Progress in Histo- and Cytochemistry, Vol. 26 W. Graumann / J. Drukker (Eds.). Histochemistry of Receptors © Fischer Verlag· Stuttgart· Jena· New York· !992
6.6 Phosphotyrosine localization in the mature rat brain E. MARANI!, J. A. MAASSEN2 I Neuroregulation Group, Department of Physiology, and Department of Medical Biochemistry, Sylvius Laboratory, Leiden Medical Faculty, Leiden (The Netherlands)
2
Introduction Action by hormones and neurotransmitters in the brain is initiated by binding of the ligand to its receptor. Most receptors (except those for steroids and thyroid hormones) are located on the plasma membrane. Brain membrane receptors are able to exercize their effect in the cell nucleus (steroid and thyroid hormones) or in the cytoplasm (adrenaline and peptide hormones). The intracellular part of the membrane receptor can deploy a second messenger system (c.AMP, inositol triphosphate or diacylglycerol; GILMAN 1987; LEVITZKI 1988; BERRIDGE 1987) to interact with the cell metabolism or else intervene using a tyrosine kinase activity inside the cell (CARPENTER 1987). In the study of the brain, both the nerve growth factor (NGF) receptor and the epidermal growth factor (EGF) receptor have been focused on (KAPLAN et al. 1991; SOPPET et al. 1991; see also Table 1). Both types of receptors - and also the insulin receptor (COBB and ROSEN 1984)perform their activities via phosphorylation of tyrosine. In the case of the brain insulin receptor, the tyrosine residues of the beta subunit are phosphorylated (COBB and ROSEN 1984; GAROFALO and ROSEN 1989). In the clinical field, this autophosphorylation of the insulin receptor has been Abbreviations AC(A) Acb AHi AI
(anterior) part of anterior commissure nucleus accumbens amygdalohippocampal area agranular insular cortex
BL BM
basolateral amygdaloid nucleus basomedial amygdaloid nucleus
CA1-3-4 c.c. Cg CG CPu
Ammon's horn fields 1-3-4 corpus callosum cingulate cortex central gray caudate putamen (striatum)
FL
forelimb area
IL Ip
infralimbic area nucleus interpeduncularis
10 LS
lateral olfactory tract nucleus septalis lateralis
Oc
occipital cortex
Pir
primary olfactory cortex
RSG
granular retrosplenial cortex
SC SN
superior colliculus substantia nigra
TE Tu VO
temporal cortex olfactory tubercle ventral orbital area
272 . E. Marani, J. A. Maassen
applied to prenatal diagnosis involving the chorionic villi of patients with severe insulin resistance like leprechaunism (MAASSEN et al. 1990). Insulin receptors are present in most areas of the mammalian and avian brain. The role of these receptors is still being debated, likewise that of the proto-oncogenes (see Table 1). During development, the concentrations of insulin receptors are higher in rat, man and bird immature brains than in the respective adult ones. Insulin seems to promote dendritic outgrowth in dorsal root ganglion cells. The olfactory bulb contains the highest concentration of insulin receptors in the period in which the olfactory bulbs are continuously receiving new axonal input. Based on these data a role is postulated for insulin like hormones in neural growth (see GAROFALO and ROSEN 1989). NGF is not the only neurotrophic factor. Recently brain-derived neurotrophic factor and neurotrophin-3 have been cloned, their biological properties having been found to be similar to those of NGF. The signal transduction pathways they take from the neuronal membrane to the inside of the cell was previously unknown. The tyrosine kinase gene family seems though to fulfil this role. The receptors have, therefore, been assigned to the tyrosine kinase receptor class (SOPPET et al. 1991; see also Table 1). Indeed, the increase in phosphotyrosine could be related to the NGF receptor in PC 12 cells (KAPLAN et al. 1991). Activation of the kinase was performed in these cells by hormone binding to the extracellular part of the receptor. The EGF receptor contains a similar signal transduction pathway as that observed with insulin or NGF (BERRIDGE 1987). The net result of all actions using a cytosolic or receptor tyrosine kinase is the increase in phosphotyrosine inside the cell. Tyrosine phosphorylation occurs within one minute after NGF treatment of PC 12 cells; it reaches maximum levels after 5 min and declines thereafter (KAPLAN et al. 1991), indicating a slow process (see also COBB and ROSEN 1984). This makes it possible to Table 1. Cytosolic and receptor tyrosine kinases present in the nervous system a . I. Cytosolic tyrosine kinases al) C-src proto-oncogene (C-src product pp60 C-src+) b2 ) C-src proto-oncogene (a novel 33 nucleotide exon) b) C-yes gene product c) fyn gene d) c-abl gene
II. Receptor tyrosine kinases a) Epidermal growth factor receptor b) Fibroblast growth factor receptor c) Nerve growth factor receptor d) Insulin receptor e) Insulin-like growth factor I receptor f) Platelet-derived growth factor receptor gl) trk proto-oncogenes i) trk B proto-oncogenes h) ltk proto-oncogenes i) brain derived neurotropic factor receptor j) neurotrophin-3 receptors a
e)
Several receptors or cytosolic kinases and 2)] can be identical or partially identical like trkB and nerve growth factor receptor (see WAGNER et al. 1991).
Phosphotyrosine localization in the brain . 273
morphologically detect the phosphotyrosine within the rat brain using antibodies against phosphotyrosine (BURGERING et al. 1991; PANG et al. 1985). Addition of phosphatase inhibitors will increase the amount of phosphotyrosine present, favoring its location in brain tissue. This paper describes the architectonic localization of phosphotyrosine in the normal rat brain as evidenced by rabbit polyclonal and monoclonal antibodies.
Material and methods Animals (see Table 2) Mature WAG male and female rats (n = 24) were anaesthetized with ether. The thorax was opened via the abdominal cavity and 0.2 ml of heparinR 5000 IU/ml: 1% sodium nitrite (1 : 1) was injected intracardially. After opening the right atrium, perfusion through the aorta was performed at 20 mllmin for 5 min with physiologic saline followed by buffered formaldehyde 4% (pH 7.2) for 15 minutes. To both solutions were added the phosphatase inhibitors: 20mM NaF and 5mM Na Vanadate (Sigma, USA). All rats obtained food ad libitum. Four rats were starved for 24h, and two rats before perfusion had 10 ml of 5% sucrose solution administered to their stomachs for 30 min. Insulin was then given intravenously: O.5ml of a 40 Elml solution, 10 min before perfusion. A few rats received a colchicine application in their lateral ventricle (see MARANI et al. 1988). Table 2. Series treated with tyrosine phosphate antibodies". Number
Properties
Experiments carried out
C4145 C4146 C4147"· C4148* C4149 C4150" C4151 C4154 C4166 C4184 C4190 C4232* C4242* C4251* C4259 C4288 C4298 C4299* C4309A C4309B C4310A C4310B C4310C C4331
6.6 Phosphotyrosine localization in the mature rat brain E. MARANI!, J. A. MAASSEN2 I Neuroregulation Group, Department of Physiology, and Department of Medical Biochemistry, Sylvius Laboratory, Leiden Medical Faculty, Leiden (The Netherlands)
2
Introduction Action by hormones and neurotransmitters in the brain is initiated by binding of the ligand to its receptor. Most receptors (except those for steroids and thyroid hormones) are located on the plasma membrane. Brain membrane receptors are able to exercize their effect in the cell nucleus (steroid and thyroid hormones) or in the cytoplasm (adrenaline and peptide hormones). The intracellular part of the membrane receptor can deploy a second messenger system (c.AMP, inositol triphosphate or diacylglycerol; GILMAN 1987; LEVITZKI 1988; BERRIDGE 1987) to interact with the cell metabolism or else intervene using a tyrosine kinase activity inside the cell (CARPENTER 1987). In the study of the brain, both the nerve growth factor (NGF) receptor and the epidermal growth factor (EGF) receptor have been focused on (KAPLAN et al. 1991; SOPPET et al. 1991; see also Table 1). Both types of receptors - and also the insulin receptor (COBB and ROSEN 1984)perform their activities via phosphorylation of tyrosine. In the case of the brain insulin receptor, the tyrosine residues of the beta subunit are phosphorylated (COBB and ROSEN 1984; GAROFALO and ROSEN 1989). In the clinical field, this autophosphorylation of the insulin receptor has been Abbreviations AC(A) Acb AHi AI
(anterior) part of anterior commissure nucleus accumbens amygdalohippocampal area agranular insular cortex
BL BM
basolateral amygdaloid nucleus basomedial amygdaloid nucleus
CA1-3-4 c.c. Cg CG CPu
Ammon's horn fields 1-3-4 corpus callosum cingulate cortex central gray caudate putamen (striatum)
FL
forelimb area
IL Ip
infralimbic area nucleus interpeduncularis
10 LS
lateral olfactory tract nucleus septalis lateralis
Oc
occipital cortex
Pir
primary olfactory cortex
RSG
granular retrosplenial cortex
SC SN
superior colliculus substantia nigra
TE Tu VO
temporal cortex olfactory tubercle ventral orbital area
272 . E. Marani, J. A. Maassen
applied to prenatal diagnosis involving the chorionic villi of patients with severe insulin resistance like leprechaunism (MAASSEN et al. 1990). Insulin receptors are present in most areas of the mammalian and avian brain. The role of these receptors is still being debated, likewise that of the proto-oncogenes (see Table 1). During development, the concentrations of insulin receptors are higher in rat, man and bird immature brains than in the respective adult ones. Insulin seems to promote dendritic outgrowth in dorsal root ganglion cells. The olfactory bulb contains the highest concentration of insulin receptors in the period in which the olfactory bulbs are continuously receiving new axonal input. Based on these data a role is postulated for insulin like hormones in neural growth (see GAROFALO and ROSEN 1989). NGF is not the only neurotrophic factor. Recently brain-derived neurotrophic factor and neurotrophin-3 have been cloned, their biological properties having been found to be similar to those of NGF. The signal transduction pathways they take from the neuronal membrane to the inside of the cell was previously unknown. The tyrosine kinase gene family seems though to fulfil this role. The receptors have, therefore, been assigned to the tyrosine kinase receptor class (SOPPET et al. 1991; see also Table 1). Indeed, the increase in phosphotyrosine could be related to the NGF receptor in PC 12 cells (KAPLAN et al. 1991). Activation of the kinase was performed in these cells by hormone binding to the extracellular part of the receptor. The EGF receptor contains a similar signal transduction pathway as that observed with insulin or NGF (BERRIDGE 1987). The net result of all actions using a cytosolic or receptor tyrosine kinase is the increase in phosphotyrosine inside the cell. Tyrosine phosphorylation occurs within one minute after NGF treatment of PC 12 cells; it reaches maximum levels after 5 min and declines thereafter (KAPLAN et al. 1991), indicating a slow process (see also COBB and ROSEN 1984). This makes it possible to Table 1. Cytosolic and receptor tyrosine kinases present in the nervous system a . I. Cytosolic tyrosine kinases al) C-src proto-oncogene (C-src product pp60 C-src+) b2 ) C-src proto-oncogene (a novel 33 nucleotide exon) b) C-yes gene product c) fyn gene d) c-abl gene
II. Receptor tyrosine kinases a) Epidermal growth factor receptor b) Fibroblast growth factor receptor c) Nerve growth factor receptor d) Insulin receptor e) Insulin-like growth factor I receptor f) Platelet-derived growth factor receptor gl) trk proto-oncogenes i) trk B proto-oncogenes h) ltk proto-oncogenes i) brain derived neurotropic factor receptor j) neurotrophin-3 receptors a
e)
Several receptors or cytosolic kinases and 2)] can be identical or partially identical like trkB and nerve growth factor receptor (see WAGNER et al. 1991).
Phosphotyrosine localization in the brain . 273
morphologically detect the phosphotyrosine within the rat brain using antibodies against phosphotyrosine (BURGERING et al. 1991; PANG et al. 1985). Addition of phosphatase inhibitors will increase the amount of phosphotyrosine present, favoring its location in brain tissue. This paper describes the architectonic localization of phosphotyrosine in the normal rat brain as evidenced by rabbit polyclonal and monoclonal antibodies.
Material and methods Animals (see Table 2) Mature WAG male and female rats (n = 24) were anaesthetized with ether. The thorax was opened via the abdominal cavity and 0.2 ml of heparinR 5000 IU/ml: 1% sodium nitrite (1 : 1) was injected intracardially. After opening the right atrium, perfusion through the aorta was performed at 20 mllmin for 5 min with physiologic saline followed by buffered formaldehyde 4% (pH 7.2) for 15 minutes. To both solutions were added the phosphatase inhibitors: 20mM NaF and 5mM Na Vanadate (Sigma, USA). All rats obtained food ad libitum. Four rats were starved for 24h, and two rats before perfusion had 10 ml of 5% sucrose solution administered to their stomachs for 30 min. Insulin was then given intravenously: O.5ml of a 40 Elml solution, 10 min before perfusion. A few rats received a colchicine application in their lateral ventricle (see MARANI et al. 1988). Table 2. Series treated with tyrosine phosphate antibodies". Number
Properties
Experiments carried out
C4145 C4146 C4147"· C4148* C4149 C4150" C4151 C4154 C4166 C4184 C4190 C4232* C4242* C4251* C4259 C4288 C4298 C4299* C4309A C4309B C4310A C4310B C4310C C4331
