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Localization of vasopressin mRNA-containing neurones in the hypothalamus of the monkey
Molecular Brain Research, 4 (1988) 81-85 Elsevier
81
BRM 80026
Localization of vasopressin mRNA-containing neurones in the hypothalamus of the monkey Yosuke Ichimiya 1, Piers C. Emson 1 and Fraser D. Shaw 2 IMRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U.K.) and 2Department of Anatomy, University of Cambridge, Cambridge ( U. K. )
(Accepted 12 April 1988) Key words: In situ hybridization; Vasopressin; Neurophysin; Hypothalamus; Monkey
Neuronal perikarya containing vasopressin mRNA were detected in cryostat sections of cynomolgus monkey brains by using an in situ hybridization technique. The neurones were observed in hypothalamic regions (supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus and accessory supraoptic nucleus). These findings are in agreement with previous reports using immunohistochemical methods.
The technique of in situ hybridization histochemistry allows the localization of sites in gene expression in the central nervous system ~2. Although the in situ method is reasonably well established in rat or mouse brains, the method has not been widely applied to primate or human brain material. In this study, we have applied an in situ hybridization technique to cryostat sections of monkey brains using oligonucleotide probes specific for vasopressin (AVP) mRNA. AVP-positive neurones have been found in the hypothalamus and extrahypothalamic region of monkeys 4,6,1° and human brains 2-5 using immunohistochemical techniques. In order to complement these findings, we used a selective AVP probe and here describe the location of AVP m R N A containing neurones in the brain of the monkey (cynomolgous monkey). For detection of AVP m R N A , an anti-sense oligonucleotide probe (36 mer) was used. The probe was specific for the sequence of the first 12 amino acids of rat neurophysin (NP) II glycopeptide sequence (AlaSer-Asp-Ser-Asn-Ala-Thr-Leu-Asp-Gly). The probe was synthesized using a Biosearch 8750 Multiple Column D N A Synthesizer with the fl-cyanoethyl phos-
phoramidite chemistry. The probe was labelled (spec. act. >1.0 x 10s dpm//~g) with 35S using [35S]dCTP (Amersham, SJ 305; >600 ci/mmol) terminal deoxynucleotidyl transferase (Pharmacia) and tailing buffer (100 mM potassium cacodylate; pH 7.2, 1 mM CoC12 and 0.2 mM dithiothreitol). Adult cynomolgus monkeys of both sexes were anesthetized, the brains removed and frozen immediately in liquid nitrogen, sections (20/~m) were cut on a cryostat at -20 °C and stored at - 7 0 °C for subsequent in situ hybridization. For in situ hybridization, the sections were briefly fixed in 4% paraformaldehyde in phosphate buffer for 15 min, and then rinsed in 0.1 M phosphate buffer saline (PBS) containing 0.1% (v/v) diethylpyrocarbonate (PBS/DEPC). Following dehydration and rehydration through graded alcohols, the sections were rinsed in PBS/DEPC and then prehybridized in pi'ehybridization solution (50% deionised formamide, 2 x SSC (1 x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.4), 0.2% bovine serum albumin, polyvinylpyrrolidone and Ficoll 400, 100/~g/ml salmon sperm D N A and 0.3% fl-mercaptoethanol) for 60 min at 37 °C.
Correspondence: P.C. Emson, MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, CB2 4AT, U.K.
82 After draining off the prehybridization solution, hybridization solution (identical to the prehybridization solution but with the addition of 5% dextran sulphate) containing labelled probe (0.6-1.0 x 105 cpm/50/zl of hybridization solution) was poured on each section. The sections were incubated overnight in a moist chamber at 37 °C. Next day, the sections were rinsed in 2 x SSC, 1 × SSC and 0.5 x SSC for 30 min each at 37 °C and quickly washed in sterile H20/ DEPC. After dehydration through graded alcohol, the sections were allowed to air dry. In order to visualize labelled areas, the sections were opposed to X-ray film before coating with autoradiography emulsion (Ilford K-2) to allow the cellular localization of labelled cells. For control experiments some sections were pretreated for 60 min with 20 ktg/ml RNase A (Sigma) in 0.5 NTE buffer (0.5 M sodium chloride, 10 mM TrisHCI and 1 mM EDTA; pH 8.0) at 37 °C. After enzyme pretreatment, the sections were rinsed in
0.5 NTE buffer for 60 min at 37 °C (the buffer was changed 5 times) and then hybridized as described above. Neurophysin (NP) immunohistochemistry was used to check the localization of AVP neurones. The NP antiserum which has been previously characterized and recognises NP 9, was used at a dilution of 1:500. To visualise the NP immunoreactivity, the peroxidase-antiperoxidase (PAP) method of Sternberger 11was used. In the sections including the hypothalamic region, magnocellular AVP m R N A containing neurones were observed in the supraoptic nucleus (SON; Figs. 1 and 2A), paraventricular nucleus (PVN; Figs. 1 and 2C) and accessory supraoptic nucleus (Fig. 1). In addition to these magnocellular neurones, parvo (and medio) cellular AVP mRNA-containing neurones were found in the suprachiasmatic nucleus (Fig. 2D). In the sections treated with RNase A before hybridization, to digest cellular mRNA, positive neurones
Fig. l. AVP mRNA signal in the hypothalamus. Low-power, bright-field image derived from X-ray film image.
83
E
Fig. 2. Autoradiographic localization of AVP mRNA-containing neurons in the supraoptic nucleus (A, bright-field photomicrograph, × 35), the paraventricular nucleus (C, dark field, x 70) and the suprachiasmatic nucleus (D, bright field, x 350). No AVP mRNA-containing neurons were detectable in the RNase A pretreated section (B, × 140). Neurophysin immunoreactivity was also observed in the monkey hypothalamus (E, the paraventricular nucleus, × 28). OT, optic tract; SON, supraoptic nucleus; III, 3rd ventricle.
84
A. ~
(3.
D° Ill
Fig. 3. A schema of the distribution of AVP containing neurons through the monkey hypothalamus. From A (at the level of the anterior commissure) to D, the pictures are arranged at intervals of 800~tm from rostral to caudal. Black dots represent AVP mRNA neurons. III, 3rd ventricle; OC, optic chiasm; OT, optic tract.
were not detectable (Fig. 2B). As expected, the distribution of NP-immunoreactive neurones revealed by immunocytochemistry coincided with those areas containing neurons hybridizing with the AVP mRNA probe (Fig. 2E). A schema showing the distribution of AVP mRNA containing neurones throughout the hypothalamus is presented in Fig. 3. To our knowledge, this report is the first to demonstrate the presence of AVP mRNA-containing neurones in the monkey brain using an in situ hybridization technique. We used a probe for the rat NP II glycopeptide sequence. This part of the sequence of the
AVP mRNA was chosen as it is very similar to the corresponding human sequence 7`s. The homology between rat and human sequence is 86%; however, from 3'-prime end to 25th base, the sequence is identical and this homology is clearly sufficient for reliable detection of AVP m R N A in the monkey. In confirmation of this suggestion, our findings are in good agreement with previous reports of the distribution of AVP containing neurons in monkey brain j'6'l°. Further, staining with NP antiserum revealed that the location of NP immunoreactivity and AVP mRNA-containing neurons coincided. The distribution of AVP mRNA containing neurons in the monkey brain, reported here, is in good agreement with previous reports 1'6'1° which used immunochemical methods. Kawata and Sano 6 described the distribution of AVP-positive neurons in monkey brain and provided a detailed map. The localization of AVP mRNA-containing neurons observed in the present study is essentially identical to that described by Kawata and Sano 6. In particular, the number and location of AVP neurons in the SON and PVN, are similar, as would be expected. In addition to these two principal locations, the AVP m R N A neurons were also observed in the suprachiasmatic nucleus, the accessory supraoptic nucleus, periventricular area and the lateral hypothalamic area. It is important to note that in this study of expression of the AVP gene, fresh frozen cryostat sections of monkey brain were used. However, in the future we will be able to use the monkey brain and this relatively abundant message (AVP) to consider in detail the variables involved in applying the in situ hybridization techniques to human postmortem material. Thus the monkey brain will be used to investigate such questions as postmortem stability of mRNA, to optimise processing of sections and to investigate the possibility of using formalin-fixed human material. We would like to thank Mrs. B.A. Waters for help in preparing the manuscript and Mr. T. Buss for photographic work. Y.I. is a visiting research fellow from the Juntendo University School of Medicine, Tokyo, Japan.
85 1 Antunes, J.L. and Zimmerman, E.A., The hypothalamic magnocellular system of the rhesus monkey: An immunohistochemical study, J. Comp. Neurol., 181 (1978) 539-566. 2 Dierickx, K. and Vandessande, F., Immunocytochemical localization of the vasopressinergic and the oxytocinergic neurons in the human hypothalamus, Cell Tissue Res., 184 (1977) 15-27. 3 Dierickx, K. and Vandessande, F., Immunocytochemical demonstration of separate vasopressin-neurophysin and oxytocin-neurophysin neurons in the human hypothalamus, Cell Tissue Res., 196 (1979) 203-212. 4 Fliers, E., Guldenaar, S.E.F., Wal, N.V.D. and Swaab, D.F., Extrahypothalamic vasopressin and oxytocin in the human brain; Presence of vasopressin cells in the bed nucleus of the stria terminalis, Brain Res., 375 (1986) 363-367. 5 Fliers, F., Swaab, D.F., Pool, Chr. W. and Verwer, R.W.H., The vasopressin and oxytocin neurons in the human supraoptic and paraventricular nucleus; changes with aging and in senile dementia, Brain Res., 342 (1985) 45-53. 6 Kawata, M. and Sano, Y., Immunohistochemical identification of the oxytocin and vasopressin neurons in the hypothalamus of the monkey (Macacafuscata), Anat. Embryol.,
165 (1982) 151-167. 7 Mohr, E., Hillers, M., Ivell, R., Haulica, I.D. and Richter, D., Expression of the vasopressin and oxytocin genes in human hypothalamus, FEBS Lett., 193 (1985) 12-16. 8 Rehbein, M., Hillers, M., Mohr, E., Ivell, R., Morley, S., Schmale, H. and Richter, D., The neurohypophyseal hormones vasopressin and oxytocin, precursor structure, synthesis and regulation, Biol. Chem. Hoppe-Seyler, 367 (1986) 695-704. 9 Sofroniew, M.V. and Glasmann, W., Golgi-like immunoperoxidase staining of hypothalamic magnocellular neurons that contain vasopressin, oxytocin or neurophysin in the rat, Neuroscience, 6 (1981) 619-643. 10 Sofroniew, M.V. and Weindl, A., Identification of parvocellular vasopressin and neurophysin neurons in the surachiasmatic nucleus of a variety of mammals including primates, J. Comp. Neurol., 193 (1980) 659-675. 11 Sternberger, L.A., Immunohistochemistry, 2nd edn., Wiley, New York, 1979. 12 Uhl, G.R., Evans, J., Parta, M., Walworth, C., Hill, K., Sasek, C., Voigt, M. and Reppert, S., Vasopressin and somatostatin mRNA in situ hybridization. In G.R. Uhl (Ed.), In Situ Hybridization in Brain, Plenum, New York, 1986, pp. 21-47.
81
BRM 80026
Localization of vasopressin mRNA-containing neurones in the hypothalamus of the monkey Yosuke Ichimiya 1, Piers C. Emson 1 and Fraser D. Shaw 2 IMRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U.K.) and 2Department of Anatomy, University of Cambridge, Cambridge ( U. K. )
(Accepted 12 April 1988) Key words: In situ hybridization; Vasopressin; Neurophysin; Hypothalamus; Monkey
Neuronal perikarya containing vasopressin mRNA were detected in cryostat sections of cynomolgus monkey brains by using an in situ hybridization technique. The neurones were observed in hypothalamic regions (supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus and accessory supraoptic nucleus). These findings are in agreement with previous reports using immunohistochemical methods.
The technique of in situ hybridization histochemistry allows the localization of sites in gene expression in the central nervous system ~2. Although the in situ method is reasonably well established in rat or mouse brains, the method has not been widely applied to primate or human brain material. In this study, we have applied an in situ hybridization technique to cryostat sections of monkey brains using oligonucleotide probes specific for vasopressin (AVP) mRNA. AVP-positive neurones have been found in the hypothalamus and extrahypothalamic region of monkeys 4,6,1° and human brains 2-5 using immunohistochemical techniques. In order to complement these findings, we used a selective AVP probe and here describe the location of AVP m R N A containing neurones in the brain of the monkey (cynomolgous monkey). For detection of AVP m R N A , an anti-sense oligonucleotide probe (36 mer) was used. The probe was specific for the sequence of the first 12 amino acids of rat neurophysin (NP) II glycopeptide sequence (AlaSer-Asp-Ser-Asn-Ala-Thr-Leu-Asp-Gly). The probe was synthesized using a Biosearch 8750 Multiple Column D N A Synthesizer with the fl-cyanoethyl phos-
phoramidite chemistry. The probe was labelled (spec. act. >1.0 x 10s dpm//~g) with 35S using [35S]dCTP (Amersham, SJ 305; >600 ci/mmol) terminal deoxynucleotidyl transferase (Pharmacia) and tailing buffer (100 mM potassium cacodylate; pH 7.2, 1 mM CoC12 and 0.2 mM dithiothreitol). Adult cynomolgus monkeys of both sexes were anesthetized, the brains removed and frozen immediately in liquid nitrogen, sections (20/~m) were cut on a cryostat at -20 °C and stored at - 7 0 °C for subsequent in situ hybridization. For in situ hybridization, the sections were briefly fixed in 4% paraformaldehyde in phosphate buffer for 15 min, and then rinsed in 0.1 M phosphate buffer saline (PBS) containing 0.1% (v/v) diethylpyrocarbonate (PBS/DEPC). Following dehydration and rehydration through graded alcohols, the sections were rinsed in PBS/DEPC and then prehybridized in pi'ehybridization solution (50% deionised formamide, 2 x SSC (1 x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.4), 0.2% bovine serum albumin, polyvinylpyrrolidone and Ficoll 400, 100/~g/ml salmon sperm D N A and 0.3% fl-mercaptoethanol) for 60 min at 37 °C.
Correspondence: P.C. Emson, MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, CB2 4AT, U.K.
82 After draining off the prehybridization solution, hybridization solution (identical to the prehybridization solution but with the addition of 5% dextran sulphate) containing labelled probe (0.6-1.0 x 105 cpm/50/zl of hybridization solution) was poured on each section. The sections were incubated overnight in a moist chamber at 37 °C. Next day, the sections were rinsed in 2 x SSC, 1 × SSC and 0.5 x SSC for 30 min each at 37 °C and quickly washed in sterile H20/ DEPC. After dehydration through graded alcohol, the sections were allowed to air dry. In order to visualize labelled areas, the sections were opposed to X-ray film before coating with autoradiography emulsion (Ilford K-2) to allow the cellular localization of labelled cells. For control experiments some sections were pretreated for 60 min with 20 ktg/ml RNase A (Sigma) in 0.5 NTE buffer (0.5 M sodium chloride, 10 mM TrisHCI and 1 mM EDTA; pH 8.0) at 37 °C. After enzyme pretreatment, the sections were rinsed in
0.5 NTE buffer for 60 min at 37 °C (the buffer was changed 5 times) and then hybridized as described above. Neurophysin (NP) immunohistochemistry was used to check the localization of AVP neurones. The NP antiserum which has been previously characterized and recognises NP 9, was used at a dilution of 1:500. To visualise the NP immunoreactivity, the peroxidase-antiperoxidase (PAP) method of Sternberger 11was used. In the sections including the hypothalamic region, magnocellular AVP m R N A containing neurones were observed in the supraoptic nucleus (SON; Figs. 1 and 2A), paraventricular nucleus (PVN; Figs. 1 and 2C) and accessory supraoptic nucleus (Fig. 1). In addition to these magnocellular neurones, parvo (and medio) cellular AVP mRNA-containing neurones were found in the suprachiasmatic nucleus (Fig. 2D). In the sections treated with RNase A before hybridization, to digest cellular mRNA, positive neurones
Fig. l. AVP mRNA signal in the hypothalamus. Low-power, bright-field image derived from X-ray film image.
83
E
Fig. 2. Autoradiographic localization of AVP mRNA-containing neurons in the supraoptic nucleus (A, bright-field photomicrograph, × 35), the paraventricular nucleus (C, dark field, x 70) and the suprachiasmatic nucleus (D, bright field, x 350). No AVP mRNA-containing neurons were detectable in the RNase A pretreated section (B, × 140). Neurophysin immunoreactivity was also observed in the monkey hypothalamus (E, the paraventricular nucleus, × 28). OT, optic tract; SON, supraoptic nucleus; III, 3rd ventricle.
84
A. ~
(3.
D° Ill
Fig. 3. A schema of the distribution of AVP containing neurons through the monkey hypothalamus. From A (at the level of the anterior commissure) to D, the pictures are arranged at intervals of 800~tm from rostral to caudal. Black dots represent AVP mRNA neurons. III, 3rd ventricle; OC, optic chiasm; OT, optic tract.
were not detectable (Fig. 2B). As expected, the distribution of NP-immunoreactive neurones revealed by immunocytochemistry coincided with those areas containing neurons hybridizing with the AVP mRNA probe (Fig. 2E). A schema showing the distribution of AVP mRNA containing neurones throughout the hypothalamus is presented in Fig. 3. To our knowledge, this report is the first to demonstrate the presence of AVP mRNA-containing neurones in the monkey brain using an in situ hybridization technique. We used a probe for the rat NP II glycopeptide sequence. This part of the sequence of the
AVP mRNA was chosen as it is very similar to the corresponding human sequence 7`s. The homology between rat and human sequence is 86%; however, from 3'-prime end to 25th base, the sequence is identical and this homology is clearly sufficient for reliable detection of AVP m R N A in the monkey. In confirmation of this suggestion, our findings are in good agreement with previous reports of the distribution of AVP containing neurons in monkey brain j'6'l°. Further, staining with NP antiserum revealed that the location of NP immunoreactivity and AVP mRNA-containing neurons coincided. The distribution of AVP mRNA containing neurons in the monkey brain, reported here, is in good agreement with previous reports 1'6'1° which used immunochemical methods. Kawata and Sano 6 described the distribution of AVP-positive neurons in monkey brain and provided a detailed map. The localization of AVP mRNA-containing neurons observed in the present study is essentially identical to that described by Kawata and Sano 6. In particular, the number and location of AVP neurons in the SON and PVN, are similar, as would be expected. In addition to these two principal locations, the AVP m R N A neurons were also observed in the suprachiasmatic nucleus, the accessory supraoptic nucleus, periventricular area and the lateral hypothalamic area. It is important to note that in this study of expression of the AVP gene, fresh frozen cryostat sections of monkey brain were used. However, in the future we will be able to use the monkey brain and this relatively abundant message (AVP) to consider in detail the variables involved in applying the in situ hybridization techniques to human postmortem material. Thus the monkey brain will be used to investigate such questions as postmortem stability of mRNA, to optimise processing of sections and to investigate the possibility of using formalin-fixed human material. We would like to thank Mrs. B.A. Waters for help in preparing the manuscript and Mr. T. Buss for photographic work. Y.I. is a visiting research fellow from the Juntendo University School of Medicine, Tokyo, Japan.
85 1 Antunes, J.L. and Zimmerman, E.A., The hypothalamic magnocellular system of the rhesus monkey: An immunohistochemical study, J. Comp. Neurol., 181 (1978) 539-566. 2 Dierickx, K. and Vandessande, F., Immunocytochemical localization of the vasopressinergic and the oxytocinergic neurons in the human hypothalamus, Cell Tissue Res., 184 (1977) 15-27. 3 Dierickx, K. and Vandessande, F., Immunocytochemical demonstration of separate vasopressin-neurophysin and oxytocin-neurophysin neurons in the human hypothalamus, Cell Tissue Res., 196 (1979) 203-212. 4 Fliers, E., Guldenaar, S.E.F., Wal, N.V.D. and Swaab, D.F., Extrahypothalamic vasopressin and oxytocin in the human brain; Presence of vasopressin cells in the bed nucleus of the stria terminalis, Brain Res., 375 (1986) 363-367. 5 Fliers, F., Swaab, D.F., Pool, Chr. W. and Verwer, R.W.H., The vasopressin and oxytocin neurons in the human supraoptic and paraventricular nucleus; changes with aging and in senile dementia, Brain Res., 342 (1985) 45-53. 6 Kawata, M. and Sano, Y., Immunohistochemical identification of the oxytocin and vasopressin neurons in the hypothalamus of the monkey (Macacafuscata), Anat. Embryol.,
165 (1982) 151-167. 7 Mohr, E., Hillers, M., Ivell, R., Haulica, I.D. and Richter, D., Expression of the vasopressin and oxytocin genes in human hypothalamus, FEBS Lett., 193 (1985) 12-16. 8 Rehbein, M., Hillers, M., Mohr, E., Ivell, R., Morley, S., Schmale, H. and Richter, D., The neurohypophyseal hormones vasopressin and oxytocin, precursor structure, synthesis and regulation, Biol. Chem. Hoppe-Seyler, 367 (1986) 695-704. 9 Sofroniew, M.V. and Glasmann, W., Golgi-like immunoperoxidase staining of hypothalamic magnocellular neurons that contain vasopressin, oxytocin or neurophysin in the rat, Neuroscience, 6 (1981) 619-643. 10 Sofroniew, M.V. and Weindl, A., Identification of parvocellular vasopressin and neurophysin neurons in the surachiasmatic nucleus of a variety of mammals including primates, J. Comp. Neurol., 193 (1980) 659-675. 11 Sternberger, L.A., Immunohistochemistry, 2nd edn., Wiley, New York, 1979. 12 Uhl, G.R., Evans, J., Parta, M., Walworth, C., Hill, K., Sasek, C., Voigt, M. and Reppert, S., Vasopressin and somatostatin mRNA in situ hybridization. In G.R. Uhl (Ed.), In Situ Hybridization in Brain, Plenum, New York, 1986, pp. 21-47.