with the results of the receptor binding studies2,8 as well as with our present data by showing AT1 receptor expression in the OVLT, the MnPO, the SFO, the locus coeruleus, the mesencephalic and motor nuclei of the trigeminal nerve, the cochlear nucleus and superior olivary nuclei, the nucleus of the
solitary tract, the nucleus hypoglossus, the rostroventrolateral medullary area and the nucleus ambiguus. However, very strong immunohistochemical staining has also been reported in the magnocellular neurons of the paraventricular and supraoptic nuclei, devoid of angiotensin II binding sites2,8 or AT1A or AT1B mRNA expression.1,12–14 One possible explanation is that this antibody also detects a non-AT1A non-AT1B receptor which does not bind radiolabelled angiotensin II analogues but shows an identity in its amino acid sequence to the portion of the 3rd cytoplasmic loop of the AT1 receptor. Structures associated with the lamina terminalis include the MnPO and the circumventricular organs SFO and OVLT. Current evidence indicates that blood-borne signals, such as angiotensin II, reach the SFO and OVLT where they are transduced. This information is then carried via neural pathways to brain nuclei, such as MnPO, PVN, supraoptic nucleus, NTS, where it is integrated with other inputs derived from systemic arterial blood pressure and volume receptors. Because of their receptive and integrative functions, lamina terminalis structures are essential for the normal control of hormone release (e.g., vasopressin), sympathetic activation and behaviours (thirst and salt appetite), which collectively contribute to maintenance of cardiovascular and body fluid homeostasis (for review see Ref. 11). In the choroid plexus, angiotensin II was shown to decrease the blood flow19,20 and the production rate of cerebrospinal fluid.21,40 This organ is rich in angiotensin converting enzyme23 and contains AT1 binding sites.36 The angiotensin II receptors can be synthetized locally as cells in the connective tissue of this organ show a moderate expression of AT1A mRNA. Alternatively, angiotensin II receptors synthesized elsewhere could be transported to this structure as cholinergic fibres from the vagus and adrenergic fibres from the superior cervical ganglion
840
Z. Lenkei et al.
Fig. 18. Distribution of AT1A receptor mRNA in the adult rat brain, sagittal section. Nuclei where the labelling was ‘‘+++’’ or ‘‘++’’ (Table 1) are marked with dark grey, nuclei with high but scattered labelling are marked with light grey. For abbreviations see list.
innervate the blood vessels and epithelia of the choroid plexus.16,17 The possible presence of AT1A mRNA in small cells of several fibre tracts (anterior commisure, optic tract) is in line with the observation that AT1 receptor expression was found in cultured glial cell lines, derived from embryonic tissues37,38,39 or from the adult corpus callosum.22 However, immunohistochemical detection of the glial marker GFAP after in situ hybridization for the AT1A mRNA in the hypothalamus, shows that the AT1A mRNA is expressed only in neurons. In fibre tracts, the small clusters of silver grains could not be associated either to GFAPpositive cells, or to other non-labelled cells. Thus, this AT1A specific labelling might be expressed by small GFAP-negative glial cells, whose small size makes the cellular localization of the labelling difficult, but further studies using glial-specific markers for double labelling studies might elucidate this question. Alternatively, we are detecting a non-specific phenomena
characteristic of the AT1A antisense riboprobe, since this labelling was detected neither with the AT1B antisense nor with the AT1A sense probe. CONCLUSION
In conclusion, by the demonstration of a relatively wide distribution pattern of AT1A mRNA in the adult rat brain we provide a basis for further investigations concerning the role of the brain angiotensin system in the control of the salt water homeostasis and blood pressure, and probably other regulatory mechanisms. Acknowledgements—The authors thank K. Bernstein for the AT1A receptor cDNA clone pCa18b, K. Sandberg for AT1B receptor cDNA clone RAG6D4.60, J. Helfferich for skilfull technical assistence. This work received the financial support of Bristol–Myers–Squibb Laboratories and of a French–Hungarian cooperation (Balaton) managed by the french ‘‘Ministere des Affaires Etrange`res’’ and OMFB.
REFERENCES
1. 2. 3. 4. 5. 6. 7.
Aguilera G., Young W. S., Kiss A. and Bathia A. (1995) Direct regulation of hypothalamic corticotropin-releasing hormone neurons by angiotensin II. Neuroendocrinology 61, 437–444. Allen A. M., Paxinos G., Song K. F. and Mendelsohn F. A. O. (1992) Localization of angiotensin receptor binding sites in the rat brain. In Handbook of Chemical Neuroanatomy Vol. 11: Neuropeptide Receptors in the CNS (eds A. Bjo¨rklund, T. Ho¨kfelt and M. J. Kuhar), Vol. 11, pp. 1–37. Elsevier, Amsterdam. Bunnemann B., Iwai N., Metzger R., Fuxe K., Inagami T. and Ganten D. (1992) The distribution of angiotensin II AT1 receptor subtype mRNA in the rat brain. Neurosci. Lett. 142, 155–158. Clauser E., Curnow K. M., Conchon S., Davies E., Teutsch B., Vianello B., Monnot C. and Corvol P. (1995) Molecular structure and mechanisms of action of mammalian angiotensin receptors. Curr. Opin. Endocr. Diab. 2, 404–411. Elton T. S., Stephan C. C., Taylor G. R., Kimball M. G., Martin M. M., Durand J. N. and Oparil S. (1992) Isolation of two distinct type I angiotensin II receptor genes. Biochem. biophys. Res. Commun. 184, 1067–1073. Ganong W. F. (1993) Blood, pituitary, and brain renin–angiotensin systems and regulation of secretion of anterior pituitary gland. Front. Neuroendocrinol. 14, 233–249. Ganong W. F. (1995) Reproduction and the renin–angiotensin system. Neurosci. Biobehav. Rev. 19, 241–250.
AT1 receptor mRNA expression in the adult rat brain 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
841
Gehlert D. R., Gackenheimer S. L. and Schober D. A. (1991) Autoradiographic localization of subtypes of angiotensin II antagonist binding in the rat brain. Neuroscience 44, 501–514. Hein L., Barsh G., Pratt R. E., Dzau V. J. and Koblika B. K. (1995) Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor gene in mice. Nature 377, 744–747. Ichiki T., Labosky P. A., Shiota C., Okuyama S., Imagawa Y., Fogo A., Niimura F., Ichikawa I., Hogan B. L. and Inagami T. (1995) Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature 377, 748–750. Johnson A. K., Cunningham J. T. and Thunhorst R. L. (1996) Integrative role of the lamina terminalis in the regulation of cardiovascular and body fluid homeostasis. Clin. exp. Pharm. Physiol. 23, 183–191. Jo¨hren O. and Saavedra J. M. (1996) Expression of AT1A and AT1B angiotensin II receptor messenger RNA in forebrain of 2-wk-old rats. Am. J. Physiol. – Endocrinol. Met. 34, E104–E112. Lenkei Z., Corvol P. and Llorens-Cortes C. (1995) The angiotensin receptor subtype AT1A predominates in rat forebrain areas involved in blood pressure, body fluid homeostasis and neuroendocrine control. Molec. Brain Res. 30, 53–60. Lenkei Z., Corvol P. and Llorens-Cortes C. (1995) Comparative expression of vasopressin and angiotensin type-1 receptor mRNA in rat hypothalamic nuclei: a double in situ hybridization study. Molec. Brain Res. 34, 135–142. Lind R. W. and Ganten D. (1990) Angiotensin. In Handbook of Chemical Neuroanatomy (eds A. Bjo¨rklund, T. Ho¨kfelt and M. J. Kuhar), Vol. 9, Part II, pp. 165–286. Elsevier, Amsterdam. Lindvall M., Edvinsson L. and Owman C. (1978) Sympathetic nervous control of cerebrospinal fluid production from the choroid plexus. Science 201, 176–178. Lindvall M. and Owman C. (1981) Autonomic nerves in the mammalian choroid plexus and their influence on the formation of cerebrospinal fluid. J. cerebr. Blood Flow Metab. 1, 245–266. Lippoldt A., Bunnemann B., Iwai N., Metzger R., Inagami T., Fuxe K. and Ganten D. (1993) Cellular localization of angiotensin type 1 receptor and angiotensinogen mRNAs in the subfornical organ of the rat brain. Neurosci. Lett. 150, 153–158. Maktabi M. A., Heistad D. D. and Faraci F. M. (1990) Effects of angiotensin II on blood flow to choroid plexus. Am. J. Physiol. 258, H414–H418. Maktabi M. A., Heistad D. D. and Faraci F. M. (1991) Effects of central and intravascular angiotensin I and II on the choroid plexus. Am. J. Physiol. 261, R1126–R1132. Maktabi M. A., Stachovic G. C. and Faraci F. M. (1993) Angiotensin II decreases the rate of production of cerebrospinal fluid. Brain Res. 606, 44–49. Matute C., Pulakat L., Rı´o C., Varca´rcel C. and Miledi R. (1994) Properties of angiotensin II receptors in glial cells from the adult corpus callosum. Proc. natn. Acad. Sci. U.S.A. 91, 3774–3778. McKinley M. J., McAllen R. M., Mendelsohn F. A. O., Allen A. M., Chai S. Y. and Oldfield B. J. (1990) Circumventricular organs: neuroendocrine interfaces between the brain and the hemal milieu. Front. Neuroendocrinol. 11, 91–127. Murphy T. J., Alexander R. W., Griendling K. K., Runge M. S. and Bernstein K. E. (1991) Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature 351, 233–236. Oldfield B. J., Ganten D. and McKinley M. J. (1989) An ultrastructural analysis of the distribution of angiotensin II in the rat brain. J. Neuroendocrinol. 1, 121–128. Oldfield B. O. and McKinley M. J. (1994) Circumventricular organs. In The Rat Nervous System, 2nd edn (ed. G. Paxinos). Academic, London. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic, Sydney. Phillips M. I., Shen L., Richards E. M. and Raizada M. K. (1993) Immunohistochemical mapping of angiotensin AT1 receptors in the brain. Regul. Pept. 44, 95–107. Saavedra J. M. (1992) Brain and pituitary angiotensin. Endocr. Rev. 13, 329–380. Saavedra J. M. (1994) Brain angiotensin II receptor subtypes. In Angiotensin Receptors (eds J. M. Saavedra and P. B. M. W. M. Timmermans), pp. 151–175. Plenum, New York. Sandberg K. (1994) Structural analysis and regulation of angiotensin II receptors. Trends Endocrinol. Metab. 5, 28–35. Sandberg K., Ji H., Clark A. J., Shapira H. and Catt K. J. (1992) Cloning and expression of a novel angiotensin II receptor subtype. J. biol. Chem. 267, 9455–9458. Sasamura H., Hein L., Krieger J. E., Pratt R. E., Kobilka B. K. and Dzau V. J. (1992) Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome. Biochem. biophys. Res. Commun. 185, 253–259. Shanmugam S., Monnot C., Corvol P. and Gasc J.-M. (1994) Distribution of type 1 angiotensin II receptor subtype messenger RNAs in the rat fetus. Hypertension 23, 137–141. Sibony M., Commo F., Callard P. and Gasc J. M. (1995) Enhancement of mRNA in situ hybridization signal by microwave heating. Lab. Invest. 73, 586–591. Song K., Allen A. M., Paxinos G. and Mendelsohn F. A. (1992) Mapping of angiotensin II receptor subtype heterogeneity in rat brain. J. comp. Neurol. 316, 467–484. Sumners C., Myers L. M., Kalberg C. J. and Raizada M. K. (1990) Physiological and pharmacological comparisons of angiotensin II receptors in neuronal and astrocyte glial cultures. Prog. Neurobiol. 34, 355–385. Sumners C., Tang W., Paulding W. and Raizada M. K. (1994) Peptide receptors in astroglia: focus on angiotensin II and atrial natriuretic peptide. Glia 11, 110–116. Sumners C., Tang W., Zelezna B. and Raizada M. K. (1991) Angiotensin II receptor subtypes are coupled with distinct signal-transduction mechanisms in neurons and astrocytes from rat brain. Proc. natn. Acad. Sci. U.S.A. 88, 7567–7571. Vogh B. P. and Godman D. R. (1989) Effects of inhibition of angiotensin converting enzyme and carbonic anhydrase on fluid production by ciliary process, choroid plexus, and pancreas. J. ocul. Pharmac. 5, 303–311. Wright J. W. and Harding J. W. (1992) Regulatory role of brain angiotensins in the control of physiological and behavioral responses. Brain Res. Rev. 17, 227–262. (Accepted 10 June 1997)