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Neurotensin-like immunoreactivity and neurotensin receptors in the rat hypothalamus and in the neurointermediate lobe of the pituitary gland
Brain Research, 358 (1985) 59-69 Elsevier
59
BRE 11174
Neurotensin-Like Immunoreactivity and Neurotensin Receptors in the Rat Hypothalamus and in the Neurointermediate Lobe of the Pituitary Gland M. GOEDERT 1, S.L. LIGHTMAN2, P.W. MANTYH1, S.P. HUNT 1and P.C. EMSON 1 1MRC Neurochemical Pharmacology Unit Medical Research Council Centre, Medical School Cambridge CB2 2QH and 2Department of Medicine, Westminster Hospital London (U. K. ) (Accepted February 19th, 1985) Key words: neurotensin-like immunoreactivity-- neurotensin receptor-- hypothalamus - - neurointermediate pituitary gland-- monosodium glutamate - - pituitary stalk transection
In the rat hypothalamus, cell bodies containing neurotensin-like immunoreactivity were mainly found in the medial preoptic area, the periventrieular nucleus, the paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumulation of silver grains over the paraventricular nucleus, the arcuate nucleus and the median eminence region. By radioimmunoassay neurotensin-like immunoreactivity was also found in the neurointermediate lobe of the pituitary gland of various mammalian species and in human postmortem posterior pituitary glands. In the rat studies involving pituitary stalk transections and the neurotoxin monosodium glutamate indicated the presence of a neurotensinergic pathway from the arcuate nucleus to the neurointermediate lobe of the pituitary gland. [3H]neurotensin binding sites were found to be concentrated over the intermediate lobe of the pituitary gland and their presence was not affected by pituitary stalk transection, indicating their localization on endocrine cells of the intermediate lobe of the pituitary gland.
INTRODUCTION Neurotensin is a 13 amino acid peptide first isolated from bovine hypothalamus and later from both bovine and human small intestine7,9, 23. It is widely distributed throughout the central nervous system of various mammalian species, where it probably functions as a neurotransmitter 41. In all species examined to date, the highest concentration of radioimmunoassayable neurotensin-like immunoreactivity is found in the hypothalamusS,lOA2,13A7,3t,40,48. By immunohistochemistry neurotensin-like immunoreactivity has been found in cell bodies in several hypothalamic nuclei, including the parvocellular portion of the paraventricular nucleus and the arcuate nucleus25,27,28,30; it has been shown to co-exist with corticotropin-releasing factor-like immunoreactivity in the paraventricular nucleus and with tyrosine hydroxylase-like immunoreactivity in the arcuate nucleus25,27, 43. In addition, neurotensin-like immunoreactivity is present in
high concentrations in nerve fibres in the external layer of the median eminence25,28,30. By electron microscopy, the latter have been shown to contact portal blood vessels, suggesting that neurotensin may function as a hypothalamic releasing-hormone; this is supported by the finding that neurotensin influences the release of several anterior pituitary hormones, both at the hypothalamic and the pituitary levels 14,39,50,51. High levels of neurotensin-like immunoreactivity are also found in the pituitary gland, where the immunoreactive material is found in cells in the anterior lobe and in nerve fibres in the neurointermediate lobe16,20,49. To date, the function of neurotensin-like immunoreactivity in the pituitary gland is unknown. In the present communication, we have investigated the immunohistochemical distribution of neurotensin-like immunoreactivity and the autoradiographic distribution of specific [3H]neurotensin binding sites in both the rat hypothalamus and the neu-
Correspondence: M. Goedert, MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Cambridge CB2 2QH, U.K. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
60 rointermediate lobe of the pituitary gland. MATERIALS AND METHODS
Extraction of tissues and radioimmunoassay Rat, guinea-pig, rabbit and cat neurointermediate pituitary glands were dissected and immediately frozen on dry-ice; pig and bovine tissues were obtained from a local abattoir and frozen within 1 h following the death of the animals. Postmortem human pituitary gland posterior lobes were obtained from patients who had died of peripheral vascular disorders (age group 69-79 years). At autopsy there wasno evidence for either endocrinological or neurological diseases. The mean-time elapsed between death and tissue removal amounted to 28 h (18-48 h). The tissues were weighed and extracted using 10 vol. boiling 1 M acetic acid. They were homogenized in glass-teflon homogenizers and left at 4 °C for 20 min in order to ensure complete extraction. They were then centrifuged at 2000 g for 10 min and the supernatants freeze-dried. The lyophilized tissues were reconstituted in assay buffer, centrifuged at 2000 g for 10 min in order to remove insoluble material and assayed in duplicate for neurotensin-like immunoreactivity, using a previously described antiserum selective for the aminoterminus of synthetic neurotensinlL
heparin followed by 400 ml paraformaldehyde/lysine/sodium periodate 3s. Forty-/~m sections were cut on a freezing microtome and treated with 1% hydrogen peroxide for 10 min in order to reduce endogenous peroxidase activity. The antibodies were diluted with 100 mM sodium phosphate buffer (pH 7.4) containing 0.1% Triton X-100 and 1% sheep serum. Free floating sections were incubated with the previously described primary antiseralS. 26 (diluted 1:400 for neurotensin and 1:500 for tyrosine hydroxylase) for 24 h at 4 °C. Sections were then washed in 100 mM sodium phosphate buffer for 30 min and incubated with sheep anti-rabbit antiserum (Miles), diluted 1:20, for 1 h. Following this, the sections were washed in 100 mM sodium phosphate buffer for 30 min, incubated with rabbit peroxidase anti-peroxidase conjugate (Miles) at a dilution of 1:100 for t h and washed again in 100 mM sodium phosphate buffer for 30 min. The tissue then was incubated for 5-10 min in 100 mM sodium phosphate buffer contaning 0.09% 3,3-diaminobenzidine and 0.005% hydrogen peroxide and subsequently washed in 100 mM sodium phosphate buffer. In control experiments, the primary antisera were omitted or in the case of the neurotensin antiserum, preincubated for 2 h at room temperature with 10/zM neurotensin.
Receptor autoradiography High-performance liquid chromatography Lyophilyzed rat neurointermediate pituitary gland tissue extracts were reconstituted in 0.1 M acetic acid and subjected to high-performance liquid chromatography (HPLC) on reverse phase by using a/~-Bondapak C18 column and a linear 5-30% acetonitrile gradient in 10 mM ammonium acetate, pH 4.0, as the aqueous phase. One ml fractions were collected and their neurotensin-like immunoreactivity content determined by using the previously described aminoand carboxyterminus directed antisera 12.
Immunohistochemistry The immunohistochemical distribution of neurotensin-like immunoreactivity was investigated in the rat hypothalamus. In order to enhance neuronal cell body staining adult male Sprague-Dawley rats (150200 g) received an intraventricular injection of 100/~g colchicine 24 h prior to intracardial perfusion with 100 ml physiological saline containing 800 U/litre
Adult male Sprague-Dawley rats (200-250 g) were stunned, the hypothalamus and the pituitary gland dissected, frozen and sectioned in the transverse plane at 13/~m using a cryostat. The sections were pressed onto gelatin-coated slides, melted in place and dried overnight at 4 °C in air-tight boxes with desiccant. The thawed sections were incubated for 10 min at 25 °C in 2 nM [3,11-tyrosyl-3,5-3H]neurotensin (56.3 Ci/mmol, New England Nuclear Corporation) in 50 mM Tris-HCl, pH 7.4, containing 0.1% bovine serum albumin (radioimmunoassay grade, Sigma Fine Chemicals), 40 mg/litre bacitracin (Sigma Fine Chemicals) and 1 mM EDTA. Non-specific binding was defined in adjacent sections as binding in the presence of 1/~M unlabelled neurotensin (Cambridge Research Biochemicals). Following the incubation the sections were washed twice in cold buffer (2 min each time), dipped into cold water and dried under a stream of cold air. They were then apposed to emulsion-coated coverslips or a tritium-sen-
61 sitive film (3H-Ultrofilm, LKB Laboratories)19,22 and kept at -20 °C for 2 months. The coverslips and the tritium-sensitive film were developed in safe-light conditions using Kodak D 19 developer and the tissue sections put into Carnoy's fixative, Nissl stained and mounted using Depex. Dark-field photomicrographs were taken of the silver grains and light-field photomicrographs of the Nissl stain.
Test-tube binding assay The test-tube binding assay for [3H]neurotensin was performed essentially as described before 21. Briefly, the pituitary glands from adult male Sprague-Dawley rats (150-200 g) were dissected, weighed and homogenized in 10 vol. (w/v) of cold 50 mM Tris-HCl, pH 7.4, with a Polytron at setting 7 for 15 s. After 20 min centrifugation at 50,000 g the pellet was resuspended in 10 vol. 50 mM Tris-HCI, incubated at 37 °C for 30 min and centrifuged again. The washed pellet was resuspended in 50 mM Tris-HCl, pH 7.4, containing 0.1% bovine serum albumin, 40 mg/litre bacitracin and 1 mM E D T A to yield a concentration of 10 mg tissue/ml buffer. One ml of homogenate was incubated in the presence of [3H]neurotensin at 25 °C for 10 min. At the end of the incubation period the tubes were transferred to ice and filtered immediately under reduced pressure through GF/B glass fibre filters (Whatman) pretreated with 0.2% polyethylenimine (Sigma Fine Chemicals) in water. Each of the filters was washed 4 times with 5 ml cold buffer and the radioactivity determined by liquid scintillation spectrometry. Non-specific binding was defined as binding in the presence of 1 ,uM neurotensin, and specific binding was obtained by subtracting the non-specific from the total binding. Proteins were determined according to Lowry et al. 37 using bovine serum albumin as the standard. Monosodium glutamate treatment and pituitary stalk transection Neonatal Sprague-Dawley rats were injected for 10 days on alternate days wit h 4 mg/kg monosodium glutamate (Sigma Fine Chemicals) dissolved in distilled water; control animals received the same volume of vehicle. Both controls and monosodium glutamate-treated animals were killed at the age of 6 months and male animals selected for the present study. Animals treated with monosodium glutamate
demonstrated the previously-described abnormalities, such as stunted growth, obesity and atrophy of the optic nerve 42,44. Knife lesions of the pituitary stalk were made in adult male Sprague-Dawley rats (150-200 g), as described previously35. The extent of the lesions was assessed 8 days after surgery by measuring the water intake of the animals over 3 successive 24-h periods. The mean water intake of the sham-operated controls was 36 _ 8.4 ml/kg b.wt./24 h, whereas it amounted to 278 + 56.6* ml/kg b.wt./24 h for the stalk-transected animals (n = 6* P < 0.001 Student's t-test). Visual inspection during the dissection verified that the pituitary gland was undamaged. RESULTS
Neurotensin-like immunoreactivity and [3H]neurotensin binding sites in the hypothalamus Perikarya containing neurotensin-like immunoreactivity were concentrated in the medial preoptic area, the periventricular nucleus, the paraventricular nucleus and the arcuate nucleus, with smaller numbers in the anterior and posterior hypothalamus and in the dorsomedial nucleus of the hypothalamus. No cell bodies positive for neurotensin-like immunoreactivity were observed in either the suprachiasmatic nucleus or the ventromedial hypothalamus. Nerve fibres positive for neurotensin-like immunoreactivity were observed in the same regions as the cell bodies; in addition, a dense band of nerve fibres was found in the external zone of the median eminence. Some of these findings are illustrated in Fig. 1. Note the labelling of a large number of cell bodies in the medial parvocellular part of the paraventricular nucleus with only occasional cells stained in the magnocellular portion of the nucleus (Fig. la); Fig. lb shows a higher magnification of a, demonstrating the presence of positive cell bodies and nerve fibres. Fig. lc and d illustrates the presence of cell bodies positive for neurotensin-like immunoreactivity in the supraoptic nucleus, as well as in both the arcuate and the periventricular nuclei. No staining was observed in the absence of the primary antiserum and the staining was completely abolished by preadsorption of the diluted primary antiserum with 10 ~M neurotensin. [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumu-
62
~! j ili i! i!!
ii!i i!! ili
Fig. 1. Immunohistochemical localization of neurotensin-like immunoreactivity (a-d) and autoradiographic localization of 13H]neuro tensin binding sites in the rat hypothalamus (e, f). a-d: light-field photomicrographs of the neurotensin-positive cell bodies and nerve fibres in different regions of the hypothalamus. Note the large number of neurotensin-positive cell bodies in the parvocellular part of
63 NT (1-13)
lation of silver grains o v e r the p a r a v e n t r i c u l a r nucleus, the arcuate nucleus, the periventricular nucleus and the region of the m e d i a n eminence (Fig. l e ) .
Neurotensin-like immunoreactivity and [3H]neurotensin binding sites in the neurointermediate lobe o f the pituitary gland By r a d i o i m m u n o a s s a y neurotensin-like i m m u n o reactivity was d e t e c t e d in variable amounts in the n e u r o i n t e r m e d i a t e lobe of several m a m m a l i a n species and in the posterior lobe of p o s t - m o r t e m h u m a n pituitary glands (Table I). High levels were found in the cat, rabbit and pig neurointermediate lobes, medium amounts in rat and guinea-pig tissues and low levels in both the bovine n e u r o i n t e r m e d i a t e lobe and the h u m a n posterior pituitary gland. The i m m u n o r e a c tive material present in the n e u r o i n t e r m e d i a t e lobe of the rat pituitary gland was characterized by reverse-phase H P L C and it was found to co-elute with synthetic neurotensin (Fig. 2). Lesions of the pituitary stalk r e d u c e d the n e u r o i n t e r m e d i a t e lobe content of neurotensin-like immunoreactivity by m o r e than 90% (Table II). The question of a h y p o t h a l a m i c origin o f n e u r o i n t e r m e d i a t e lobe neurotensin-like
TABLE I
Each value represents the mean + S.E.M. of the number of determinations shown within parentheses.
Rat Guinea-pig Rabbit Cat Pig Cow Human
~
3
0
2O
#z
3 2
10
o o
lo
20 30 Elution Volume ( m l )
40
50
Fig. 2. Reverse-phase HPLC of neurotensin-like immunoreactivity in rat neurointermediate pituitarygland. Fractions were assayed using the aminoterminus (O) and the carboxyterminus (V) directed neurotensin radioimmunoassays. The elution position of synthetic neurotensin (1-13) is indicated by the arrow.
immunoreactivity was further investigated by using the neurotoxin m o n o s o d i u m glutamate. The success of the lesion was controlled by staining the anterior portion of the arcuate nucleus with the p e r o x i d a s e a n t i p e r o x i d a s e for neurotensin-like immunoreac-
TABLE II
Concentration of neurotensin-like immunoreactivity in the neurointermediate lobe of the pituitary gland of various mammalian species and in the human posterior pituitary gland
Species
l
Effects of pituitary stalk transection and monosodium glutamate treatment on neurotensin-like immunoreactivity in the neurointermediate lobe of the rat pituitary gland Each value represents the mean + S.E.M. of the number of determinations shown within parentheses.
Neurotensin-like immunoreactivity (pmol/g) 32 + 35 + 154 + 349 + 55 + 6+
4.2 (12) 3.9 (4) 29.8 (4) 44.0 (5) 4.1 (4) 0.7 (5)
4 + 1.1 (4)
Neurotensin-like immunoreacitivity Controls Stalk transection
(pmol/g) 24 + 1.2 (5) 3 + 1.0" (5)
Controls Monosodium glutamate
32 + 3.0 (6) 9 + 1.9" (6)
* P < 0.001 (Student's t-test).
4.-the paraventricular nucleus in a, with a higher magnification shown in b, the neurotensin-positive cell bodies in the supraoptic nucleus in c, as well as the numerous neurotensin-positive cell bodies in the arcuate nucleus and the dense network of neurotensin-positive nerve fibres in the median eminence in d. Scale bars = 75/~m in a, c, d and 40/~m in b. e: dark-field photomicrograph of the autoradiographic localization of [3H]neurotensin binding sites in the rat hypothalamus. The tissue sections were incubated in the presence of 2 nM [3H]neurotensin. f: dark-field photomicrograph of the autoradiogram of an adjacent section to e incubated in the presence of 2 nM [3H]neurotensin and 1/~M neurotensin. Scale bars = 500/~m. Pa, paraventricular nucleus; SO, supraoptic nucleus; OX, optic chiasm; Arc, arcuate nucleus; VMH, ventromedial hypothalamus.
64
': ~< ~' ~.....~i~!i!!!!~~ ~i[,i ~i~!~~',i~i!i~!~i ~;: ~:~ii~~~ ~i~i~~~!ii,~i'~i
Fig. 3. Autoradiographic localization of [3H]neurotensin binding sites in the rat pituitary gland, a. c: dark-field photomicrographs of the autoradiograms of transverse sections through the rat pituitary gland after incubation in the presence of 2 nM [3H]neurotensin. Sections incubated in the presence of [~Hlncurotensin and 1 ~M neurotensin showed only background labelling, b, d: light-field photomicrographs of the Nissl stains of the same sections as in a and c. Note the patchy accumulation of silver grains over the intermediate lobe in a and c. Scale bars = 10(Ifan. AL. anterior lobe; IL, intermediate lobe; NL, neural lobe.
65 itary glands from 12 different animals. In the testtube binding assay [3H]neurotensin bound specifically to crude membrane fractions prepared from whole rat pituitary gland. At a radioligand concentration of 2 nM the non-specific binding amounted to 40% of the total binding and the specific binding represented 11.8 _+ 0.84 fmol/mg protein (n = 6). The question as to whether the [3H]neurotensin binding sites in the intermediate lobe of the rat pituitary gland are present on endocrine cells or on nerve fibres was investigated by receptor autoradiography performed on pituitary glands from animals taken 4 weeks after transections of the pituitary stalk. No apparent reduction in the labelling pattern of the intermediate lobe was observed (Fig. 4), indicating that the majority of [3H]neurotensin binding sites is likely to be present on endocrine cells of the intermediate lobe of the pituitary gland.
tivity and tyrosine hydroxylase-like immunoreactivity. Numerous cells in the anterior part of control arcuate nuclei were positive for neurotensin-like immunoreactivity and tyrosine hydroxylase; in contrast, in monosodium glutamate-treated animals, a very large reduction of immunoreactive cells was observed with only occasional cells stained for either neurotensinlike immunoreactivity or tyrosine hydroxylase, thereby confirming the results obtained in a recent study 29. The effect of arcuate nucleus destruction consequent to monosodium glutamate treatment was investigated by measuring the neurotensin-like immunoreactivity content of the neurointermediate lobe of control and treated animals. As shown in Table II, a significant reduction in neurotensin-like immunoreactivity was observed following monosodium glutamate treatment, indicating that a large percentage of the neurointermediate lobe neurotensinlike immunoreactivity originates from the arcuate nucleus of the hypothalamus. By receptor autoradiography, the vast majority of [3H]neurotensin binding sites was localized in the intermediate lobe of the rat pituitary gland with much smaller amounts in anterior and neural lobes (Fig. 3). In the intermediate lobe the silver grains were distributed in an heterogeneous manner with local patches of dense staining embedded in a uniform labelling of the tissue. The staining was completely abolished in the presence of 1/~M unlabelled neurotensin; identical results were obtained for the pitu-
DISCUSSION The present results on the immunohistochemical distribution of neurotensin-like immunoreactivity and the autoradiographic localization of [3H]neurotensin binding sites in the rat hypothalamus confirm and extend previous studies30.55. lmmunohistochemically, cell bodies positively stained for neurotensin-like immunoreactivity were mainly found in the medial preoptic area; the medial parvocellular portion of the paraventricular nucleus,
| Fig. 4. The effect of pituitary stalk transection on the autoradiographic localization of [3H]neurotensin binding sites in the rat pituitary gland. A: dark-field photomicrograph of the autoradiogram of a transverse section through a control rat pituitary gland after incubation in the presence of 2 nM [3H]neurotensin. Sections incubated in the presence of [3H]neurotensin and 1 pM neurotensin showed only background labelling. B: dark-field photomicrograph of the autoradiogram of a transverse section through a rat pituitary gland 4 weeks after transection of the pituitary stalk; the section was incubated in the presence of 2 nM [3H]neurotensin. Sections incubated in the presence of [3H]neurotensin and 1aM neurotensin showed only background labelling. Scale bar = 800 ~m. AL, anterior lobe; IL, intermediate lobe; NL, neural lobe. Note the presence of silver grains over the intermediate lobe in A and B.
66 the supraoptic nucleus, the arcuate nucleus and the periventricular nucleus. These results are in good agreement with the findings obtained in a previous investigation, with the exception of the supraoptic nucleus where no neurotensin-positive cell bodies were found 30. This discrepancy may be explained by the different neurotensin antiserum used or by a more effective complete blockade of axonal transport achieved in the present study. Autoradiographically, [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumulation of silver grains over the paraventricular nucleus, the arcuate nucleus, the periventricular nucleus and the median eminence region. Similar results were obtained when the distribution of neurotensinlike immunoreactivity and the localization of [3H]neurotensin binding sites were investigated in the hypothalamus of the cat (data not shown). Neurotensin-like immunoreactivity is thus present in several hypothalamic nuclei that are known to be important for the function of the anterior pituitary gland. Retrograde labelling studies have established that cells of the medial preoptic area, the periventricular nucleus, the parvocellular portion of the paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus project to the median eminence of the hypothalamus 33,54. It would be interesting to investigate which of these cell body regions contribute to the neurotensin-positive nerve fibres present in the external layer of the median eminence. At present it is largely unknown whether neurotensin-like immunoreactivity co-exists with other neurotransmitters and neuropeptides in the different hypothalamic nuclei. The only available information concerns the co-existence of neurotensin-like immunoreactivity and dopamine in the arcuate nucleus and the periventricular nucleus, and of neurotensin-like immunoreactivity and corticotropin-releasing factorlike immunoreactivity in the parvocellular portion of the paraventricular nucleus 25,27,43. The arcuate nucleus is known to contain material immunoreactive with antibodies raised against several neuropeptides, such as adrenocorticotropin and fl-endorphin, somatostatin, growth hormone-releasing hormone, thyrotropin-releasing hormone and neuropeptide y2,4,5, 15,32. A co-existence of neurotensin-like immunoreactivity with either adrenocorticotropin and/3-endorphin or neuropeptide Y appears unlikely, since the
latter are known not to co-exist with dopamine 4,15. The finding that all cells of the supraoptic nucleus are neurophysin-positive 46 implies the co-existence of neurotensin-like immunoreactivity with either oxytocin or vasopressin. In addition, material immunoreactive with antibodies raised against cholecystokinin26_33, Met-enkephalin, Leu-enkephalin, dynorphin1_17 and glucagon has been found in the supraoptic nucleus of various mammalian speciesL, 47,52,53. In the paraventricular nucleus, a co-existence of neurotensin-like immunoreactivity with oxytocin or vasopressin seems less likely, since neurotensin-positive cells are mostly found in the medial parvocellular nucleus and oxytocin and vasopressin mainly in cells of the lateral magnocellular nucleus. Neurotensin-like immunoreactivity may, however, coexist with somatostatin, Met-enkephalin, Leu-enkephalin, substance P and peptide histidine-isoleucine which are all found in the parvocellular portion of the paraventricular nucleus2,6,11, 24,32,36.
Neurotensin-like immunoreactivity was found by radioimmunoassay in the neurointermediate lobe of the pituitary gland of various mammalian species and in the human posterior pituitary gland. In the rat, transection of the pituitary stalk resulted in a large reduction in the levels of neurotensin-tike immunoreactivity, indicating that the immunoreactive material originated in the hypothalamus. Since the arcuate nucleus is known to project to the neurointermediate lobe of the pituitary gland 3 and since a large number of neurotensin-positive cells were present in that nucleus, the effects of monosodium glutamate on the levels of neurotensin-like immunoreactivity in the rat pituitary gland neurointermediate lobe were investigated. Monosodium glutamate has been shown to produce a selective, although incomplete lesion of the arcuate nucleus 44. It was found that neurotensinlike immunoreactivity was greatly reduced following monosodium glutamate treatment, indicating the existence of a neurotensinergic pathway from the arcuate nucleus to the neurointermediate lobe of the pituitary gland. Monosodium glutamate treatment, however, did not result in a complete disappearance of neurotensin-like immunoreactivity and it is at present not known whether this was due to the incompleteness of the lesion or whether hypothalamic nuclei projecting to the posterior lobe of the pituitary gland, such as the paraventricular and supraoptic nu-
67 clei45,54, contribute neurotensin-positive nerve fibres to the neurointermediate lobe of the rat pituitary gland. [3H]neurotensin binding sites were found to be concentrated in the intermediate lobe of the rat pituitary gland by autoradiography, with smaller amounts in both anterior and neural lobes. Similar results were obtained in the cat pituitary gland (data not shown). In the rat, the [3H]neurotensin binding sites did not appear to be affected by pituitary stalk transection, indicating that they are localized on cells of the intermediate lobe. Using the test-tube binding assay for [3H]neurotensin, low levels of specific binding sites were found in whole rat pituitary glands. However, considering that the intermediate lobe comprises less than 5% of the total pituitary gland, these results indicate that the local concentration of [3H]neurotensin binding sites is comparable with that found in the central nervous system 2~. Interestingly, the staining pattern for [3H]neurotensin was similar
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to the one obtained for dopamine receptors when [3H]spiperone was used as the radioligand34. However, the present findings argue against a localization of the majority of [3H]neurotensin binding sites on dopaminergic nerve fibres in the intermediate lobe and they suggest that both types of receptors are present on endocrine cells of the intermediate lobe. Hopefully, future experiments will investigate the effects of neurotensin on the synthesis and the release of intermediate lobe hormones. In conclusion, the present results indicate that, in addition to being a modulator of anterior pituitary function, neurotensin may also be an important regulator of the neurointermediate lobe of the pituitary gland. ACKNOWLEDGEMENTS We thank Mrs. M. Wynn for her great help in the preparation of the present manuscript.
9 Carraway, R., Kitabgi, P. and Leeman, S.E., The amino acid sequences of radioimmunoassayable neurotensin from bovine intestine, J. Biol. Chem., 253 (1978) 7996-7998. 10 Cooper, P.E., Fernstrom, M.H., Rorstad, O.P., Leeman, S.E. and Martin, J.B., The regional distribution of somatostatin, substance P and neurotensin in human brain, Brain Research, 218 (1981) 219-232. 11 Dierickx, K. and Vandesande, F., Immunocytochemical localization of somatostatin-containing neurons in the rat hypothalamus, Cell Tissue Res., 201 (1979) 349-359. 12 Emson, P.C., Goedert, M., Horsfield, P., Rioux, F. and St Pierre, S., The regional distribution and chromatographic characterisation of neurotensin-like immunoreactivity in the rat central nervous system, J. Neurochem., 38 (1982) 992-999. 13 Emson, P.C., Horsfield, P., Goedert, M., Rossor, M.N. and Hawkes, C.H., Neurotensin in human brain: regional distribution and effects of neurological, Brain Research, in press. 14 Enjalbert, A., Arancibia, S., Priam, M., Bluet-Pajot, M.T. and Kordon, C., Neurotensin stimulation of prolactin secretion in vitro, Neuroendocrinology, 34 (1982) 95-98. 15 Everitt, B.J., H6kfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M., Differential co-existence of neuropeptide Y-like immunoreactivity with catecholamines in the central nervous system of the rat, Neuroscience, 11 (1984) 443-462. 16 Goedert, M., Lightman, S.L., Nagy, J.I., Marley, P.D. and Emson, P.C., Neurotensin in the rat anterior pituitary gland, Nature (London), 298 (1982) 163-165. 17 Goedert, M. and Emson, P.C., The regional distribution of neurotensin-like immunoreactivity in central and peripheral tissues of the cat, Brain Research, 272 (1983) 291-297. 18 Goedert, M., Mantyh, P.W., Hunt, S.P. and Emson, P.C., Mosaic distribution of neurotensin-like immunoreactivity
68 in the cat striatum, Brain Research, 274 (1983) 176-179. 19 Goedert, M., Mantyh, P.W., Emson, P.C. and Hunt, S.P., Inverse relationship between neurotensin receptors and neurotensin-like immunoreactivity in the cat striatum, Nature (London), 307 (1984) 543-546. 20 Goedert, M., Lightman, S.L. and Emson, P.C., Neurotensin in the rat anterior pituitary gland: effects of endocrinological manipulations, Brain Research, 299 (1984) 160-163. 21 Goedert, M., Pittaway, K., Williams, B.J. and Emson, P.C., Specific binding of tritiated neurotensin in rat brain: characterisation and regional distribution, Brain Research, 304 (1984) 7t-81• 22 Goedert, M., Hunter, J.C. and Ninkovic, M., Evidence for neurotensin as a non-adrenergic, non-cholinergic neurotransmitter in guinea pig ileum, Nature (London). 311 (1984) 59-62• 23 Hammer, R.A., Leeman, S.E., Carraway, R. and Williams, R.H., Isolation of human intestinal neurotensin, J. Biol. Chem•, 255 (1980) 2476-2480. 24 HOkfelt, T., Fahrenkrug, J., Tatemoto, K., Mutt, V., Werner, S., Hulting, A.L., Terenius, L. and Chang, K.J., The PHI/corticotropin-releasing factor/enkephalin immunoreactive hypothalamic neuron: possible morphological basis for integrated control of prolactin, corticotropin, and growth hormone secretion, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 895-898. 25 H6kfelt, T., Everitt, B.J., Theodorsson-Norheim, E. and Goldstein, M., Occurrence of neurotensin-like immunoreactivity in subpopulations of hypothalamic, mesencephalic and medullary catecholamine neurons, J. Comp. Neurol., 222 (1984) 543-560. 26 Hunt, S.P., Kelly, J.S., Emson, P.C., Kimmel, J.R., Miller, R.J. and Wu, J.Y., An immunohistochemical study of neuronal populations containing neuropeptides and y-aminobutyric acid within the superficial layers of the dorsal horn, Neuroscience, 6 (1981) 1883-1889• 27 Ibata, Y., Fukui, K., Okamura, H., Kawakami, T., Tanaka, M., Obata, H.L., Tsuto, T., Terubayashi, H., Yanaihara, C. and Yanaihara, N., Co-existence of dopamine and neurotensin in hypothalamic arcuate and periventricular neurons, Brain Research, 169 (1983) 177-179. 28 Ibata, Y., Kawakami, F., Fukui, K., Obata-Tsuto, H.L., Tanaka, M., Kubo, T., Okamura, H., Morimoto, N., Yanaihara, C. and Yanaihara, N., Light and electron microscopic immunocytochemistry of neurotensin-like immunoreactivity neurons in the rat hypothalamus, Brain Research, 302 (1984) 221-230. 29 Jennes, L., Stumpf, W.E., Bissette, G. and Nemeroff, C.B., Monosodium glutamate lesions in rat hypothalamus studies by immunohistochemistry for gonadotropin releasing hormone, neurotensin, tyrosine hydroxylase, and glutamic acid decarboxylase and by autoradiography for [3H]estradiol, Brain Research, 308 (1984) 245-253. 30 Kahn, D., Abrams, G.M., Zimmerman, E.A., Carraway, R. and Leeman, S.E., Neurotensin neurons in the rat hypothalamus: An immunocytochemical study, Endocrinology, 107 (1980) 47-53. 31 Kobayashi, R.M., Brown, M. and Vale, W., Regional distribution of neurotensin and somatostatin in rat brain, Brain Research, 126 (1977) 584-588. 32 Lechan, R.M. and Jackson, I.M.D., Immunohistochemieal localization of thyrotropin-releasing hormone in the rat hypothalamus and pituitary, Endocrinology, l l l (1982) 55-65•
33 Lechan, R.M., Nestler, J.L. and Jacobson, S., The tuberoinfundibular system of the rat as demonstrated by immunohistochemicat localization of retrogradely transported wheat germ agglutinin from the median eminence. Brain Research. 145 (1982) 1-15. 34 Lightman, S.L., Ninkovic, M. and Hunt, S.P., Localization of [3H]spiperone binding sites in the intermediate lobe of the rat pituitary gland, Neurosci. Lett., 32 (1982) 99-102. 35 Lightman, S.L., Ninkovic, M., Hunt, S.P. and Iversen, L.L., Evidence for opiate receptors on pituicytes. Nature (London), 305 (1983) 235-237. 36 Ljungdahl, A., H6kfelt. T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-944. 37 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193 (1951) 265-275. 38 McLean, I.W. and Nakane, P.D., Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. 39 Maeda, K. and Frohman, L.A., Dissociation of systemic and central effects of neurotensin on the secretion of growth hormone, prolactin, and thyrotropin, Endocrinology, 103 (1978) 1903-1908. 40 Manberg, P.J., Youngblood, W.W., Nemeroff, C.B., Rossor, M.N., Iversen, L.L., Prange, A.J. and Kizer, J.S., Regional distribution of neurotensin in human brain. J. Neurochem., 38 (1982) 1777-1780. 41 Nemeroff, C.B., Luttinger, D. and Prange, A.J., Neurotensin and bombesin. In L.L. Iversen, S.D. Iversen and S.H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 16, Plenum Press, New York 1983, pp. 363-466• 42 Olney, J.W., Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate, Science, 164 (1969) 719-721. 43 Sawchenko, P.E., Swanson, L.W. and Vale, W.W., Corticotropin-releasing factor: co-expression within distinct subsets of oxytocin-, vasopressin-, and neurotensin-immunoreactive neurons in the hypothalamus of the male rat, J. Neurosci., 4 (1984) 1118-1129. 44 Seress, L., Divergent effects of acute and chronic monosodim k-glutamate treatment on the anterior and posterior parts of the arcuate nucleus, Neuroscience, 7 (1982) 2207- 2216. 45 Sherlock, D.A., Field, P.M. and Raisman, G., Retrograde transport of horseradish peroxidase in the magnocellular neurosecretory system of the rat, Brain Research, 88 (1975) 403-414. 46 Sokol, H.W., Zimmerman, E.A., Sawyer, W.H. and Robinson, A.G., The hypothalamo-neurohypophyseal system of the rat: Localization and quantitation of neurophysin by light microscopic immunocytochemistry in normal rat and in Brattleboro rats deficient in vasopressin and a neurophysin, Endocrinology, 98 (1976) 1176-1188. 47 Tager, H., Hohenboken, M., Markese, J. and Dinerstein, R.J., Identification and localization of glucagon-related peptides in rat brain, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 6229-6233. 48 Uhl, G.R. and Snyder, S.H., Regional and subcellular distribution of brain neurotensin, Life Sci., 19 (1976) 1827-1232. 49 Uhl, G.R., Kuhar, M. and Snyder, S.H., Neurotensin: Ira-
69 munohistochemical localization in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 1059-1063. 50 Vijayan, E. and McCann, S.M., In vivo and in vitro effects of substance P and neurotensin on gonadotropin and prolactin release, Endocrinology,. 105 (1979) 64-68. 51 Vijayan, E. and McCann, S.M., Effect of substance P and neurotensin on growth hormone and thyrotropin release in vivo and in vitro, Life Sci., 26 (1980) 321-327. 52 Watkins, W.B., Presence of adrenocorticotropin and fl-endorphin immunoreactivities in the magnocellular neurosecretory system of the rat hypothalamus, Cell Tissue Res.,
207 (1980) 65-80. 53 Watson, S.J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, E.A., Nilaver, G. and Van Wiersma Greidanus, T.B., Dynorphin and vasopressin: common localization in magnocellular neurons, Science, 216 (1983) 85-87. 54 Wiegand, S.J. and Price, J.L., Cells of origin of the afferent fibers to the median eminence in the rat, I. comp. Neurol., 192 (1980) 1-19. 55 Young, W.S. and Kuhar, M.J., Neurotensin receptor localization by light microscopic autoradiography in rat brain, Brain Research, 206 (1981) 273-285.
59
BRE 11174
Neurotensin-Like Immunoreactivity and Neurotensin Receptors in the Rat Hypothalamus and in the Neurointermediate Lobe of the Pituitary Gland M. GOEDERT 1, S.L. LIGHTMAN2, P.W. MANTYH1, S.P. HUNT 1and P.C. EMSON 1 1MRC Neurochemical Pharmacology Unit Medical Research Council Centre, Medical School Cambridge CB2 2QH and 2Department of Medicine, Westminster Hospital London (U. K. ) (Accepted February 19th, 1985) Key words: neurotensin-like immunoreactivity-- neurotensin receptor-- hypothalamus - - neurointermediate pituitary gland-- monosodium glutamate - - pituitary stalk transection
In the rat hypothalamus, cell bodies containing neurotensin-like immunoreactivity were mainly found in the medial preoptic area, the periventrieular nucleus, the paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumulation of silver grains over the paraventricular nucleus, the arcuate nucleus and the median eminence region. By radioimmunoassay neurotensin-like immunoreactivity was also found in the neurointermediate lobe of the pituitary gland of various mammalian species and in human postmortem posterior pituitary glands. In the rat studies involving pituitary stalk transections and the neurotoxin monosodium glutamate indicated the presence of a neurotensinergic pathway from the arcuate nucleus to the neurointermediate lobe of the pituitary gland. [3H]neurotensin binding sites were found to be concentrated over the intermediate lobe of the pituitary gland and their presence was not affected by pituitary stalk transection, indicating their localization on endocrine cells of the intermediate lobe of the pituitary gland.
INTRODUCTION Neurotensin is a 13 amino acid peptide first isolated from bovine hypothalamus and later from both bovine and human small intestine7,9, 23. It is widely distributed throughout the central nervous system of various mammalian species, where it probably functions as a neurotransmitter 41. In all species examined to date, the highest concentration of radioimmunoassayable neurotensin-like immunoreactivity is found in the hypothalamusS,lOA2,13A7,3t,40,48. By immunohistochemistry neurotensin-like immunoreactivity has been found in cell bodies in several hypothalamic nuclei, including the parvocellular portion of the paraventricular nucleus and the arcuate nucleus25,27,28,30; it has been shown to co-exist with corticotropin-releasing factor-like immunoreactivity in the paraventricular nucleus and with tyrosine hydroxylase-like immunoreactivity in the arcuate nucleus25,27, 43. In addition, neurotensin-like immunoreactivity is present in
high concentrations in nerve fibres in the external layer of the median eminence25,28,30. By electron microscopy, the latter have been shown to contact portal blood vessels, suggesting that neurotensin may function as a hypothalamic releasing-hormone; this is supported by the finding that neurotensin influences the release of several anterior pituitary hormones, both at the hypothalamic and the pituitary levels 14,39,50,51. High levels of neurotensin-like immunoreactivity are also found in the pituitary gland, where the immunoreactive material is found in cells in the anterior lobe and in nerve fibres in the neurointermediate lobe16,20,49. To date, the function of neurotensin-like immunoreactivity in the pituitary gland is unknown. In the present communication, we have investigated the immunohistochemical distribution of neurotensin-like immunoreactivity and the autoradiographic distribution of specific [3H]neurotensin binding sites in both the rat hypothalamus and the neu-
Correspondence: M. Goedert, MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Cambridge CB2 2QH, U.K. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
60 rointermediate lobe of the pituitary gland. MATERIALS AND METHODS
Extraction of tissues and radioimmunoassay Rat, guinea-pig, rabbit and cat neurointermediate pituitary glands were dissected and immediately frozen on dry-ice; pig and bovine tissues were obtained from a local abattoir and frozen within 1 h following the death of the animals. Postmortem human pituitary gland posterior lobes were obtained from patients who had died of peripheral vascular disorders (age group 69-79 years). At autopsy there wasno evidence for either endocrinological or neurological diseases. The mean-time elapsed between death and tissue removal amounted to 28 h (18-48 h). The tissues were weighed and extracted using 10 vol. boiling 1 M acetic acid. They were homogenized in glass-teflon homogenizers and left at 4 °C for 20 min in order to ensure complete extraction. They were then centrifuged at 2000 g for 10 min and the supernatants freeze-dried. The lyophilized tissues were reconstituted in assay buffer, centrifuged at 2000 g for 10 min in order to remove insoluble material and assayed in duplicate for neurotensin-like immunoreactivity, using a previously described antiserum selective for the aminoterminus of synthetic neurotensinlL
heparin followed by 400 ml paraformaldehyde/lysine/sodium periodate 3s. Forty-/~m sections were cut on a freezing microtome and treated with 1% hydrogen peroxide for 10 min in order to reduce endogenous peroxidase activity. The antibodies were diluted with 100 mM sodium phosphate buffer (pH 7.4) containing 0.1% Triton X-100 and 1% sheep serum. Free floating sections were incubated with the previously described primary antiseralS. 26 (diluted 1:400 for neurotensin and 1:500 for tyrosine hydroxylase) for 24 h at 4 °C. Sections were then washed in 100 mM sodium phosphate buffer for 30 min and incubated with sheep anti-rabbit antiserum (Miles), diluted 1:20, for 1 h. Following this, the sections were washed in 100 mM sodium phosphate buffer for 30 min, incubated with rabbit peroxidase anti-peroxidase conjugate (Miles) at a dilution of 1:100 for t h and washed again in 100 mM sodium phosphate buffer for 30 min. The tissue then was incubated for 5-10 min in 100 mM sodium phosphate buffer contaning 0.09% 3,3-diaminobenzidine and 0.005% hydrogen peroxide and subsequently washed in 100 mM sodium phosphate buffer. In control experiments, the primary antisera were omitted or in the case of the neurotensin antiserum, preincubated for 2 h at room temperature with 10/zM neurotensin.
Receptor autoradiography High-performance liquid chromatography Lyophilyzed rat neurointermediate pituitary gland tissue extracts were reconstituted in 0.1 M acetic acid and subjected to high-performance liquid chromatography (HPLC) on reverse phase by using a/~-Bondapak C18 column and a linear 5-30% acetonitrile gradient in 10 mM ammonium acetate, pH 4.0, as the aqueous phase. One ml fractions were collected and their neurotensin-like immunoreactivity content determined by using the previously described aminoand carboxyterminus directed antisera 12.
Immunohistochemistry The immunohistochemical distribution of neurotensin-like immunoreactivity was investigated in the rat hypothalamus. In order to enhance neuronal cell body staining adult male Sprague-Dawley rats (150200 g) received an intraventricular injection of 100/~g colchicine 24 h prior to intracardial perfusion with 100 ml physiological saline containing 800 U/litre
Adult male Sprague-Dawley rats (200-250 g) were stunned, the hypothalamus and the pituitary gland dissected, frozen and sectioned in the transverse plane at 13/~m using a cryostat. The sections were pressed onto gelatin-coated slides, melted in place and dried overnight at 4 °C in air-tight boxes with desiccant. The thawed sections were incubated for 10 min at 25 °C in 2 nM [3,11-tyrosyl-3,5-3H]neurotensin (56.3 Ci/mmol, New England Nuclear Corporation) in 50 mM Tris-HCl, pH 7.4, containing 0.1% bovine serum albumin (radioimmunoassay grade, Sigma Fine Chemicals), 40 mg/litre bacitracin (Sigma Fine Chemicals) and 1 mM EDTA. Non-specific binding was defined in adjacent sections as binding in the presence of 1/~M unlabelled neurotensin (Cambridge Research Biochemicals). Following the incubation the sections were washed twice in cold buffer (2 min each time), dipped into cold water and dried under a stream of cold air. They were then apposed to emulsion-coated coverslips or a tritium-sen-
61 sitive film (3H-Ultrofilm, LKB Laboratories)19,22 and kept at -20 °C for 2 months. The coverslips and the tritium-sensitive film were developed in safe-light conditions using Kodak D 19 developer and the tissue sections put into Carnoy's fixative, Nissl stained and mounted using Depex. Dark-field photomicrographs were taken of the silver grains and light-field photomicrographs of the Nissl stain.
Test-tube binding assay The test-tube binding assay for [3H]neurotensin was performed essentially as described before 21. Briefly, the pituitary glands from adult male Sprague-Dawley rats (150-200 g) were dissected, weighed and homogenized in 10 vol. (w/v) of cold 50 mM Tris-HCl, pH 7.4, with a Polytron at setting 7 for 15 s. After 20 min centrifugation at 50,000 g the pellet was resuspended in 10 vol. 50 mM Tris-HCI, incubated at 37 °C for 30 min and centrifuged again. The washed pellet was resuspended in 50 mM Tris-HCl, pH 7.4, containing 0.1% bovine serum albumin, 40 mg/litre bacitracin and 1 mM E D T A to yield a concentration of 10 mg tissue/ml buffer. One ml of homogenate was incubated in the presence of [3H]neurotensin at 25 °C for 10 min. At the end of the incubation period the tubes were transferred to ice and filtered immediately under reduced pressure through GF/B glass fibre filters (Whatman) pretreated with 0.2% polyethylenimine (Sigma Fine Chemicals) in water. Each of the filters was washed 4 times with 5 ml cold buffer and the radioactivity determined by liquid scintillation spectrometry. Non-specific binding was defined as binding in the presence of 1 ,uM neurotensin, and specific binding was obtained by subtracting the non-specific from the total binding. Proteins were determined according to Lowry et al. 37 using bovine serum albumin as the standard. Monosodium glutamate treatment and pituitary stalk transection Neonatal Sprague-Dawley rats were injected for 10 days on alternate days wit h 4 mg/kg monosodium glutamate (Sigma Fine Chemicals) dissolved in distilled water; control animals received the same volume of vehicle. Both controls and monosodium glutamate-treated animals were killed at the age of 6 months and male animals selected for the present study. Animals treated with monosodium glutamate
demonstrated the previously-described abnormalities, such as stunted growth, obesity and atrophy of the optic nerve 42,44. Knife lesions of the pituitary stalk were made in adult male Sprague-Dawley rats (150-200 g), as described previously35. The extent of the lesions was assessed 8 days after surgery by measuring the water intake of the animals over 3 successive 24-h periods. The mean water intake of the sham-operated controls was 36 _ 8.4 ml/kg b.wt./24 h, whereas it amounted to 278 + 56.6* ml/kg b.wt./24 h for the stalk-transected animals (n = 6* P < 0.001 Student's t-test). Visual inspection during the dissection verified that the pituitary gland was undamaged. RESULTS
Neurotensin-like immunoreactivity and [3H]neurotensin binding sites in the hypothalamus Perikarya containing neurotensin-like immunoreactivity were concentrated in the medial preoptic area, the periventricular nucleus, the paraventricular nucleus and the arcuate nucleus, with smaller numbers in the anterior and posterior hypothalamus and in the dorsomedial nucleus of the hypothalamus. No cell bodies positive for neurotensin-like immunoreactivity were observed in either the suprachiasmatic nucleus or the ventromedial hypothalamus. Nerve fibres positive for neurotensin-like immunoreactivity were observed in the same regions as the cell bodies; in addition, a dense band of nerve fibres was found in the external zone of the median eminence. Some of these findings are illustrated in Fig. 1. Note the labelling of a large number of cell bodies in the medial parvocellular part of the paraventricular nucleus with only occasional cells stained in the magnocellular portion of the nucleus (Fig. la); Fig. lb shows a higher magnification of a, demonstrating the presence of positive cell bodies and nerve fibres. Fig. lc and d illustrates the presence of cell bodies positive for neurotensin-like immunoreactivity in the supraoptic nucleus, as well as in both the arcuate and the periventricular nuclei. No staining was observed in the absence of the primary antiserum and the staining was completely abolished by preadsorption of the diluted primary antiserum with 10 ~M neurotensin. [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumu-
62
~! j ili i! i!!
ii!i i!! ili
Fig. 1. Immunohistochemical localization of neurotensin-like immunoreactivity (a-d) and autoradiographic localization of 13H]neuro tensin binding sites in the rat hypothalamus (e, f). a-d: light-field photomicrographs of the neurotensin-positive cell bodies and nerve fibres in different regions of the hypothalamus. Note the large number of neurotensin-positive cell bodies in the parvocellular part of
63 NT (1-13)
lation of silver grains o v e r the p a r a v e n t r i c u l a r nucleus, the arcuate nucleus, the periventricular nucleus and the region of the m e d i a n eminence (Fig. l e ) .
Neurotensin-like immunoreactivity and [3H]neurotensin binding sites in the neurointermediate lobe o f the pituitary gland By r a d i o i m m u n o a s s a y neurotensin-like i m m u n o reactivity was d e t e c t e d in variable amounts in the n e u r o i n t e r m e d i a t e lobe of several m a m m a l i a n species and in the posterior lobe of p o s t - m o r t e m h u m a n pituitary glands (Table I). High levels were found in the cat, rabbit and pig neurointermediate lobes, medium amounts in rat and guinea-pig tissues and low levels in both the bovine n e u r o i n t e r m e d i a t e lobe and the h u m a n posterior pituitary gland. The i m m u n o r e a c tive material present in the n e u r o i n t e r m e d i a t e lobe of the rat pituitary gland was characterized by reverse-phase H P L C and it was found to co-elute with synthetic neurotensin (Fig. 2). Lesions of the pituitary stalk r e d u c e d the n e u r o i n t e r m e d i a t e lobe content of neurotensin-like immunoreactivity by m o r e than 90% (Table II). The question of a h y p o t h a l a m i c origin o f n e u r o i n t e r m e d i a t e lobe neurotensin-like
TABLE I
Each value represents the mean + S.E.M. of the number of determinations shown within parentheses.
Rat Guinea-pig Rabbit Cat Pig Cow Human
~
3
0
2O
#z
3 2
10
o o
lo
20 30 Elution Volume ( m l )
40
50
Fig. 2. Reverse-phase HPLC of neurotensin-like immunoreactivity in rat neurointermediate pituitarygland. Fractions were assayed using the aminoterminus (O) and the carboxyterminus (V) directed neurotensin radioimmunoassays. The elution position of synthetic neurotensin (1-13) is indicated by the arrow.
immunoreactivity was further investigated by using the neurotoxin m o n o s o d i u m glutamate. The success of the lesion was controlled by staining the anterior portion of the arcuate nucleus with the p e r o x i d a s e a n t i p e r o x i d a s e for neurotensin-like immunoreac-
TABLE II
Concentration of neurotensin-like immunoreactivity in the neurointermediate lobe of the pituitary gland of various mammalian species and in the human posterior pituitary gland
Species
l
Effects of pituitary stalk transection and monosodium glutamate treatment on neurotensin-like immunoreactivity in the neurointermediate lobe of the rat pituitary gland Each value represents the mean + S.E.M. of the number of determinations shown within parentheses.
Neurotensin-like immunoreactivity (pmol/g) 32 + 35 + 154 + 349 + 55 + 6+
4.2 (12) 3.9 (4) 29.8 (4) 44.0 (5) 4.1 (4) 0.7 (5)
4 + 1.1 (4)
Neurotensin-like immunoreacitivity Controls Stalk transection
(pmol/g) 24 + 1.2 (5) 3 + 1.0" (5)
Controls Monosodium glutamate
32 + 3.0 (6) 9 + 1.9" (6)
* P < 0.001 (Student's t-test).
4.-the paraventricular nucleus in a, with a higher magnification shown in b, the neurotensin-positive cell bodies in the supraoptic nucleus in c, as well as the numerous neurotensin-positive cell bodies in the arcuate nucleus and the dense network of neurotensin-positive nerve fibres in the median eminence in d. Scale bars = 75/~m in a, c, d and 40/~m in b. e: dark-field photomicrograph of the autoradiographic localization of [3H]neurotensin binding sites in the rat hypothalamus. The tissue sections were incubated in the presence of 2 nM [3H]neurotensin. f: dark-field photomicrograph of the autoradiogram of an adjacent section to e incubated in the presence of 2 nM [3H]neurotensin and 1/~M neurotensin. Scale bars = 500/~m. Pa, paraventricular nucleus; SO, supraoptic nucleus; OX, optic chiasm; Arc, arcuate nucleus; VMH, ventromedial hypothalamus.
64
': ~< ~' ~.....~i~!i!!!!~~ ~i[,i ~i~!~~',i~i!i~!~i ~;: ~:~ii~~~ ~i~i~~~!ii,~i'~i
Fig. 3. Autoradiographic localization of [3H]neurotensin binding sites in the rat pituitary gland, a. c: dark-field photomicrographs of the autoradiograms of transverse sections through the rat pituitary gland after incubation in the presence of 2 nM [3H]neurotensin. Sections incubated in the presence of [~Hlncurotensin and 1 ~M neurotensin showed only background labelling, b, d: light-field photomicrographs of the Nissl stains of the same sections as in a and c. Note the patchy accumulation of silver grains over the intermediate lobe in a and c. Scale bars = 10(Ifan. AL. anterior lobe; IL, intermediate lobe; NL, neural lobe.
65 itary glands from 12 different animals. In the testtube binding assay [3H]neurotensin bound specifically to crude membrane fractions prepared from whole rat pituitary gland. At a radioligand concentration of 2 nM the non-specific binding amounted to 40% of the total binding and the specific binding represented 11.8 _+ 0.84 fmol/mg protein (n = 6). The question as to whether the [3H]neurotensin binding sites in the intermediate lobe of the rat pituitary gland are present on endocrine cells or on nerve fibres was investigated by receptor autoradiography performed on pituitary glands from animals taken 4 weeks after transections of the pituitary stalk. No apparent reduction in the labelling pattern of the intermediate lobe was observed (Fig. 4), indicating that the majority of [3H]neurotensin binding sites is likely to be present on endocrine cells of the intermediate lobe of the pituitary gland.
tivity and tyrosine hydroxylase-like immunoreactivity. Numerous cells in the anterior part of control arcuate nuclei were positive for neurotensin-like immunoreactivity and tyrosine hydroxylase; in contrast, in monosodium glutamate-treated animals, a very large reduction of immunoreactive cells was observed with only occasional cells stained for either neurotensinlike immunoreactivity or tyrosine hydroxylase, thereby confirming the results obtained in a recent study 29. The effect of arcuate nucleus destruction consequent to monosodium glutamate treatment was investigated by measuring the neurotensin-like immunoreactivity content of the neurointermediate lobe of control and treated animals. As shown in Table II, a significant reduction in neurotensin-like immunoreactivity was observed following monosodium glutamate treatment, indicating that a large percentage of the neurointermediate lobe neurotensinlike immunoreactivity originates from the arcuate nucleus of the hypothalamus. By receptor autoradiography, the vast majority of [3H]neurotensin binding sites was localized in the intermediate lobe of the rat pituitary gland with much smaller amounts in anterior and neural lobes (Fig. 3). In the intermediate lobe the silver grains were distributed in an heterogeneous manner with local patches of dense staining embedded in a uniform labelling of the tissue. The staining was completely abolished in the presence of 1/~M unlabelled neurotensin; identical results were obtained for the pitu-
DISCUSSION The present results on the immunohistochemical distribution of neurotensin-like immunoreactivity and the autoradiographic localization of [3H]neurotensin binding sites in the rat hypothalamus confirm and extend previous studies30.55. lmmunohistochemically, cell bodies positively stained for neurotensin-like immunoreactivity were mainly found in the medial preoptic area; the medial parvocellular portion of the paraventricular nucleus,
| Fig. 4. The effect of pituitary stalk transection on the autoradiographic localization of [3H]neurotensin binding sites in the rat pituitary gland. A: dark-field photomicrograph of the autoradiogram of a transverse section through a control rat pituitary gland after incubation in the presence of 2 nM [3H]neurotensin. Sections incubated in the presence of [3H]neurotensin and 1 pM neurotensin showed only background labelling. B: dark-field photomicrograph of the autoradiogram of a transverse section through a rat pituitary gland 4 weeks after transection of the pituitary stalk; the section was incubated in the presence of 2 nM [3H]neurotensin. Sections incubated in the presence of [3H]neurotensin and 1aM neurotensin showed only background labelling. Scale bar = 800 ~m. AL, anterior lobe; IL, intermediate lobe; NL, neural lobe. Note the presence of silver grains over the intermediate lobe in A and B.
66 the supraoptic nucleus, the arcuate nucleus and the periventricular nucleus. These results are in good agreement with the findings obtained in a previous investigation, with the exception of the supraoptic nucleus where no neurotensin-positive cell bodies were found 30. This discrepancy may be explained by the different neurotensin antiserum used or by a more effective complete blockade of axonal transport achieved in the present study. Autoradiographically, [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumulation of silver grains over the paraventricular nucleus, the arcuate nucleus, the periventricular nucleus and the median eminence region. Similar results were obtained when the distribution of neurotensinlike immunoreactivity and the localization of [3H]neurotensin binding sites were investigated in the hypothalamus of the cat (data not shown). Neurotensin-like immunoreactivity is thus present in several hypothalamic nuclei that are known to be important for the function of the anterior pituitary gland. Retrograde labelling studies have established that cells of the medial preoptic area, the periventricular nucleus, the parvocellular portion of the paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus project to the median eminence of the hypothalamus 33,54. It would be interesting to investigate which of these cell body regions contribute to the neurotensin-positive nerve fibres present in the external layer of the median eminence. At present it is largely unknown whether neurotensin-like immunoreactivity co-exists with other neurotransmitters and neuropeptides in the different hypothalamic nuclei. The only available information concerns the co-existence of neurotensin-like immunoreactivity and dopamine in the arcuate nucleus and the periventricular nucleus, and of neurotensin-like immunoreactivity and corticotropin-releasing factorlike immunoreactivity in the parvocellular portion of the paraventricular nucleus 25,27,43. The arcuate nucleus is known to contain material immunoreactive with antibodies raised against several neuropeptides, such as adrenocorticotropin and fl-endorphin, somatostatin, growth hormone-releasing hormone, thyrotropin-releasing hormone and neuropeptide y2,4,5, 15,32. A co-existence of neurotensin-like immunoreactivity with either adrenocorticotropin and/3-endorphin or neuropeptide Y appears unlikely, since the
latter are known not to co-exist with dopamine 4,15. The finding that all cells of the supraoptic nucleus are neurophysin-positive 46 implies the co-existence of neurotensin-like immunoreactivity with either oxytocin or vasopressin. In addition, material immunoreactive with antibodies raised against cholecystokinin26_33, Met-enkephalin, Leu-enkephalin, dynorphin1_17 and glucagon has been found in the supraoptic nucleus of various mammalian speciesL, 47,52,53. In the paraventricular nucleus, a co-existence of neurotensin-like immunoreactivity with oxytocin or vasopressin seems less likely, since neurotensin-positive cells are mostly found in the medial parvocellular nucleus and oxytocin and vasopressin mainly in cells of the lateral magnocellular nucleus. Neurotensin-like immunoreactivity may, however, coexist with somatostatin, Met-enkephalin, Leu-enkephalin, substance P and peptide histidine-isoleucine which are all found in the parvocellular portion of the paraventricular nucleus2,6,11, 24,32,36.
Neurotensin-like immunoreactivity was found by radioimmunoassay in the neurointermediate lobe of the pituitary gland of various mammalian species and in the human posterior pituitary gland. In the rat, transection of the pituitary stalk resulted in a large reduction in the levels of neurotensin-tike immunoreactivity, indicating that the immunoreactive material originated in the hypothalamus. Since the arcuate nucleus is known to project to the neurointermediate lobe of the pituitary gland 3 and since a large number of neurotensin-positive cells were present in that nucleus, the effects of monosodium glutamate on the levels of neurotensin-like immunoreactivity in the rat pituitary gland neurointermediate lobe were investigated. Monosodium glutamate has been shown to produce a selective, although incomplete lesion of the arcuate nucleus 44. It was found that neurotensinlike immunoreactivity was greatly reduced following monosodium glutamate treatment, indicating the existence of a neurotensinergic pathway from the arcuate nucleus to the neurointermediate lobe of the pituitary gland. Monosodium glutamate treatment, however, did not result in a complete disappearance of neurotensin-like immunoreactivity and it is at present not known whether this was due to the incompleteness of the lesion or whether hypothalamic nuclei projecting to the posterior lobe of the pituitary gland, such as the paraventricular and supraoptic nu-
67 clei45,54, contribute neurotensin-positive nerve fibres to the neurointermediate lobe of the rat pituitary gland. [3H]neurotensin binding sites were found to be concentrated in the intermediate lobe of the rat pituitary gland by autoradiography, with smaller amounts in both anterior and neural lobes. Similar results were obtained in the cat pituitary gland (data not shown). In the rat, the [3H]neurotensin binding sites did not appear to be affected by pituitary stalk transection, indicating that they are localized on cells of the intermediate lobe. Using the test-tube binding assay for [3H]neurotensin, low levels of specific binding sites were found in whole rat pituitary glands. However, considering that the intermediate lobe comprises less than 5% of the total pituitary gland, these results indicate that the local concentration of [3H]neurotensin binding sites is comparable with that found in the central nervous system 2~. Interestingly, the staining pattern for [3H]neurotensin was similar
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