Neuroscience Letters, 114 (1990) 147-153
147
Elsevier Scientific Publishers Ireland Ltd.
NSL 06936
Autoradiographic localization of m a s proto-oncogene mRNA in adult rat brain using in situ hybridization B. Bunnemann I, K. Fuxe 1, R. Metzger 2, J. Mullins 2, T.R. Jackson 4, M.R. Hanley 3 and D. Ganten 2 1Department of Histology and Neurobiology, Karolinska Institute, Stockholm (Sweden), :Department of Pharmacology, University of Heidelberg, Heidelberg ( F.R.G.), 3Medical Research Council Molecular Neurobiology Unit and ~Laboratory of Molecular Biology, MRC Centre, Cambridge ( U.K.) (Received 29 January 1990; Revised version received 15 February 1990; Accepted 21 February 1990)
Key words: mas proto-oncogene; Rat; In situ hybridization; Angiotensin peptide The cellular localization and the distribution of the mas proto-oncogene/angiotensin receptor mRNA have been studied in the male rat brain using in situ hybridization with radiolabelled mas cRNA probes. Neuronal cell populations in the forebrain were selectively labelled. A strong specific labelling was demonstrated in the dentate gyrus, the CA3 and CA4 areas of the hippocampus, the olfactory tubercle (medial part), the piriform cortex and the olfactory bulb, while a weak to moderate labelling was present all over the neocortex and especially in the frontal lobe.
Many proto-oncogenes are now recognised to be expressed at high level s in mature nerve cells; including neurons, a non-dividing cell type [5]. In this regard, the m a s proto-oncogene exhibited the highest level of mRNA expression in the hippocampus and cerebral cortex of the rat brain, whereas it appeared to be absent from most, if not all, rat peripheral organs [16]. Functional analysis identified that the m a s oncogene encoded an angiotensin-sensitive receptor, which, in human brain, is also found preferentially expressed in hippocampus and cerebral cortex [8]. Against this background, the cellular localization and regional distribution of m a s proto-oncogene mRNA was studied in adult rat brain using in situ hybridization. The present study reports a well-defined distribution of mas transcripts in mature rat brain linked to discrete neuronal populations in the forebrain. The RNA-probes used for the in situ hybridization experiments were synthesized using a 615 base pairs long Barn H I/Hinc II rat m a s cDNA fragment subcloned into the vector pGEM 4 and representing positions 258-873 of the m a s mRNA. The Correspondence: K. Fuxe, Department of Histology and Neurobiology, Karolinska Institutet, Box 60400, S-10401 Stockholm, Sweden.
148 probes were labelled with [35S]~-UTP (Dupont N E N , Boston, MA, U.S.A.) by SP 6 or T 7 in vitro transcription, purified on Nensorb cartridges (Dupont N E N , Boston, MA, U.S.A.) and used directly for the hybridization reactions. The specific activity of the probes was in the order of 5 x 108 dpm//zg RNA. Five adult male SpragueDawley rats (300 g body weight) were anesthetized with sodium pentobarbital (60 mg/ kg body weight) and perfused transcardially with 200 ml of ice-cold 0.9% saline. The brains were immediately removed and snap-frozen with COz, 10/zm thick sagittal sections were made in a cryostat, thaw-mounted onto poly-L-lysine coated slides and fixed for 15 min in 4% paraformaldehyde in PBS pH 7.0. After 3 short washes in PBS they were stored in 70% ethanol at 4°C until further use. After a deproteination with 0. I M HCI for 10 min and an acetylation step in 0.1 M triethanolamine/0.25% acetic anhydride for 20 min, the slides were dehydrated in graded ethanol and air-dried, 150/11 prehybridization buffer (50% deionized formamide, 50 m M Tris-HC1 pH 7.6, 25 m M EDTA, p H 8.0, 20 m M NaCI, 0.25 mg/ml yeast t R N A , 2.5 x Denhardt's solution) were applied to each section and the slides were incubated in a humidified chamber for 2 h at 37°C.
Fig. 1. Microphotographs of emulsion-coated sagittal sections (lateral level = 3.9 mm) of the rat brain hybridized with [35S]~-UTPlabelled mas cRNA and counterstained with cresyl violet. Large numbers of strongly labelled neuronal perikarya are found in the followingareas: (a) CA4 region of the hippocampus (pyramidal cell layer); (b) area indicated by rectangle in (a) in higher magnification: note the neuron free of signal (arrowhead); (c) CA3 region of the hippocampus (pyramidal cell layer); (d) control. Same area as (e) but hybridized with radiolabelledmas mRNA; (e) area indicated by rectangle in (c) in higher magnification; (f) outer layer of the piriform cortex. Bars in a, c, d = 50/lm; Bars in b, e, f = 25/zm.
149
Fig. 2. Microphotographsof emulsion-coatedsagittal sections [lateral levels = 2.4 mm (a, b) and 0.4 mm (c, d)] of the rat brain hybridizedwith [35S]~-UTPlabelledm a s cRNA and counterstained with cresylviolet. Large numbers of strongly labelled neuronal perikarya are found in the followingareas: (a) olfactory bulb (outer layer); (b) area indicated by rectangle in (a) in higher magnification; (c) olfactory tubercle (outer layer); (d) area indicated by rectangle in (c) in higher magnification; Bars in a, c = 50/zm; Bars in b, d = 25/zm.
The prehybridization buffer was drained off the slides and subsequently 15 /zl hybridization buffer (50% deionized formamide, 20 mM Tris-HC1, pH 7.6, 1 mM EDTA, pH 8.0, 0.3 M NaCI, 0.1 M DTT, 0.5 mg/ml yeast tRNA, 0.1 mg/ml poly-A, 1 x Denhardt's solution, 10% dextransulphate) were applied per section. This buffer also contained 60 pg//d [35S]~t-UTP labelled m a s c R N A (or m a s m R N A for control experiments). The sections were covered with siliconized coverslips and incubated in a humidified chamber at 37°C for 16-18 h. To remove the coverslips the slides were immersed in 2 x SSC at 48°C for 30 min. After that step the slides were washed in 1 x SSC/50% formamide at 48°C for 6 h. The washing solution was changed every 90 min. After 2 short washes in 1 × SSC for 10 min each, the slides were dehydrated in graded ethanol and air-dried. The slides were exposed to [3H]LKB-Ultrofilm at - 7 0 ° C or covered with Kodak NTB 2 photographic emulsion. Counterstaining of sections was performed with cresyl violet. Hybridization with the respective [35S]0t-UTP labelled m a s m R N A at the same specific activity and concentration as the m a s cRNA served as control in all experiments, The specific labelling found after incubation with the m a s c R N A probe was exclusively located over neuronal profiles in all areas analyzed as seen following counterstaining with cresyl violet (Figs. 1 and 2). In the analysis of the distribution of the m a s proto-oncogene m R N A levels within the rat brain a strong labelling was exclusively linked to the dentate gyrus, the CA3 and CA4 areas of the gyrus hippocampi, the olfactory tubercle (medial part), the piriform cortex and the olfactory bulb (Fig. 3). Within the hippocampal formation the strong labelling was mainly located over
150
o i-r~_
~4"t
Fig. 3. Autoradiograms of sagittal sections of the rat brain hybridized with [3~S]ct-UTPlabelled r n a s cRNA (a,c,e) and m a s mRNA (b,d,t): note the specific moderate (CA1, CA2) to strong signal (CA3, CA4, Pir) in the indicated areas compared to the control sections. Bars = 2 ram. Lateral levels = 3.9 mm (a,b), 2.4 mm (c,d), 0.4 mm (e,f). ACo, amygdaloid cortex; DG, dentate gyrus; FCo, frontal cortex; OB, olfactory bulb: Tu, olfactory tubercle; Pir, piriform cortex.
the granular cell layer o f the dentate gyrus and over the pyramidal cell layers o f the CA3 and C A 4 areas, while a moderate labelling was present in the C A I and CA2 areas. Within the piriform cortex, the olfactory tubercle (medial part) and the olfactory bulb the strong labelling was mainly found over the outer layers o f these regions. The lateral part o f the olfactory tubercle only contained a weak to moderate labelling. A weak to moderate labelling was also demonstrated over the entire part o f the cerebral cortex, the labelling being mainly located in the outer and inner layers o f the neocortex (Fig. 3). A rostrocaudal gradient was also observed within the cerebral cortex, the highest labelling being present within the frontal cortex. A weak to m o d erate labelling was noted within the amygdaloid cortex (Fig. 3a). N o specific labelling
151
was found within the diencephalon, the mesencephalon, the pons, the medulla oblongata and the cerebellar cortex. In the present paper evidence has been provided that the m a s proto-oncogene mRNA levels within the rat brain are mainly present within neuronal cell populations of the hippocampal formation, of the piriform cortex, the olfactory tubercle and the olfactory bulb. Also of interest is to notice the wide-spread distribution of low levels of m a s proto-oncogene mRNA all over the neocortex. Thus, within the rat brain the r n a s proto-oncogene may be selectively expressed within the forebrain and especially within parts of the limbic system, that is the dentate gyrus, the CA3 and CA4 areas of the gyrus hippocampi and in the outer layers of the ventral limbic cortex and of the olfactory bulb. These results indicate that the m a s gene product may have a special role in information handling and in trophic regulation within the forebrain and especially within discrete limbic subcortical and cortical areas including the olfactory bulb. Several independent techniques - Northern blots [8], S I nuclease protection mRNA assays [16], and in situ hybridization (this work) - confirm that the m a s proto-oncogene is expressed in mammalian brain with a novel and distinctive distribution. The current report now indicates that this regional pattern arises from selective expression in neurons. Previous work has established that m a s encodes a specific molecular species of angiotensin receptor, as assessed by X e n o p u s oocyte or stable transfectant expression [6,8], or by transient expression in COS cells [10]. Thus, an overlap between m a s gene expression and other markers of angiotensin pathways would be expected. However, the pattern of r n a s transcripts revealed by in situ hybridization is quite unlike the regional distribution of either immunoreactive angiotensin II [1-3,7] or [125I]angiotensin II binding sites [1,3,13]. There are several possibilities for the origin of this disagreement. First, m a s may be transcribed, but not translated, in the mature brain. This would raise the interesting speculation that m a s functional expression might not be constitutive, but rather could be inducible by translational control, such as occurs for mRNAs activated during early oocyte development. In particular, alterations in m a s expression during neural injury and recovery should be assessed. A second possibility is that m a s may respond in vivo to an 'angiotensin-like' peptide, which is not detected immunocytochemically by angiotensin II antisera. There is support for this speculation in that endogenous brain 'angiotensin II' has been reported to behave differently from authentic angiotensin II [7, 12]. Indeed, recent work, using a highly specific radioreceptor assay based on [3H]angiotensin II binding, has detected highest levels of biologically active 'angiotensin II-like' material in the rat cerebral cortex, whereas a highly specific radioimmunoassay detected highest levels of authentic angiotensin II in the rat hippocampus [11]. A third possibility is that the physiological source for the angiotensins acting on the m a s gene product is the angiotensinogen found widely in the brain in astrocytes [1, 14]. Based on the present observations, experiments must be performed to clarify if stored or transiently produced angiotensin peptides have novel biological roles in the limbic areas and in the neocortex where the mas proto-oncogene appears to be selectively expressed. Of special interest will be the anatomically predicted interaction
152 between the D2 d o p a m i n e receptor a n d the m a s gene p r o d u c t within regions wherein their d i s t r i b u t i o n overlaps, i.e., olfactory tubercle, piriform cortex a n d frontal cortex. This analysis might pay unexpected dividends in view of the hypothetical involvem e n t o f D2 d o p a m i n e receptors in these regions in schizophrenia. It m a y also be speculated that d i s t u r b a n c e s in m a s p r o t o - o n c o g e n e activity d u r i n g d e v e l o p m e n t m a y lead to alterations in the cellular proliferation a n d differentiation of the cortical areas, especially the limbic system [4-6, 15].
This work has been supported by a g r a n t (04X-715) from the Swedish Medical Research Council, the Deutsche F o r s c h u n g s g e m e i n s c h a f t (SFB 317), N I H grant 1 R O I HL-35821-01 a n d the Medical Research C o u n c i l (U.K.). T.R.J. is the recipient of the Royal Society Mr. a n d Mrs. J o h n Jaffe D o n a t i o n Research Fellowship. The a u t h o r s would like to t h a n k Drs. D a l l a n Y o u n g a n d Michael Wigler for initial provision of genetic constructions. 1 Bunnemann, B., Fuxe, K., Bjelke, B. and Ganten, D., The brain renin-angiotensin system and its possible involvement in volume transmission, In: K. Fuxe and L.F. Agnati (Ed.), Frontiers in Neuroscience, Vol. 1, Raven Press, New York, NY, in press. 2 Fuxe, K., Ganten, D., H6kfelt, T. and Bolme, P. lmmunohistochemical evidence for the existence of angiotensin II containing nerve terminals in the brain and spinal cord of the rat, Neurosci. Lett., 2 (1976) 229-234. 3 Fuxe, K., Bunnemann, B., Aronsson, M., Tinner, B., Cintra, A., von Euler, G., Agnati, L.F., Nakanishi, S., Okhubo, H. and Ganten, D., Pre- and postsynaptic features of the central angiotensin systems. Indications for a role of angiotensin peptides in volume transmission and for interactions with central monoamine neurons, Clin. Exp. Hypertens. Part A Theory Pract., AI0 (Suppl. 1) (1988) 143168. 4 Fuxe, K., Agnati, L.F., Zoli, M., et al., Studies on the neuroplasticity of mesotelencephalicdopamine neurons at the network and receptor level give new aspects on the role of dopamine in schizophrenia and possible pharmacological treatments. In Proceedingsof the International Congress on Schizophrenia Research, Raven Press, New York, NY, in press. 5 Hanley,M.R., Proto-oncogenes in the nervous system, Neuron, 1 (1988) 175-182. 6 Hanley, M.R., Cheung, W.T., Hawkins, P., Poyner, D., Benton, H.P., Blair, L., Jackson, T.R. and Goedert, M., The m a s oncogene as a neural peptide receptor: expression, regulation and mechanism of action. In J. Marsh (Ed.), Proto-Oncogenes in Cell Development (CIBA Symposium 150), Wiley, Chichester, 1990, pp. 23-46. 7 Horvath, J.S., Baxter, C., Furby, F. and Tiller, D.J., Endogenous angiotensin in the brain. In W. de Jong, A.P. Provoost and A.P. Shapiro (Eds.), Hypertension and Brain Mechanisms (Progress in Brain Research, Vol. 47), Elsevier,Amsterdam, 1977, pp. 161 165. 8 Jackson, T,R., Blair, L.A.C., Marshall, J., Goedert, M. and Hanley, M.R., The m a s oncogene encodes an angiotensin receptor, Nature, 335 (1988) 437-440. 9 Lind, R.W., Swanson, L.W. and Ganten, D., Organization of angiotensin II immunoreactivecells and fibres in the rat central nervous system, Neuroendocrinology, 40 (1985) 2-24. 10 McGillis, J.P., Sudduth-Klinger, J., Harrowe, G., Mitsuhashi, M. and Payan, D.G., Transient expression of the angiotensin II receptor: a rapid and functional analysis of a calcium-mobilizingseven-transmembrane domain receptor in COS-7 cells, Biochem. Biophys. Res. Commun., 165 (1989) 935-941. I 1 Pohl, M., Carayon, A., Cesselin, F. and Hamon, M., Angiotensin II-like material extracted from the rat brain is distinct from authentic angiotensin II, J. Neuroehem., 51 (1988) 1407-1413. 12 Sirett, N.E., Bray, J.J. and Hubbard, J.I., Brain angiotensin II: discrepancy between estimates by a radioreceptor assay and a radioimmunoassay, Proc. Univ. Otago Med. School, 58 (1980) 80--82.
153 13 Sirett, N.E., McLean, A.S., Bray, J.J. and Hubbard, J.I., Distribution of angiotensin II receptors in rat brain, Brain Res., 122 (1977) 299-312. 14 Stornetta, R.L., Hawelu-Johnson, C.L., Guyenet, P.G. and Lynch, K.R., Astrocytes synthesize angiotensinogen in brain, Science, 242 (1988) 1444-1446. 15 Weinberger, D.R., Implications of normal brain development for the pathogenesis of schizophrenia, Arch. Gen. Psychiatry, 44 (1987) 660-669. 16 Young, D., O'Neill, K., Jessell, T. and Wigler, M., Characterization of the rat mas oncogene and its high-level expression in the hippocampus and cerebral cortex of rat brain, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 5339-5342.
147
Elsevier Scientific Publishers Ireland Ltd.
NSL 06936
Autoradiographic localization of m a s proto-oncogene mRNA in adult rat brain using in situ hybridization B. Bunnemann I, K. Fuxe 1, R. Metzger 2, J. Mullins 2, T.R. Jackson 4, M.R. Hanley 3 and D. Ganten 2 1Department of Histology and Neurobiology, Karolinska Institute, Stockholm (Sweden), :Department of Pharmacology, University of Heidelberg, Heidelberg ( F.R.G.), 3Medical Research Council Molecular Neurobiology Unit and ~Laboratory of Molecular Biology, MRC Centre, Cambridge ( U.K.) (Received 29 January 1990; Revised version received 15 February 1990; Accepted 21 February 1990)
Key words: mas proto-oncogene; Rat; In situ hybridization; Angiotensin peptide The cellular localization and the distribution of the mas proto-oncogene/angiotensin receptor mRNA have been studied in the male rat brain using in situ hybridization with radiolabelled mas cRNA probes. Neuronal cell populations in the forebrain were selectively labelled. A strong specific labelling was demonstrated in the dentate gyrus, the CA3 and CA4 areas of the hippocampus, the olfactory tubercle (medial part), the piriform cortex and the olfactory bulb, while a weak to moderate labelling was present all over the neocortex and especially in the frontal lobe.
Many proto-oncogenes are now recognised to be expressed at high level s in mature nerve cells; including neurons, a non-dividing cell type [5]. In this regard, the m a s proto-oncogene exhibited the highest level of mRNA expression in the hippocampus and cerebral cortex of the rat brain, whereas it appeared to be absent from most, if not all, rat peripheral organs [16]. Functional analysis identified that the m a s oncogene encoded an angiotensin-sensitive receptor, which, in human brain, is also found preferentially expressed in hippocampus and cerebral cortex [8]. Against this background, the cellular localization and regional distribution of m a s proto-oncogene mRNA was studied in adult rat brain using in situ hybridization. The present study reports a well-defined distribution of mas transcripts in mature rat brain linked to discrete neuronal populations in the forebrain. The RNA-probes used for the in situ hybridization experiments were synthesized using a 615 base pairs long Barn H I/Hinc II rat m a s cDNA fragment subcloned into the vector pGEM 4 and representing positions 258-873 of the m a s mRNA. The Correspondence: K. Fuxe, Department of Histology and Neurobiology, Karolinska Institutet, Box 60400, S-10401 Stockholm, Sweden.
148 probes were labelled with [35S]~-UTP (Dupont N E N , Boston, MA, U.S.A.) by SP 6 or T 7 in vitro transcription, purified on Nensorb cartridges (Dupont N E N , Boston, MA, U.S.A.) and used directly for the hybridization reactions. The specific activity of the probes was in the order of 5 x 108 dpm//zg RNA. Five adult male SpragueDawley rats (300 g body weight) were anesthetized with sodium pentobarbital (60 mg/ kg body weight) and perfused transcardially with 200 ml of ice-cold 0.9% saline. The brains were immediately removed and snap-frozen with COz, 10/zm thick sagittal sections were made in a cryostat, thaw-mounted onto poly-L-lysine coated slides and fixed for 15 min in 4% paraformaldehyde in PBS pH 7.0. After 3 short washes in PBS they were stored in 70% ethanol at 4°C until further use. After a deproteination with 0. I M HCI for 10 min and an acetylation step in 0.1 M triethanolamine/0.25% acetic anhydride for 20 min, the slides were dehydrated in graded ethanol and air-dried, 150/11 prehybridization buffer (50% deionized formamide, 50 m M Tris-HC1 pH 7.6, 25 m M EDTA, p H 8.0, 20 m M NaCI, 0.25 mg/ml yeast t R N A , 2.5 x Denhardt's solution) were applied to each section and the slides were incubated in a humidified chamber for 2 h at 37°C.
Fig. 1. Microphotographs of emulsion-coated sagittal sections (lateral level = 3.9 mm) of the rat brain hybridized with [35S]~-UTPlabelled mas cRNA and counterstained with cresyl violet. Large numbers of strongly labelled neuronal perikarya are found in the followingareas: (a) CA4 region of the hippocampus (pyramidal cell layer); (b) area indicated by rectangle in (a) in higher magnification: note the neuron free of signal (arrowhead); (c) CA3 region of the hippocampus (pyramidal cell layer); (d) control. Same area as (e) but hybridized with radiolabelledmas mRNA; (e) area indicated by rectangle in (c) in higher magnification; (f) outer layer of the piriform cortex. Bars in a, c, d = 50/lm; Bars in b, e, f = 25/zm.
149
Fig. 2. Microphotographsof emulsion-coatedsagittal sections [lateral levels = 2.4 mm (a, b) and 0.4 mm (c, d)] of the rat brain hybridizedwith [35S]~-UTPlabelledm a s cRNA and counterstained with cresylviolet. Large numbers of strongly labelled neuronal perikarya are found in the followingareas: (a) olfactory bulb (outer layer); (b) area indicated by rectangle in (a) in higher magnification; (c) olfactory tubercle (outer layer); (d) area indicated by rectangle in (c) in higher magnification; Bars in a, c = 50/zm; Bars in b, d = 25/zm.
The prehybridization buffer was drained off the slides and subsequently 15 /zl hybridization buffer (50% deionized formamide, 20 mM Tris-HC1, pH 7.6, 1 mM EDTA, pH 8.0, 0.3 M NaCI, 0.1 M DTT, 0.5 mg/ml yeast tRNA, 0.1 mg/ml poly-A, 1 x Denhardt's solution, 10% dextransulphate) were applied per section. This buffer also contained 60 pg//d [35S]~t-UTP labelled m a s c R N A (or m a s m R N A for control experiments). The sections were covered with siliconized coverslips and incubated in a humidified chamber at 37°C for 16-18 h. To remove the coverslips the slides were immersed in 2 x SSC at 48°C for 30 min. After that step the slides were washed in 1 x SSC/50% formamide at 48°C for 6 h. The washing solution was changed every 90 min. After 2 short washes in 1 × SSC for 10 min each, the slides were dehydrated in graded ethanol and air-dried. The slides were exposed to [3H]LKB-Ultrofilm at - 7 0 ° C or covered with Kodak NTB 2 photographic emulsion. Counterstaining of sections was performed with cresyl violet. Hybridization with the respective [35S]0t-UTP labelled m a s m R N A at the same specific activity and concentration as the m a s cRNA served as control in all experiments, The specific labelling found after incubation with the m a s c R N A probe was exclusively located over neuronal profiles in all areas analyzed as seen following counterstaining with cresyl violet (Figs. 1 and 2). In the analysis of the distribution of the m a s proto-oncogene m R N A levels within the rat brain a strong labelling was exclusively linked to the dentate gyrus, the CA3 and CA4 areas of the gyrus hippocampi, the olfactory tubercle (medial part), the piriform cortex and the olfactory bulb (Fig. 3). Within the hippocampal formation the strong labelling was mainly located over
150
o i-r~_
~4"t
Fig. 3. Autoradiograms of sagittal sections of the rat brain hybridized with [3~S]ct-UTPlabelled r n a s cRNA (a,c,e) and m a s mRNA (b,d,t): note the specific moderate (CA1, CA2) to strong signal (CA3, CA4, Pir) in the indicated areas compared to the control sections. Bars = 2 ram. Lateral levels = 3.9 mm (a,b), 2.4 mm (c,d), 0.4 mm (e,f). ACo, amygdaloid cortex; DG, dentate gyrus; FCo, frontal cortex; OB, olfactory bulb: Tu, olfactory tubercle; Pir, piriform cortex.
the granular cell layer o f the dentate gyrus and over the pyramidal cell layers o f the CA3 and C A 4 areas, while a moderate labelling was present in the C A I and CA2 areas. Within the piriform cortex, the olfactory tubercle (medial part) and the olfactory bulb the strong labelling was mainly found over the outer layers o f these regions. The lateral part o f the olfactory tubercle only contained a weak to moderate labelling. A weak to moderate labelling was also demonstrated over the entire part o f the cerebral cortex, the labelling being mainly located in the outer and inner layers o f the neocortex (Fig. 3). A rostrocaudal gradient was also observed within the cerebral cortex, the highest labelling being present within the frontal cortex. A weak to m o d erate labelling was noted within the amygdaloid cortex (Fig. 3a). N o specific labelling
151
was found within the diencephalon, the mesencephalon, the pons, the medulla oblongata and the cerebellar cortex. In the present paper evidence has been provided that the m a s proto-oncogene mRNA levels within the rat brain are mainly present within neuronal cell populations of the hippocampal formation, of the piriform cortex, the olfactory tubercle and the olfactory bulb. Also of interest is to notice the wide-spread distribution of low levels of m a s proto-oncogene mRNA all over the neocortex. Thus, within the rat brain the r n a s proto-oncogene may be selectively expressed within the forebrain and especially within parts of the limbic system, that is the dentate gyrus, the CA3 and CA4 areas of the gyrus hippocampi and in the outer layers of the ventral limbic cortex and of the olfactory bulb. These results indicate that the m a s gene product may have a special role in information handling and in trophic regulation within the forebrain and especially within discrete limbic subcortical and cortical areas including the olfactory bulb. Several independent techniques - Northern blots [8], S I nuclease protection mRNA assays [16], and in situ hybridization (this work) - confirm that the m a s proto-oncogene is expressed in mammalian brain with a novel and distinctive distribution. The current report now indicates that this regional pattern arises from selective expression in neurons. Previous work has established that m a s encodes a specific molecular species of angiotensin receptor, as assessed by X e n o p u s oocyte or stable transfectant expression [6,8], or by transient expression in COS cells [10]. Thus, an overlap between m a s gene expression and other markers of angiotensin pathways would be expected. However, the pattern of r n a s transcripts revealed by in situ hybridization is quite unlike the regional distribution of either immunoreactive angiotensin II [1-3,7] or [125I]angiotensin II binding sites [1,3,13]. There are several possibilities for the origin of this disagreement. First, m a s may be transcribed, but not translated, in the mature brain. This would raise the interesting speculation that m a s functional expression might not be constitutive, but rather could be inducible by translational control, such as occurs for mRNAs activated during early oocyte development. In particular, alterations in m a s expression during neural injury and recovery should be assessed. A second possibility is that m a s may respond in vivo to an 'angiotensin-like' peptide, which is not detected immunocytochemically by angiotensin II antisera. There is support for this speculation in that endogenous brain 'angiotensin II' has been reported to behave differently from authentic angiotensin II [7, 12]. Indeed, recent work, using a highly specific radioreceptor assay based on [3H]angiotensin II binding, has detected highest levels of biologically active 'angiotensin II-like' material in the rat cerebral cortex, whereas a highly specific radioimmunoassay detected highest levels of authentic angiotensin II in the rat hippocampus [11]. A third possibility is that the physiological source for the angiotensins acting on the m a s gene product is the angiotensinogen found widely in the brain in astrocytes [1, 14]. Based on the present observations, experiments must be performed to clarify if stored or transiently produced angiotensin peptides have novel biological roles in the limbic areas and in the neocortex where the mas proto-oncogene appears to be selectively expressed. Of special interest will be the anatomically predicted interaction
152 between the D2 d o p a m i n e receptor a n d the m a s gene p r o d u c t within regions wherein their d i s t r i b u t i o n overlaps, i.e., olfactory tubercle, piriform cortex a n d frontal cortex. This analysis might pay unexpected dividends in view of the hypothetical involvem e n t o f D2 d o p a m i n e receptors in these regions in schizophrenia. It m a y also be speculated that d i s t u r b a n c e s in m a s p r o t o - o n c o g e n e activity d u r i n g d e v e l o p m e n t m a y lead to alterations in the cellular proliferation a n d differentiation of the cortical areas, especially the limbic system [4-6, 15].
This work has been supported by a g r a n t (04X-715) from the Swedish Medical Research Council, the Deutsche F o r s c h u n g s g e m e i n s c h a f t (SFB 317), N I H grant 1 R O I HL-35821-01 a n d the Medical Research C o u n c i l (U.K.). T.R.J. is the recipient of the Royal Society Mr. a n d Mrs. J o h n Jaffe D o n a t i o n Research Fellowship. The a u t h o r s would like to t h a n k Drs. D a l l a n Y o u n g a n d Michael Wigler for initial provision of genetic constructions. 1 Bunnemann, B., Fuxe, K., Bjelke, B. and Ganten, D., The brain renin-angiotensin system and its possible involvement in volume transmission, In: K. Fuxe and L.F. Agnati (Ed.), Frontiers in Neuroscience, Vol. 1, Raven Press, New York, NY, in press. 2 Fuxe, K., Ganten, D., H6kfelt, T. and Bolme, P. lmmunohistochemical evidence for the existence of angiotensin II containing nerve terminals in the brain and spinal cord of the rat, Neurosci. Lett., 2 (1976) 229-234. 3 Fuxe, K., Bunnemann, B., Aronsson, M., Tinner, B., Cintra, A., von Euler, G., Agnati, L.F., Nakanishi, S., Okhubo, H. and Ganten, D., Pre- and postsynaptic features of the central angiotensin systems. Indications for a role of angiotensin peptides in volume transmission and for interactions with central monoamine neurons, Clin. Exp. Hypertens. Part A Theory Pract., AI0 (Suppl. 1) (1988) 143168. 4 Fuxe, K., Agnati, L.F., Zoli, M., et al., Studies on the neuroplasticity of mesotelencephalicdopamine neurons at the network and receptor level give new aspects on the role of dopamine in schizophrenia and possible pharmacological treatments. In Proceedingsof the International Congress on Schizophrenia Research, Raven Press, New York, NY, in press. 5 Hanley,M.R., Proto-oncogenes in the nervous system, Neuron, 1 (1988) 175-182. 6 Hanley, M.R., Cheung, W.T., Hawkins, P., Poyner, D., Benton, H.P., Blair, L., Jackson, T.R. and Goedert, M., The m a s oncogene as a neural peptide receptor: expression, regulation and mechanism of action. In J. Marsh (Ed.), Proto-Oncogenes in Cell Development (CIBA Symposium 150), Wiley, Chichester, 1990, pp. 23-46. 7 Horvath, J.S., Baxter, C., Furby, F. and Tiller, D.J., Endogenous angiotensin in the brain. In W. de Jong, A.P. Provoost and A.P. Shapiro (Eds.), Hypertension and Brain Mechanisms (Progress in Brain Research, Vol. 47), Elsevier,Amsterdam, 1977, pp. 161 165. 8 Jackson, T,R., Blair, L.A.C., Marshall, J., Goedert, M. and Hanley, M.R., The m a s oncogene encodes an angiotensin receptor, Nature, 335 (1988) 437-440. 9 Lind, R.W., Swanson, L.W. and Ganten, D., Organization of angiotensin II immunoreactivecells and fibres in the rat central nervous system, Neuroendocrinology, 40 (1985) 2-24. 10 McGillis, J.P., Sudduth-Klinger, J., Harrowe, G., Mitsuhashi, M. and Payan, D.G., Transient expression of the angiotensin II receptor: a rapid and functional analysis of a calcium-mobilizingseven-transmembrane domain receptor in COS-7 cells, Biochem. Biophys. Res. Commun., 165 (1989) 935-941. I 1 Pohl, M., Carayon, A., Cesselin, F. and Hamon, M., Angiotensin II-like material extracted from the rat brain is distinct from authentic angiotensin II, J. Neuroehem., 51 (1988) 1407-1413. 12 Sirett, N.E., Bray, J.J. and Hubbard, J.I., Brain angiotensin II: discrepancy between estimates by a radioreceptor assay and a radioimmunoassay, Proc. Univ. Otago Med. School, 58 (1980) 80--82.
153 13 Sirett, N.E., McLean, A.S., Bray, J.J. and Hubbard, J.I., Distribution of angiotensin II receptors in rat brain, Brain Res., 122 (1977) 299-312. 14 Stornetta, R.L., Hawelu-Johnson, C.L., Guyenet, P.G. and Lynch, K.R., Astrocytes synthesize angiotensinogen in brain, Science, 242 (1988) 1444-1446. 15 Weinberger, D.R., Implications of normal brain development for the pathogenesis of schizophrenia, Arch. Gen. Psychiatry, 44 (1987) 660-669. 16 Young, D., O'Neill, K., Jessell, T. and Wigler, M., Characterization of the rat mas oncogene and its high-level expression in the hippocampus and cerebral cortex of rat brain, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 5339-5342.
