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Partial coexistence of growth hormone-releasing hormone and tyrosine hydroxylase in paraventricular neurons in rats
Peptides.Vol. 10, pp. 791-795. ©Pergamon Press plc. 1989. Printed in the U.S.A.
0196-9781/89 $3.00 + .00
Partial Coexistence of Growth Hormone-Releasing Hormone and Tyrosine Hydroxylase in Paraventricular Neurons in Rats S , ~ N D O R H O R V / i , T H , * t I I~VA M E Z E Y * ~ A N D M I K L O S PALKOVITS*:]:
*Laboratory of Cell Biology, NIMH, Bethesda, MD "?Department of Neurology and ¢First Department of Anatomy Semmelweis University Medical School, Budapest, Hungary R e c e i v e d 31 January 1989
HORV,/~TH, S., 1~. MEZEY AND M. PALKOVITS. Partial coexistence of growth hormone-releasing hormone and tyrosine hydroxylase in paraventricular neurons in rats. PEPTIDES 10(4) 791-795, 1989.--lmmunocytochemistry revealed growth hormone-releasing hormone (GRF)-containing cells (4-10/50 v,m thick coronal sections) in the ventral portion of the medial parvicellular subdivision of the paraventricular nucleus (PVN) in the rat. In the same region we also detected about the same number of neurons containing the mRNA that encodes GRF using in situ hybridization histochemistry. Fluorescence double labelling immunohistochemistry showed the presence of GRF and tyrosine hydroxylase (TH) in the same PVN neurons. The overlap between the two populations of cells is only partial: less than half of TH-positive cells contain GRF and vice versa. Growth hormone-releasing hormone In situ hybridization histochemistry
Tyrosine hydroxylase
Paraventricular nucleus
NEURONS containing GRF in the central nervous system of the rat have been mapped by the mid-80's (2, 5, 9, 12, 16). Unlike other hypothalamic releasing hormones growth hormone-releasing hormone (GRF) neurons occupy a relatively small area of the brain; they are located exclusively in the middle part of the hypothalamus. Most of the GRF-containing cells are located in the arcuate nucleus, a considerable number of cells surround the ventromedial nucleus and scattered GRF cells are in the territory of the perifornical and dorsomedial nuclei as well as in the lateral hypothalamus. In early studies (2,12) a few cells that immunostained for GRF have also been reported in the hypothalamic paraventricular nucleus (PVN). Recently, a substantial number of GRF-like immunoreactive neurons have been reported here using a monoclonal antibody directed against rat GRF (I). The first part of our study was designed to precisely localize these GRFimmunopositive neurons and verify their character by detecting the mRNA encoding for GRF in the same region using in situ hybridization histochemistry. During the past few years tyrosine hydroxylase (TH) activity has been demonstrated in various types of neuropeptide-containing neurons (3). Thus colocalization of GRF and TH immunostaining has been reported in the arcuate nucleus, where almost a 100%
GRF-TH coexistence
overlap between neurons containing the two substances has been demonstrated (3, 8, 10). In our study we examined if there is any overlap between GRF- and TH-containing cells in the PVN. METHOD Young adult (150-200 g. b.wt.) male Sprague-Dawley rats (Taconic Farms, Germantown, NY) were used.
lmmunostaining Six animals were colchicine treated (120 v,g/12 V.I saline, intracerebroventricularly) two days prior to perfusion with ice-cold fixative [4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB), pH 7.4]. Brains (n = 3) were removed and postfixed in the same fixative overnight at 4°C. Fifty v,m thick coronal sections were cut on a Vibratome (Oxford), at the level of the paraventricular nucleus (between 1.5-2.5 mm caudal to the bregma level). The sections were rinsed overnight in several changes of PB, pretreated with 3% H20 2 (15 rain) to block the endogenous peroxidase activity and immersed in 0.4% Triton-X
~Requests for reprints should be addressed to Sfindor Horvatth, M.D., First Department of Anatomy, Semmelweis University Medical School, 1450 Budapest, Tilzolt6 utca 58, Hungary.
791
792
HORV,~TH. MEZEY AND PALKOVITS
FIG. 1. Light microscopic immunostaining of GRF-positive neurons in the hypothalamic paraventricular nucleus. Fifty ~m thick coronal section. Ill--third ventricle. Scale: 100 p.m. 100 in 0.1 M PB (3 hours) to improve penetration of antibodies. After incubation in 2% normal goat serum (NGS) for 30 min, the sections were incubated in a 1:15000 dilution of polyclonal anti-GRF serum [raised in rabbits, has been characterized earlier (4)] in PB containing 1% NGS for 2 days at 4°C. The immunostaining was done with the avidin-biotin (ABC) method as described previously (4). For double immunostaining, following the same perfusion the brains (n = 3) were immersed in sucrose/ PBS (10%--I hour, 20%--3 hours, 30%--overnight) for cryoprotection, and frozen on dry ice. Coronal plane 12 I.Lm thick sections were cut in a cryostat, collected on gelatin-coated slides, and washed several times in phosphate buffered saline (PBS: 0.1 M, pH 7.6) containing 0.6% Triton-X 100 and 0.1% bovine serum albumin (BSA) to improve penetration and reduce background, respectively. The sections were then incubated in the first primary antibody [monoclonal (mouse) antityrosine hydroxylase (Boehringer-Mannheim)], at a 1:50 dilution at 4°C overnight, rinsed in several changes of PBS and then incubated in the second primary antibody (anti-GRF) at a 1:750 dilution in PBS/q'riton/BSA at 4°C overnight and rinsed in PBS. The sections were then incubated in a mixture of antimouse IgG conjugated rhodamine (1:400, Boehringer-Mannbeim) and antirabbit lgG conjugated FITC (1:100, Boehringer-Mannheim) in PBS/Triton for two hours at room temperature. They were then rinsed in PBS, air dried and mounted in Cytoseal (VWR). The fluorescence was examined with a Leitz Dialux microscope and pictures were taken using rhodamine and F r r c filters to visualize TH and GRF, respectively.
Controls The working dilution of the GRF antiserum was preabsorbed with 10 I,Lg,/l ml of the GRF peptide. The preabsorbtion totally abolished immunostaining. Omitting the primary or secondary antibodies resulted in no staining.
In Situ Hybridization The brains of 3 animals were removed after decapitation and
frozen on dry ice. Sections were cut in a cryostat (12 Ixm) and thaw-mounted onto gelatin-coated slides. The sections were then postfixed with 4% formaldehyde, rinsed in PBS, treated with acetic anhydride and dehydrated in ethanol and chloroform. Tissue sections were processed for hybridization as previously described (18). The sections were air dried and 500,000 cpm of 3sS-labelled oligonucleotide probe (corresponding to the GRF mRNA sequence between bases 195-242) in 40 vd of hybridization buffer was applied on each slide. Hybridization was performed at 37°C overnight then the sections were washed in 2 x SSC and 50% formamide for four times 15 rain at 40°C and twice in l x SSC at room temperature and air dried. The sections were put against Kodak XAR 1 film and after developing the film they were dipped into Kodak NTB3 nuclear track emulsion and exposed for four weeks with desiccant. After they were developed, the sections were counterstained with 0.5% toluidine blue and examined under a Leitz Dialux microscope. RESULTS
Immunostaining with GRF antiserum revealed positively labelled neurons in the ventromedial part of the paraventricular hypothalamic nucleus. The cells were seen at all rostro-caudal levels of the PVN, but more frequently in the middle and posterior sections of the nucleus. The immunoreactive cells were medium sized, fusiform or polygonal located close to the ventral border of the PVN. On the average 4--10 cells were observed in one 50 l~m thick section (Fig. l). In situ hybridization revealed neurons containing GRF mRNA in the PVN in a similar distribution and number to what was seen with immunostainlng (Fig. 2). Double immunostaining revealed a few cells in the PVN that were positive with both GRF and TH antibodies. The number of these cells was 1-3 in a section (Fig. 3). There were several neurons that were positive with one of the above antibodies only. DISCUSSION
Among a large number of neuropeptides GRF is synthesized in
GRH-TH COEXISTENCE IN RAT PVN
793
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FIG. 2. In situ hybridization histochemistry shows several labelled cells containing GRF mRNA in the PVN. Twelve ~rn thick coronal section. (A) Dark-field illumination and (B) bright-field illumination of the same area. The arrows indicate silver grains above labelled neurons, lII--third ven~cle. Scale: 100 ~.m.
paraventricular nucleus neurons. In agreement with previous studies (l, 2, 12), a group of GRF immunoreactive neurons was found in the medial parviceilular subdivision of the magnocellular secretory neurons. In comparison to the number of GRF immunoreactive cells in the arcuate nucleus, relatively few GRFpositive neurons are located in the PVN. GRF mRNA has already been visualized in the arcuate nucleus by in situ hybridization histochemistry (7,18). Here, we demonstrate the presence of mRNA encoding GRF in some paraventricular nucleus neurons. In the PVN the distribution of the cells
hybridizing to the probe is very similar to the distIibution of the immunopositive GRF cells. In the arcuate nucleus virtually all GRF positive cells were demonstrated to contain TH as well (3, 8, 10). In the paraventricular nucleus, however, only a minor portion of GRF-positive cells seems to also contain TH. Similarly, only a small portion of the (more numerous) TH-immunopositive cells in the PVN seems to contain GRF, too. The arcuate GRF cells (tuberoinfundibular GRF neurons) are involved in the regulation of growth hormone secretion from the
794
HORV,,~TH. MEZEY AND PALKOVITS
FIG. 3. Fluorescent microphotograph of a coronal section immunostained for tyrosine hydroxyl&se and growth hormone-releasing hormone. The figure shows the same section viewed through the rhodamine filter (TH) and the FITC filter (GRF). The large arrows indicate cells that are positive for both antigens. The small arrows (TH) and the arrowheads (GRF) point to neurons that seem to contain only one of the two antigens. The asterisk shows a cross section of a vessel, as a landmark. The photograph corresponds to the empty rectangle in the insert. NPV--paraventricular nucleus. Scale: 50 Ixm.
anterior pituitary [see (2, 3, 12)]. The functional significance of the 'extra-arcuate' GRF neurons remains to be established. These GRF cells may act on other neurons as a neurotransmitter-like substance (2), but this hypothesis needs to be proved. Neither the function nor the projections of the paraventricular GRF-containing cells is clear. Cells are located in the medial subdivision of the PVN, a region that almost exclusively projects to the median eminence (3, 13, 15). Thus, it seems to be obvious that the PVN GRF cells also participate in the hypothalamo-median eminencepituitary regulatory system, but this hypothesis needs to be proven. The vast majority of the GRF-immunopositive fibers in the median eminence originate in the arcuate nucleus (3, 10, 15). Four weeks after surgical deafferentation of the medial basal
hypothalamus the number of immunoreactive GRF fibers somewhat decreased in the lateral portion of the median eminence (2). The depleted fibers may arise in any extra-arcuate region, like GRF cells located in and around the ventromedial nucleus or in the perifornical nucleus. Transection of the paraventriculo-hypophyseal pathway in the lateral suprachiasmatic area resulted in an almost total disappearance of paraventricular origin neuropeptides in the median eminence, and a retrograde accumulation of these peptides in the neurons of the PVN [see (11)]. After transection of the paraventriculo-hypophyseal tract neither depletion of GRF in the median eminence nor accumulation of GRF in PVN neurons was observed (unpublished observations). In earlier studies the coexistence of dopamine and GRF was
GRH-TH COEXISTENCE IN RAT PVN
795
demonstrated in the arcuate nucleus (8). The authors suggested that dopamine in the GRF-containing arcuate neurons may act on GH release at the hypothalamic level. A similar function of this catecholamine in the PVN GRF cells seems possible.
ACKNOWLEIX3EMENTS The authors thank M. J. Brownstein for synthesizing the oligonucleotide probe and W. S. Young, iii and Emily Sbepard for their technical assistance with the in situ hybridization.
REFERENCES I. Bruhn, T. O.; Antony, E. L. P.; Wu, P.; Jackson, l. M. D. GRF immunoreactive neurons in the paraventricular nucleus of the rat: an immunohistochemical study with monoclonal and polyclonal antibodies. Brain Res. 424:290-298; 1987. 2. Daikoku, S.; Kawano, H.; Noguchi, M.; Nakanishi, J.; Tokuzen, M.; Chihara, K.; Nagatsu, I. GRF neurons in the rat hypothalamus. Brain Res. 399:250-261; 1986. 3. Everitt, B. J.; Meister, B.; H6kfelt, T.; Melander, T.; Terenius, L.; R6kaeus, £.; Theodorsson-Norheim, E.; Docray, G.; Edwardson, J.; Cuello, C.; Elde, R.; Goldstein, M.; Hemmings, H.; Ouimet, C.; Walaas, 1.; Greengard, P.; Vale, W.; Weber, E.; Wu, J. Y.; Chang, K. J. The hypothalamic arcuate nucleus-median eminence complex: immunohistochemistry of transmitters, peptides and DARPP-32 with special reference to coexistence in dopamine neurons. Brain Res. Rev. 11:97-155; 1986. 4. Horv,'ith, S.; Palkovits, M.; G6rcs. T.; Arimura, A. Electron microscopic immunocytochemical evidence for the existence of bidirectional synaptic connections between growth hormone-releasing hormoneand somatostatin-containing neurons in the hypothalamus of the rat. Brain Res. 481:8-15; 1989. 5. Jacobowitz, D. M.; Schulte, H.; Chrousos, G. P.; Loriaux, D. L. Localization of GRF-like immunoreactive neurons in the rat brain. Peptides 4:521-524; 1983. 6. Mayo, K. E.; Cerelli, G. M.; Rosenfeld, M. G.; Evans, R. M. Characterization of cDNA and genomic clones encoding the precursor to rat bypothalamic growth hormone-releasing factor. Nature 314: 464--467; 1985. 7. Mayo, K. E.; Evans, R. M.; Rosenfeld, M. G. Genes encoding mammalian neuroendocrine peptides: Strategies toward their identification and analysis. Annu. Rev. Physiol. 48:431-446; 1986. 8. Meister, B.; H6kfelt, T.; Vale, W. W.; Sawchenko, P. E.; Swanson, L.; Goldstein, M. Coexistence of tyrosine hydroxylase and growth hormone-releasing factor in a subpopulation of tubero-infundibular neurons of the rat. Neuroendocrinology 42:237-247; 1986. 9. Merchenthaler, I.; Thomas, C. R.; Arimura, A. Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing
10.
11. 12.
13.
14.
15. 16. 17. 18.
structures in the rat brain using anti-rat GHRF serum. Peptides 5:1071-1075; 1984. Okamura, H.; Murakami, S.; Chihara, K.; Nagatsu, I.; Ibata, Y. Coexistence of growth hormone releasing factor-like and tyrosine hydroxylase-like immunoreactivities in neurons of the rat arcuate nucleus. Neuroendocrinology 41:177-179; 1985. Palkovits, M. Neuropeptides in the median eminence of the rat. In: McCann, S. M.; Weiner, R. I., eds. Integrative neuroendocrinology: Molecular, cellular and clinical aspects. Basel: Karger; 1987:36---45. Sawchenko, P. E.; Swanson, L. W.; Rivier, J.; Vale, W. W. The distribution of growth hormone-releasing factor (GRF) immunoreactivity in the central nervous system of the rat: An immunohistochemical study using antisera directed against rat hypothalamic GRF. J. Comp. Neurol. 237:100-115; 1985. Swanson, L. W.; Kuypers, H. G. J. M. The paraventricular nucleus of the hypothalamus: cytoarehitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J. Comp. Neurol. 194:555-570; 1980. Swanson, L. W.; Sawchenko, P. E.; B6rod, A.; Hatman, B. K.; Helle, K. B.; Vanorden, D. E. An immunohistocbemical study of the organization of catecholaminergic cells and terminal fields in the paraventricular and supraoptic nuclei of the hypothalamus. J. Comp. Neurol. 196:271-285; 1981. Van den Pol, A. N. The magnocellular and parvocellular paraventricular nucleus of rat: intrinsic organization. J. Comp. Neurol. 206:317-345; 1982. VandePol, C. J.; Leidy. J. W., Jr.; Finger, T. E.; Robbins, R. J. lmmunohistochemical localization of GRF-containing neurons in rat brain. Neuroendocrinology 42:143-147; 1986. Young, W. S., I11; Bonnet, T. I.; Brann, M. R. Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc. Natl. Acad. Sci. USA 83:9827-9831; 1986. Young, W. S., III; Zoller, R. T. Neuroendocrine gene expression in the hypothalamus: in situ hybridization histochemical studies. Cell. Mol. Neurobiol. 7:353-366; 1987.
0196-9781/89 $3.00 + .00
Partial Coexistence of Growth Hormone-Releasing Hormone and Tyrosine Hydroxylase in Paraventricular Neurons in Rats S , ~ N D O R H O R V / i , T H , * t I I~VA M E Z E Y * ~ A N D M I K L O S PALKOVITS*:]:
*Laboratory of Cell Biology, NIMH, Bethesda, MD "?Department of Neurology and ¢First Department of Anatomy Semmelweis University Medical School, Budapest, Hungary R e c e i v e d 31 January 1989
HORV,/~TH, S., 1~. MEZEY AND M. PALKOVITS. Partial coexistence of growth hormone-releasing hormone and tyrosine hydroxylase in paraventricular neurons in rats. PEPTIDES 10(4) 791-795, 1989.--lmmunocytochemistry revealed growth hormone-releasing hormone (GRF)-containing cells (4-10/50 v,m thick coronal sections) in the ventral portion of the medial parvicellular subdivision of the paraventricular nucleus (PVN) in the rat. In the same region we also detected about the same number of neurons containing the mRNA that encodes GRF using in situ hybridization histochemistry. Fluorescence double labelling immunohistochemistry showed the presence of GRF and tyrosine hydroxylase (TH) in the same PVN neurons. The overlap between the two populations of cells is only partial: less than half of TH-positive cells contain GRF and vice versa. Growth hormone-releasing hormone In situ hybridization histochemistry
Tyrosine hydroxylase
Paraventricular nucleus
NEURONS containing GRF in the central nervous system of the rat have been mapped by the mid-80's (2, 5, 9, 12, 16). Unlike other hypothalamic releasing hormones growth hormone-releasing hormone (GRF) neurons occupy a relatively small area of the brain; they are located exclusively in the middle part of the hypothalamus. Most of the GRF-containing cells are located in the arcuate nucleus, a considerable number of cells surround the ventromedial nucleus and scattered GRF cells are in the territory of the perifornical and dorsomedial nuclei as well as in the lateral hypothalamus. In early studies (2,12) a few cells that immunostained for GRF have also been reported in the hypothalamic paraventricular nucleus (PVN). Recently, a substantial number of GRF-like immunoreactive neurons have been reported here using a monoclonal antibody directed against rat GRF (I). The first part of our study was designed to precisely localize these GRFimmunopositive neurons and verify their character by detecting the mRNA encoding for GRF in the same region using in situ hybridization histochemistry. During the past few years tyrosine hydroxylase (TH) activity has been demonstrated in various types of neuropeptide-containing neurons (3). Thus colocalization of GRF and TH immunostaining has been reported in the arcuate nucleus, where almost a 100%
GRF-TH coexistence
overlap between neurons containing the two substances has been demonstrated (3, 8, 10). In our study we examined if there is any overlap between GRF- and TH-containing cells in the PVN. METHOD Young adult (150-200 g. b.wt.) male Sprague-Dawley rats (Taconic Farms, Germantown, NY) were used.
lmmunostaining Six animals were colchicine treated (120 v,g/12 V.I saline, intracerebroventricularly) two days prior to perfusion with ice-cold fixative [4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB), pH 7.4]. Brains (n = 3) were removed and postfixed in the same fixative overnight at 4°C. Fifty v,m thick coronal sections were cut on a Vibratome (Oxford), at the level of the paraventricular nucleus (between 1.5-2.5 mm caudal to the bregma level). The sections were rinsed overnight in several changes of PB, pretreated with 3% H20 2 (15 rain) to block the endogenous peroxidase activity and immersed in 0.4% Triton-X
~Requests for reprints should be addressed to Sfindor Horvatth, M.D., First Department of Anatomy, Semmelweis University Medical School, 1450 Budapest, Tilzolt6 utca 58, Hungary.
791
792
HORV,~TH. MEZEY AND PALKOVITS
FIG. 1. Light microscopic immunostaining of GRF-positive neurons in the hypothalamic paraventricular nucleus. Fifty ~m thick coronal section. Ill--third ventricle. Scale: 100 p.m. 100 in 0.1 M PB (3 hours) to improve penetration of antibodies. After incubation in 2% normal goat serum (NGS) for 30 min, the sections were incubated in a 1:15000 dilution of polyclonal anti-GRF serum [raised in rabbits, has been characterized earlier (4)] in PB containing 1% NGS for 2 days at 4°C. The immunostaining was done with the avidin-biotin (ABC) method as described previously (4). For double immunostaining, following the same perfusion the brains (n = 3) were immersed in sucrose/ PBS (10%--I hour, 20%--3 hours, 30%--overnight) for cryoprotection, and frozen on dry ice. Coronal plane 12 I.Lm thick sections were cut in a cryostat, collected on gelatin-coated slides, and washed several times in phosphate buffered saline (PBS: 0.1 M, pH 7.6) containing 0.6% Triton-X 100 and 0.1% bovine serum albumin (BSA) to improve penetration and reduce background, respectively. The sections were then incubated in the first primary antibody [monoclonal (mouse) antityrosine hydroxylase (Boehringer-Mannheim)], at a 1:50 dilution at 4°C overnight, rinsed in several changes of PBS and then incubated in the second primary antibody (anti-GRF) at a 1:750 dilution in PBS/q'riton/BSA at 4°C overnight and rinsed in PBS. The sections were then incubated in a mixture of antimouse IgG conjugated rhodamine (1:400, Boehringer-Mannbeim) and antirabbit lgG conjugated FITC (1:100, Boehringer-Mannheim) in PBS/Triton for two hours at room temperature. They were then rinsed in PBS, air dried and mounted in Cytoseal (VWR). The fluorescence was examined with a Leitz Dialux microscope and pictures were taken using rhodamine and F r r c filters to visualize TH and GRF, respectively.
Controls The working dilution of the GRF antiserum was preabsorbed with 10 I,Lg,/l ml of the GRF peptide. The preabsorbtion totally abolished immunostaining. Omitting the primary or secondary antibodies resulted in no staining.
In Situ Hybridization The brains of 3 animals were removed after decapitation and
frozen on dry ice. Sections were cut in a cryostat (12 Ixm) and thaw-mounted onto gelatin-coated slides. The sections were then postfixed with 4% formaldehyde, rinsed in PBS, treated with acetic anhydride and dehydrated in ethanol and chloroform. Tissue sections were processed for hybridization as previously described (18). The sections were air dried and 500,000 cpm of 3sS-labelled oligonucleotide probe (corresponding to the GRF mRNA sequence between bases 195-242) in 40 vd of hybridization buffer was applied on each slide. Hybridization was performed at 37°C overnight then the sections were washed in 2 x SSC and 50% formamide for four times 15 rain at 40°C and twice in l x SSC at room temperature and air dried. The sections were put against Kodak XAR 1 film and after developing the film they were dipped into Kodak NTB3 nuclear track emulsion and exposed for four weeks with desiccant. After they were developed, the sections were counterstained with 0.5% toluidine blue and examined under a Leitz Dialux microscope. RESULTS
Immunostaining with GRF antiserum revealed positively labelled neurons in the ventromedial part of the paraventricular hypothalamic nucleus. The cells were seen at all rostro-caudal levels of the PVN, but more frequently in the middle and posterior sections of the nucleus. The immunoreactive cells were medium sized, fusiform or polygonal located close to the ventral border of the PVN. On the average 4--10 cells were observed in one 50 l~m thick section (Fig. l). In situ hybridization revealed neurons containing GRF mRNA in the PVN in a similar distribution and number to what was seen with immunostainlng (Fig. 2). Double immunostaining revealed a few cells in the PVN that were positive with both GRF and TH antibodies. The number of these cells was 1-3 in a section (Fig. 3). There were several neurons that were positive with one of the above antibodies only. DISCUSSION
Among a large number of neuropeptides GRF is synthesized in
GRH-TH COEXISTENCE IN RAT PVN
793
t
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""..
"
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+;+ +: "P ... • 4
+'t
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FIG. 2. In situ hybridization histochemistry shows several labelled cells containing GRF mRNA in the PVN. Twelve ~rn thick coronal section. (A) Dark-field illumination and (B) bright-field illumination of the same area. The arrows indicate silver grains above labelled neurons, lII--third ven~cle. Scale: 100 ~.m.
paraventricular nucleus neurons. In agreement with previous studies (l, 2, 12), a group of GRF immunoreactive neurons was found in the medial parviceilular subdivision of the magnocellular secretory neurons. In comparison to the number of GRF immunoreactive cells in the arcuate nucleus, relatively few GRFpositive neurons are located in the PVN. GRF mRNA has already been visualized in the arcuate nucleus by in situ hybridization histochemistry (7,18). Here, we demonstrate the presence of mRNA encoding GRF in some paraventricular nucleus neurons. In the PVN the distribution of the cells
hybridizing to the probe is very similar to the distIibution of the immunopositive GRF cells. In the arcuate nucleus virtually all GRF positive cells were demonstrated to contain TH as well (3, 8, 10). In the paraventricular nucleus, however, only a minor portion of GRF-positive cells seems to also contain TH. Similarly, only a small portion of the (more numerous) TH-immunopositive cells in the PVN seems to contain GRF, too. The arcuate GRF cells (tuberoinfundibular GRF neurons) are involved in the regulation of growth hormone secretion from the
794
HORV,,~TH. MEZEY AND PALKOVITS
FIG. 3. Fluorescent microphotograph of a coronal section immunostained for tyrosine hydroxyl&se and growth hormone-releasing hormone. The figure shows the same section viewed through the rhodamine filter (TH) and the FITC filter (GRF). The large arrows indicate cells that are positive for both antigens. The small arrows (TH) and the arrowheads (GRF) point to neurons that seem to contain only one of the two antigens. The asterisk shows a cross section of a vessel, as a landmark. The photograph corresponds to the empty rectangle in the insert. NPV--paraventricular nucleus. Scale: 50 Ixm.
anterior pituitary [see (2, 3, 12)]. The functional significance of the 'extra-arcuate' GRF neurons remains to be established. These GRF cells may act on other neurons as a neurotransmitter-like substance (2), but this hypothesis needs to be proved. Neither the function nor the projections of the paraventricular GRF-containing cells is clear. Cells are located in the medial subdivision of the PVN, a region that almost exclusively projects to the median eminence (3, 13, 15). Thus, it seems to be obvious that the PVN GRF cells also participate in the hypothalamo-median eminencepituitary regulatory system, but this hypothesis needs to be proven. The vast majority of the GRF-immunopositive fibers in the median eminence originate in the arcuate nucleus (3, 10, 15). Four weeks after surgical deafferentation of the medial basal
hypothalamus the number of immunoreactive GRF fibers somewhat decreased in the lateral portion of the median eminence (2). The depleted fibers may arise in any extra-arcuate region, like GRF cells located in and around the ventromedial nucleus or in the perifornical nucleus. Transection of the paraventriculo-hypophyseal pathway in the lateral suprachiasmatic area resulted in an almost total disappearance of paraventricular origin neuropeptides in the median eminence, and a retrograde accumulation of these peptides in the neurons of the PVN [see (11)]. After transection of the paraventriculo-hypophyseal tract neither depletion of GRF in the median eminence nor accumulation of GRF in PVN neurons was observed (unpublished observations). In earlier studies the coexistence of dopamine and GRF was
GRH-TH COEXISTENCE IN RAT PVN
795
demonstrated in the arcuate nucleus (8). The authors suggested that dopamine in the GRF-containing arcuate neurons may act on GH release at the hypothalamic level. A similar function of this catecholamine in the PVN GRF cells seems possible.
ACKNOWLEIX3EMENTS The authors thank M. J. Brownstein for synthesizing the oligonucleotide probe and W. S. Young, iii and Emily Sbepard for their technical assistance with the in situ hybridization.
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