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Prenatal ontogeny of D2 dopamine receptor and dopamine transporter gene expression in the rat mesencephalon
Neuroscience Letters, 166 (1994) 4% 50 8 1994 Elsevier Science Ireland Ltd. All rights reserved 0304-3940/94/S 07.00
48
NSL 10146
Prenatal ontogeny of D2 dopamine receptor and dopamine transporter gene expression in the rat mesencephalon Frangois Tison*, Elisabeth Normand, URA-CNRS 1200, Luboratoire d’Histologie et Embryoiogie (UFR II/,
Bertrand Bloch
Universitt! de Bordeaux II, 146 rue LPo-Saignat, 33076 Bordeaux C6de.q France
(Received 30 September 1993; Revised version received 2 November 1993; Accepted 3 November 1993) Key words:
D2 dopamine receptor; Dopamine transporter; Ontogeny; Mesencephalon; In situ hybridization
This work demonstrates the early prenatal expression (by gestational day 14) of dopamine D2 receptor and dopamine transporter mRNAs by immature dopaminergic cells of the rat mesencephalon using in situ hybridization. Our results indicate that mRNAs are detectable 3 days before the appearance of functional D2 presynaptic receptors and detectable dopamine release at striatal terminals.
The D2 dopamine receptor (D2R) and dopamine transporter (DAT) are two major components of the presynaptic regulation of the nigro-striatal dopaminergic pathway whose genes have been cloned [1,2]. The D2R acts as an inhibitory autoreceptor [l] and DAT terminates the dopaminergic transmission by re-uptake of dopamine into presynaptic terminals [2]. Little is known about the developmental pattern of gene expression of such presynaptic receptor proteins in the mesencephalon. It is not known whether their mRNAs are expressed before, concomitantly, or after functional synapses are formed and postsynaptic dopamine receptors are expressed in the striatum. This aspect may be of importance for the understanding of the maturation of the nigro-striatal dopaminergic pathway and for strategies of neurotransplantation in Parkinson’s disease. We describe here the prenatal developmental pattern of D2R and DAT mRNA expression in the rat mesencephalon using in situ hybridization (ISH). Timed pregnant Wistar rats, sperm-positive on a specific day (GO), were used. Gestational day 1 (Gl) designated the day after a successful insemination and PO the day of birth (21 days of gestation). Prenatal brains were obtained from embryos and fetuses at ages G13, G14, G15, G16, G17, G18, G20 and PO. Whole heads or dissected brains were fixed by immersion in 1% paraformaldehyde (24 h), cryopreserved, then frozen and stored at -80°C. Frozen serial coronal sections (10 ,um thick) were cut and mounted on gelatine-coated slides then *Corresponding SSDI
author. Fax: (33) (56) 98 61 82.
0304-3940(93)E0770-V
stored at -80°C until used. The following ohgonucleotidic probes were employed: a mixture of two 45 nucleotide probes (bases 854-899, 1442-1487) complementary to the rat cDNA sequence of tyrosine hydroxylase (TH) [3]; a mixture of three 45 nucleotide probes (bases 28-72, 427471, 5388582) complementary to the rat cDNA sequence of D2R [4]; a mixture of two 39 oligonucleotide probes (bases 88-127, 137-176) complementary to the rat cDNA sequence of DAT [5]. The probes were labeled by tailing using terminal deoxynucleotidyl transferase (Amersham) and [35S]dATP (New England Nuclear, specific activity: 2 x 10’ cpmlpg), as previously described [6,7]. ISH procedure was performed following a method previously described in detail elsewhere [6]. Briefly, 0.1 ng of each probe mixture per 35 ~1 of hybridization buffer was applied to tissue sections and left overnight at 40°C. The sections were then washed using decreasing concentrations of SSC buffer and dried. Sections were then coated with an Ilford K5 emulsion, exposed for 3 months, developed and stained with Toluidine blue. The region of interest was anatomically defined by the presence of TH gene expression on adjacent sections, at the anatomical level expected for the localization of immature mesencephalic dopaminergic cells, as defined by Specht et al. [8]. Densely packed immature cells expressing TH mRNA were observed during early stages (Gl3-G16). They were medially situated in the ventral part of the mesencephalic flexure with some lateral extensions (Fig. 1). By G17, TH mRNA-expressing cells could be separated into a medial and a lateral group corresponding to the ventral tegmental area (VTA) and substantia
Fig. I. ISH of TH. DAT and D2R mRNAs ventral part of mesencephalic
fexure
in developing
(AS. aqueduct
SN. At PO. anatomical
mesencephalon
of Sylvius). orgkzation
By Gl?.
at G14, Cl7
is comparable
nigra (SN), respectively (Fig. 1). At PO, the anatomical organization of TH mRNA-containing cells was comparable to that found in the adult (Fig. I). On adjacent sections, a strong DAT mRNA expression was detected by G14. The localization of DAT mRNA was situated at a similar anatomical level to that found for TH mRNA (Fig. 1). However, as already observed in the adult [3]. DAT mRNA was less expressed in the VTA during all prenatal stages. At PO. the signal in the SN was stronger than in prenatal stages. D2R mRNA was also detected by G14. The anatomical organization of the signal was similar. but weaker. to that found for TH and DAT mRNAs. As for DAT mRNA, the signal was weaker in the VTA. Much data has been accumulated on the maturation of the dopaminergic nigro-striatal pathway: TH mRNA and TH protein appears in the mesencephalon at G13 [8,9], TH-positive terminals in the striatum at G14 [IO],
and PO. DAT and D2R mRNAs
cells expressmg with that
mRNAs
are ohscr\ed
of adult. Scale
are first detected
at Cl4 in
in theta- linal po,sitton wIthin VTA or
bar. I00 jrm.
dopamine release through terminals at G17 [I I J and postsynaptic striatal D2R and D 1R mRNAs at G I4 and G17. respectively [6.7]. Little is. however. known about the presynaptic regulatory components such as D2R and DAT. Using [“?]iodosulpiride binding, Sales and coworkers [12] found that presynaptic D2R sites are detected in late embryonic stages (G 17 for the SN. G2 I for the VTA). D2R situated in the striatal terminals are able to inhibit dopamine release at (317 [I 11. We found that D2R and DAT mRNAs are esprcssed early. at G14. There is no doubt that the cells observed are immature dopaminergic cells of the future mesencephalon projecting into the striatum, taking into consideration their anatomical localization. their organization during prenatal ontogeny and their TH/DAT phenotype [2.8]. While this work was in progress, Fu.jita et al. published results demonstrating. as WC ha\,e. that DAT mRNA is first cxpressed at G14 1131. The ISH signal they observed was.
50
however, very weak whereas we observed a strong and unequivocal signal at this stage. Similar to the results of Fujita et al., we also found some difference in the expression between the VTA and SN. We also observed such a difference with D2R mRNA, but contrary to DAT, this is not found in the adult [2,14]. This may possibly be due to a differential expression of mRNA and receptor during ontogeny since Sales et al. [ 121 observed a delay between the appearence of functional D2 sites between the SN (G17) and VTA (G21), however, this needs confirmation using quantitative procedures. It is worth mentioning that the ISH procedure may have sensitivity limits for the detection of receptor mRNAs during ontogeny. Indeed, the detection of dopamine receptor mRNAs in the rat forebrain is seen much earlier using the polymerase chain reaction procedure ]15]. Conversely, there may be a delay between the expression of mRNAs and the effective expression of the functional proteins as happens for oxytocin gene expression [ 161. This possibility may explain the delay between the expression of D2R mRNA (G14) and the detection of functional D2R presynapic receptors (G17); and also the delay between the expression of DAT mRNA (G14) and detectable dopamine release through dopaminergic terminals in the striatum (G17) [I l-131. In addition, there is a possibility that quantitative changes of mRNA expression of D2R and DAT occur during development. This was not assessed in the present work and needs further investigation to address new questions about the differential functioning of maturating dopaminergic neurons in comparison with the adult. This work was supported by funds from Region d’Aquitaine and INSERM (Contrat Externe 900603). 1 Sibley, D.R. and Momma, F.J., Molecular biology of dopamine receptors, Trends in Pharm. Sci., 13 (1992) 61-69. 2 Uhl, G., Neurotransmitter transporters: a promising gene family, Trends Neurosci., 15 (1992) 265-268. 3 Grima, B., Lamouroux, A., Blunot, F., Faucon-Biguet. N. and Mallet, J., Complete coding sequence of rat tyrosine hydroxylase mRNA, Proc. Nat. Acad. Sci. USA, 82 (1985) 617-621.
4 Bunzow, J.R., Wantol, H.M., Grandy, D.K., Albert, P., Salon, J., MC Donald, C., Mashida, C.A., Neve, K.A. and Civelli, O., Cloning and expression of rat dopamine D2 receptor, Nature (London), 336 (1988) 783-787. 5 Shimada, S., Kitayama, S., Lin, C.L., Pate], A., Nanthakumar, E., Gregor, P., Kuhar, M. and Uhl, G., Cloning and expression of a cocame-sensitive dopamine transporter complementary DNA, Science, 254 (1991) 576578. 6 Guennoun, R. and Bloch, B., D2 dopamine receptor gene expression in the rat striatum during ontogeny: an in situ hybridization study, Dev. Brain Res., 60 (1991) 79-87. 7 Guennoun, R. and Bloch, B., Ontogeny of Dl and DARPP32 gene expression in the rat striatum: an in situ hybridization study, Brain Res., 12 (1992) 131-139. 8 Specht, L.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Light microscopic immunohistochemioat localization of tyrosine hydroxylase in prenatal brain: early ontogeny, J. Comp. Neural., 199 (1981) 233.. 253. 9 Burgunder, J.M. and Young, W.S., III, Ontogeny of tyrosine hydroxylase and cholecystokinin gene expression in the rat mesencephalon, Dev. Brain Res., 52 (1990) 85-93. 10 Voorn, P., Kalsbeek, A., Jonitsma-Byham, B. and Groenewegen. H.J., The pre and post natal development of the dopaminergic cell groups in the ventral mesencephaion and the dopaminergic innervation of the striatum of the rat, Neuroscience, 25 (1988) 857-887. 11 De Vries, T.J., Mulder, A.H. and Schoffelmeer, N.M., Differential ontogeny of functional dopamine and muscarinic receptors mediating presynaptic inhibition of neurotransmitter release and postsynaptic regulation of adenyl cyclase activity in rat striatum, Dev. Brain Res., 66 (1992) 91-96. 12 Sales, N., Martres, M.P., Bouthenet, M.L. and Schwartz, J.C., Ontogeny of dopaminergic M-receptors in the rat nervous system: characterization and detailed autoradiographic mapping with iodosulpiride, Neuroscience, 28 (1989) 673-700. 13 Fujita, M., Shimada, S., Nishimura, T., Uhl, G.R. and Tohyama, M., Ontogeny of dopamine transporter mRNA expression in the rat brain, Mol. Brain Res., 19 (1993) 222-226. 14 Le Moine, C. and Bloch, B., Rat striatal and mesencephalic neurons contain the long isoform of the D2 dopamine receptor, Mol. Brain Res., 10 (1991) 2833289. 15 Cadoret, A.M., Jaber, M. and Bloch B., Prenatal Dl, Dlb and D3 dopamine receptor gene expression in the rat forebrain: detection by reverse polymerase chain reaction, Neurosci. Lett., 115 (1993) 92-94. 16 Laurent, F.M., Hindelang, C., Klien, M.J., Stoeckel, M.E. and Felix, J.M., Expression of the oxytocin and vasopressin genes in the rat hypothalamus during development: an in situ hybridization study, Dev. Brain Res., 46 (1989) 145-154.
48
NSL 10146
Prenatal ontogeny of D2 dopamine receptor and dopamine transporter gene expression in the rat mesencephalon Frangois Tison*, Elisabeth Normand, URA-CNRS 1200, Luboratoire d’Histologie et Embryoiogie (UFR II/,
Bertrand Bloch
Universitt! de Bordeaux II, 146 rue LPo-Saignat, 33076 Bordeaux C6de.q France
(Received 30 September 1993; Revised version received 2 November 1993; Accepted 3 November 1993) Key words:
D2 dopamine receptor; Dopamine transporter; Ontogeny; Mesencephalon; In situ hybridization
This work demonstrates the early prenatal expression (by gestational day 14) of dopamine D2 receptor and dopamine transporter mRNAs by immature dopaminergic cells of the rat mesencephalon using in situ hybridization. Our results indicate that mRNAs are detectable 3 days before the appearance of functional D2 presynaptic receptors and detectable dopamine release at striatal terminals.
The D2 dopamine receptor (D2R) and dopamine transporter (DAT) are two major components of the presynaptic regulation of the nigro-striatal dopaminergic pathway whose genes have been cloned [1,2]. The D2R acts as an inhibitory autoreceptor [l] and DAT terminates the dopaminergic transmission by re-uptake of dopamine into presynaptic terminals [2]. Little is known about the developmental pattern of gene expression of such presynaptic receptor proteins in the mesencephalon. It is not known whether their mRNAs are expressed before, concomitantly, or after functional synapses are formed and postsynaptic dopamine receptors are expressed in the striatum. This aspect may be of importance for the understanding of the maturation of the nigro-striatal dopaminergic pathway and for strategies of neurotransplantation in Parkinson’s disease. We describe here the prenatal developmental pattern of D2R and DAT mRNA expression in the rat mesencephalon using in situ hybridization (ISH). Timed pregnant Wistar rats, sperm-positive on a specific day (GO), were used. Gestational day 1 (Gl) designated the day after a successful insemination and PO the day of birth (21 days of gestation). Prenatal brains were obtained from embryos and fetuses at ages G13, G14, G15, G16, G17, G18, G20 and PO. Whole heads or dissected brains were fixed by immersion in 1% paraformaldehyde (24 h), cryopreserved, then frozen and stored at -80°C. Frozen serial coronal sections (10 ,um thick) were cut and mounted on gelatine-coated slides then *Corresponding SSDI
author. Fax: (33) (56) 98 61 82.
0304-3940(93)E0770-V
stored at -80°C until used. The following ohgonucleotidic probes were employed: a mixture of two 45 nucleotide probes (bases 854-899, 1442-1487) complementary to the rat cDNA sequence of tyrosine hydroxylase (TH) [3]; a mixture of three 45 nucleotide probes (bases 28-72, 427471, 5388582) complementary to the rat cDNA sequence of D2R [4]; a mixture of two 39 oligonucleotide probes (bases 88-127, 137-176) complementary to the rat cDNA sequence of DAT [5]. The probes were labeled by tailing using terminal deoxynucleotidyl transferase (Amersham) and [35S]dATP (New England Nuclear, specific activity: 2 x 10’ cpmlpg), as previously described [6,7]. ISH procedure was performed following a method previously described in detail elsewhere [6]. Briefly, 0.1 ng of each probe mixture per 35 ~1 of hybridization buffer was applied to tissue sections and left overnight at 40°C. The sections were then washed using decreasing concentrations of SSC buffer and dried. Sections were then coated with an Ilford K5 emulsion, exposed for 3 months, developed and stained with Toluidine blue. The region of interest was anatomically defined by the presence of TH gene expression on adjacent sections, at the anatomical level expected for the localization of immature mesencephalic dopaminergic cells, as defined by Specht et al. [8]. Densely packed immature cells expressing TH mRNA were observed during early stages (Gl3-G16). They were medially situated in the ventral part of the mesencephalic flexure with some lateral extensions (Fig. 1). By G17, TH mRNA-expressing cells could be separated into a medial and a lateral group corresponding to the ventral tegmental area (VTA) and substantia
Fig. I. ISH of TH. DAT and D2R mRNAs ventral part of mesencephalic
fexure
in developing
(AS. aqueduct
SN. At PO. anatomical
mesencephalon
of Sylvius). orgkzation
By Gl?.
at G14, Cl7
is comparable
nigra (SN), respectively (Fig. 1). At PO, the anatomical organization of TH mRNA-containing cells was comparable to that found in the adult (Fig. I). On adjacent sections, a strong DAT mRNA expression was detected by G14. The localization of DAT mRNA was situated at a similar anatomical level to that found for TH mRNA (Fig. 1). However, as already observed in the adult [3]. DAT mRNA was less expressed in the VTA during all prenatal stages. At PO. the signal in the SN was stronger than in prenatal stages. D2R mRNA was also detected by G14. The anatomical organization of the signal was similar. but weaker. to that found for TH and DAT mRNAs. As for DAT mRNA, the signal was weaker in the VTA. Much data has been accumulated on the maturation of the dopaminergic nigro-striatal pathway: TH mRNA and TH protein appears in the mesencephalon at G13 [8,9], TH-positive terminals in the striatum at G14 [IO],
and PO. DAT and D2R mRNAs
cells expressmg with that
mRNAs
are ohscr\ed
of adult. Scale
are first detected
at Cl4 in
in theta- linal po,sitton wIthin VTA or
bar. I00 jrm.
dopamine release through terminals at G17 [I I J and postsynaptic striatal D2R and D 1R mRNAs at G I4 and G17. respectively [6.7]. Little is. however. known about the presynaptic regulatory components such as D2R and DAT. Using [“?]iodosulpiride binding, Sales and coworkers [12] found that presynaptic D2R sites are detected in late embryonic stages (G 17 for the SN. G2 I for the VTA). D2R situated in the striatal terminals are able to inhibit dopamine release at (317 [I 11. We found that D2R and DAT mRNAs are esprcssed early. at G14. There is no doubt that the cells observed are immature dopaminergic cells of the future mesencephalon projecting into the striatum, taking into consideration their anatomical localization. their organization during prenatal ontogeny and their TH/DAT phenotype [2.8]. While this work was in progress, Fu.jita et al. published results demonstrating. as WC ha\,e. that DAT mRNA is first cxpressed at G14 1131. The ISH signal they observed was.
50
however, very weak whereas we observed a strong and unequivocal signal at this stage. Similar to the results of Fujita et al., we also found some difference in the expression between the VTA and SN. We also observed such a difference with D2R mRNA, but contrary to DAT, this is not found in the adult [2,14]. This may possibly be due to a differential expression of mRNA and receptor during ontogeny since Sales et al. [ 121 observed a delay between the appearence of functional D2 sites between the SN (G17) and VTA (G21), however, this needs confirmation using quantitative procedures. It is worth mentioning that the ISH procedure may have sensitivity limits for the detection of receptor mRNAs during ontogeny. Indeed, the detection of dopamine receptor mRNAs in the rat forebrain is seen much earlier using the polymerase chain reaction procedure ]15]. Conversely, there may be a delay between the expression of mRNAs and the effective expression of the functional proteins as happens for oxytocin gene expression [ 161. This possibility may explain the delay between the expression of D2R mRNA (G14) and the detection of functional D2R presynapic receptors (G17); and also the delay between the expression of DAT mRNA (G14) and detectable dopamine release through dopaminergic terminals in the striatum (G17) [I l-131. In addition, there is a possibility that quantitative changes of mRNA expression of D2R and DAT occur during development. This was not assessed in the present work and needs further investigation to address new questions about the differential functioning of maturating dopaminergic neurons in comparison with the adult. This work was supported by funds from Region d’Aquitaine and INSERM (Contrat Externe 900603). 1 Sibley, D.R. and Momma, F.J., Molecular biology of dopamine receptors, Trends in Pharm. Sci., 13 (1992) 61-69. 2 Uhl, G., Neurotransmitter transporters: a promising gene family, Trends Neurosci., 15 (1992) 265-268. 3 Grima, B., Lamouroux, A., Blunot, F., Faucon-Biguet. N. and Mallet, J., Complete coding sequence of rat tyrosine hydroxylase mRNA, Proc. Nat. Acad. Sci. USA, 82 (1985) 617-621.
4 Bunzow, J.R., Wantol, H.M., Grandy, D.K., Albert, P., Salon, J., MC Donald, C., Mashida, C.A., Neve, K.A. and Civelli, O., Cloning and expression of rat dopamine D2 receptor, Nature (London), 336 (1988) 783-787. 5 Shimada, S., Kitayama, S., Lin, C.L., Pate], A., Nanthakumar, E., Gregor, P., Kuhar, M. and Uhl, G., Cloning and expression of a cocame-sensitive dopamine transporter complementary DNA, Science, 254 (1991) 576578. 6 Guennoun, R. and Bloch, B., D2 dopamine receptor gene expression in the rat striatum during ontogeny: an in situ hybridization study, Dev. Brain Res., 60 (1991) 79-87. 7 Guennoun, R. and Bloch, B., Ontogeny of Dl and DARPP32 gene expression in the rat striatum: an in situ hybridization study, Brain Res., 12 (1992) 131-139. 8 Specht, L.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Light microscopic immunohistochemioat localization of tyrosine hydroxylase in prenatal brain: early ontogeny, J. Comp. Neural., 199 (1981) 233.. 253. 9 Burgunder, J.M. and Young, W.S., III, Ontogeny of tyrosine hydroxylase and cholecystokinin gene expression in the rat mesencephalon, Dev. Brain Res., 52 (1990) 85-93. 10 Voorn, P., Kalsbeek, A., Jonitsma-Byham, B. and Groenewegen. H.J., The pre and post natal development of the dopaminergic cell groups in the ventral mesencephaion and the dopaminergic innervation of the striatum of the rat, Neuroscience, 25 (1988) 857-887. 11 De Vries, T.J., Mulder, A.H. and Schoffelmeer, N.M., Differential ontogeny of functional dopamine and muscarinic receptors mediating presynaptic inhibition of neurotransmitter release and postsynaptic regulation of adenyl cyclase activity in rat striatum, Dev. Brain Res., 66 (1992) 91-96. 12 Sales, N., Martres, M.P., Bouthenet, M.L. and Schwartz, J.C., Ontogeny of dopaminergic M-receptors in the rat nervous system: characterization and detailed autoradiographic mapping with iodosulpiride, Neuroscience, 28 (1989) 673-700. 13 Fujita, M., Shimada, S., Nishimura, T., Uhl, G.R. and Tohyama, M., Ontogeny of dopamine transporter mRNA expression in the rat brain, Mol. Brain Res., 19 (1993) 222-226. 14 Le Moine, C. and Bloch, B., Rat striatal and mesencephalic neurons contain the long isoform of the D2 dopamine receptor, Mol. Brain Res., 10 (1991) 2833289. 15 Cadoret, A.M., Jaber, M. and Bloch B., Prenatal Dl, Dlb and D3 dopamine receptor gene expression in the rat forebrain: detection by reverse polymerase chain reaction, Neurosci. Lett., 115 (1993) 92-94. 16 Laurent, F.M., Hindelang, C., Klien, M.J., Stoeckel, M.E. and Felix, J.M., Expression of the oxytocin and vasopressin genes in the rat hypothalamus during development: an in situ hybridization study, Dev. Brain Res., 46 (1989) 145-154.