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Insulin treatment stimulates the rat melanin-concentrating hormone-producing neurons
Neuropeptides (1994) 21,251-258 ~0 Longman Group Ltd 1994
Insulin Treatment Stimulates the Rat Melaninconcentrating Hormone-producing Neurons M. BAHJAOUI-BOUHADDI,
D. FELLMANN,
B. GRIFFOND and C. BUGNON
CNRS UA 561, Laboratoire d’Histologie Embryologie Cytog&ktique, Faculty de Mhdecine, Place Saint-Jacques, 25030 Besangon Cedex, France (Reprint requests to MB-B)
Abstract-Melanin-concentrating hormone (MCH) is involved in the regulation of body colour in teleost fish. A peptide highly homologous to salmon MCH has been found in the rat brain, but its physiological functions have not yet been precisely defined. The location of MCH neurons in the lateral hypothalamus (LHT) of the rat suggests possible implication in feeding behaviour. In the present study, immunohistochemical and in situ hybridization methods were used to investigate MCH gene expression following insulin injections. Five hours after insulin injection, a significant increase in the abundance and staining intensity of MCH immunoreactive perikarya and fibres was observed. Concurrently the level of MCH mRNA significantly increased (50%). Insulin-treatment also induced a marked and progressive increase in the number and staining intensity of nuclei detected by a Fos antiserum in LHT and other brain areas. Double labelling technique demonstrated that very few if any MCH neurons exhibited Fos-like immunoreactivity. These results demonstrate that an insulin-treatment stimulates MCH neuron activity without the mediation of the proto-oncogene c-fos. The mechanisms triggering this activation remain to be elucidated.
Introduction
Melanin-concentrating hormone is a 17 amino-acid peptide, originally isolated from salmon pituitary gland (sMCH).‘,* In teleost fish sMCH plays a role in adaptive changes in skin pigmentation.3 It may also be involved in the regulation of stress response by inhibiting corticotropic activity in trouts and ee1s.j 5 In our laboratory, immunocytochemical studies
Date received 7 March 1994 Date accepted 8 April 1994
using an antiserum (AS) raised against human somatocrinin l-37 (GRF37) demonstrated an abundant population of neurons in the lateral regions of rat and human hypothalamus.6.7 It was then demonstrated that these neurons also contain epitopes related to cc-melanotropin (cr-MSH) and to salmon MCH (sMCH).*-” The sequence of the rat peptide binding sMCH antibodies (rMCH) has a high degree of homology with sMCH.” The sequence of the mRNA encoding the rat MCH-like peptide has been determined.‘2,‘3 In addition to rMCH peptide, the rat preproMCH (ppMCH) is supposed to generate two other neuropeptides, named NE1 (neuro251
252 peptide-glutamic acid-isoleucine) and NGE (neuropeptide-glycine-glutamic acid), according to Tatemoto and Mutt’s convention.‘4 The antisera (AS) raised against these peptides stained a single neuron population.” While projections of rMCH neurons are abundant throughout the brain, the sites of synthesis of rat ppMCH are confined to the lateral hypothalamus (LHT).16 This area participates in the regulation of food and water intakes as well as in the homeostatic control of various vegetative functions.‘7m20 The low amounts of immunoreactive MCH detected in the hypophysis of most vertebrates other than teleosts and the widespread distribution of MCH fibres throughout the brain suggest that the peptide would act more as a neurotransmitter or neuromodulator than as a neurohormone.21 In the rat, recent studies have shown that MCH gene expression is negatively regulated by stress and corticosteroid withdrawal.22 It has also been suggested that rat ppMCH is implicated in processes related to nocturnal regulation and drinking behavior.23 Several electrophysiological studies have shown that LHT contains glucose-sensitive neurons, whose electrical activity is altered by application of glucose or insulin.2k27 They have not been morphologically identified yet. In the present study we examined the effects of insulin treatment on the activity of rat MCH neurons for a better understanding of the roles of this neuron population and in order to verify if it could belong to the glucosesensitive neuron population of the LHT. MCH neuron activity was checked using immunocytochemical (ICC) and in situ hybridization (ISH) techniques. In addition, the expression of Fos-like-immunoreactivity (FLI), generally considered as a marker of neuronal activation,28,29 was investigated to identify the neuron populations prominently responding to insulin-treatment.
Materials and methods Animals 38 male Sprague-Dawley rats (IFFA Credo), weighing 250-300 g, were used. Animals were housed in room with controlled temperature. They were fed and watered ad libitum with a standard food for laboratory animals (UAR).
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Experiments and tissue preparation Twenty rats received a single intraperitoneal (ip) insulin injection (Actrapid HM Novo, 20 IUjkg bw diluted in isotonic saline) and were killed 1 h. 3 h or 5 h after treatment. Nine rats were injected with an equal volume of isotonic saline and 9 untreated animals served as controls. After chloral anesthesia (70 mg/200 g bw), blood was collected by cardiac puncture and centrifuged. Plasma was frozen until determination of glucose and insulin levels. Brains and pituitary glands were perfused with 1% paraformaldehyde in O.lM phosphate buffer and removed as previously described.6 They were post-fixed in the same fixative for 2 h and then soaked overnight in 15% sucrose in O.lM phosphate buffer (pH 7.2). Serial 10 pm cryostat sections were collected on gelatin-coated slides and stored at - 70°C until ICC staining and ISH were initiated. Determination of plasma glucose and insulin levels Plasma glucose and insulin levels were determined respectively by enzymatic method according to Slein et a13’and Phasedeph Insulin RIA (Pharmacia Diagnostics AB, Uppsala, Sweden). Immunocytochemistry Following rehydration, sections were incubated overnight with an antiserum (AS) to NGE (l/100) raised in rabbits and prepared in our laboratory.” The antigen-antibody complex was revealed by fluorescein-labelled goat anti-rabbit IgG (Institut Pasteur, l/SO, 1 h). Fos immunolabelling was performed using a polyclonal sheep AS against a synthetic peptide corresponding to the 2-17 sequence of Fos (OA 11823, Cambridge Research). Sections were incubated for 24 h at room temperature with the primary AS used at a final dilution of l/2000 in phosphate-buffered-saline (PBS) containing 1% bovine serum albumin (BSA). They were then washed three times (10-15 min) in PBS and incubated for 1 h with biotinylated donkey antibody to sheep IgG diluted l/l00 (Amersham), rinsed and incubated for 1 h with streptavidin-biotinylatedperoxidase complex diluted l/l00 in PBS (Amersham). Peroxidase was detected using standard 3,3’diamino-benzidine and hydrogen peroxide solution. To determine the specificity of the staining,
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THE RAT MELANIN-CONCENTRATING
random brain and pituitary sections were treated in the same manner as described above but incubated without the Fos AS. Some of the sections stained for Fos, were thoroughly rinsed and incubated overnight at room temperature with rabbit AS to ACTH or NGE at a final dilution of l/100. NGEand ACTH-immunoreactive cells were revealed using fluorescein-conjugated goat anti-rabbit IgG as described above.
In situ hybridization We used a 27 mer oligonucleotide probe complementary to the mRNA sequence coding the 9 amino-acids of the C-terminal end of the rMCH peptide with the following sequence: 5’-GCTCA GATGGCTGGGACAACCGTTCAG-3’. The probe was 5’ labelled by kination with y 32P-ATP. Hybridization steps and buffers have been previously described.3’ Slides were incubated for 1 h in prehybridization buffer at room temperature, rinsed 15 min in 4 x SSC (standard trisodium citrate buffer) and dried in ethanol. 25 ~1 hybridization buffer containing 1 ng labelled oligonucleotide was deposited on each slide, under a sealed coverslip and incubated overnight at 42°C. The coverslips were then removed and the slides were rinsed in 4 x SSC at room temperature. They were washed in solutions containing gradually decreasing concentrations of SSC and finally put in 0.1 x SSC for 30 min at 45°C. The sections were quickly dehydrated in ethanol, coated with K5 Ilford emulsion by the classical dipping procedure,3’ exposed at 4°C for lo-15 days and revealed with Kodak D19 solution.
Quantitative data Autoradiographic rMCH-mRNA signals were quantified using a Biocom 500 image analysis system by means of a grain-counting program (RAGSOO). For each animal (treated or control), the silver grains were counted at four different levels. On each section quantification was performed on 20 randomly chosen cells displaying a nuclear profile. The number of grains was determined over cell area and expressed as a density (number of grains per 100 pm’ of cell area). The results are presented as the mean+ S.E.M. Data
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between groups were compared variance analysis (ANOVA).
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using a one-way
Results
Glycemia and insulinemia Insulin level were 620-700 pU/ml in insulin treated rats and of 7-l 1 pU/ml in vehicle-treated or uninjetted rats. Plasma glucose levels dramatically decreased in insulin treated rats. Glucose concentrations were 2.45 mM 1 h after treatment, 0.21.5 mM after 3 h and O-O.2 mM after 5 h, versus 9.8-12 mM in vehicle-treated or uninjected rats. Immunocytochemical observations NGE AS revealed fibres and perikarya of the MCH system in treated as well as in control rats (Fig. 1). The abundance of fibres and the staining intensity of neurons were increased in insulin-treated animals, particularly in those sacrificed 5 h after treatment (Fig. 2). In the latter the fibre network appeared prominent in the median eminence and in the cortex but also in all other known projection zones.16 ICC using the OA 11823 AS revealed few if any Fos immunoreactive neurons in LHT of vehicletreated and control animals. In insulin-treated rats the nuclei of many LHT neurons were stained. This pattern was already detected after 1 h but the number of FL1 nuclei and the staining intensity were strongly increased 3 h and 5 h after insulin-treatment (Fig. 3). In addition, in animals sacrificed 3 h and 5 h after treatment, FL1 was observed in multiple brain areas such as piriform cortex (Fig. 5), medial portion of gyrus dentatus (Fig. 6) the paraventricular thalamic nucleus, the infundibular nucleus, ventromedial, dorsomedial and paraventricular (PVN) hypothalamic nuclei. In the latter, FL1 was observed in different cytoarchitectonic subdivisions3* including that containing the parvocellular CRF neurons (Fig. 7). Omission of the primary AS resulted in the absence of FL1 staining in all animals. Double labelling technique demonstrated that very few MCH neurons exhibited FL1 nuclei (Fig. 4). We have recently demonstrated3’ that most of the Fos positive nuclei observed in the LHT belonged to a population of neurons colocalized
254
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Figures l-10 Effect of insulin-treatment on MCH-producing neurons immunostained with NGE antiserum. The staining intensity of MCH neurons is more intense in rats sacrificed 5 h after insulin-treatment (2) than in control rats (1). Coronal sections of hypothalamus of insulin-injected rat. 5 h after this treatment FL1 increases in LHT neurons (3). Double labelling for NGE and FL1 reveals that no NGE immunoreactive neurons exhibited FL1 nuclei (4). Fos staining is also observed in piriform cortex (5) in gyrus dentatus (6), in the PVN (7) and in anterior lobe of pituitary gland (8). Double labelling for ACTH and FLI showing that majority of ACTH-immunoreactive cells in anterior lobe contain FL1 nuclei (8). (V, third ventricle; PVN, paraventicular nucleus). Hybridization histochemical localization of MCH-mRNA in LHT rat. Darkfield photomicrographs to show the effect of insulintreatment on MCH-mRNA expression (IO). Compare with control animal (9).
Figures continued.
70
60
1
So
8
40
-
induction of FL1 was detected 3 h and 5 h after insulin-treatment. Double staining with the ACTH AS showed that most of the FL1 cells were corticotrophs (Fig. 8).
71
Hybridocytochemical observations
si i
30
Ii
20
A hybridization signal was observed on MCH cell bodies of treated (Fig. 10) and control rats (Fig. 9). The autoradiographic signal was slightly higher after 3 h and significantly higher after 5 h in insulintreated rats than in vehicle-treated and control animals (Fig. 11).
10
0
C
S
lh
3h
Sh
Fig. 11 Effect of insulin-treatment on MCH gene expression. Histogram showing the number of silver grains by 100 pm*, which reflect MCH-mRNA levels in the MCH neurons of treated rat I h. 3 h or 5 h to insulin and of the control (C) and vehicletreated rats (S).
with MCH neurons and recently characterized as prolactin immunoreactivities (PLI) and dynorphincontaining neurons.34 In the anterior lobe of the pituitary a considerable
Discussion We report here that i.p. insulin injections resulted in a strong activation of rat MCH neurons as shown by a 50% increase of the MCH-mRNA and by an increase in the abundance of NGE immunoreactive material in fibres and perikarya. This activation was noticeable after 3 h but was very marked in animals
256 sacrificed 5 h after insulin-treatment. In this study, 1 h after insulin administration, some FLI-containing neurons were detected in LHT and this response became stronger after 3-5 h. This time course is very similar to that previously described for Fos protein in different experimental conditions.35.36 The AS used is directed against 2-17 sequence of the Fos protein. It is presumed to be specific for Fos since the N-terminal peptide has little homology with known Fos-related antigens (FRA).35,36 Fos has been proposed to participate in transcriptional regulation via interaction with the protein product of c-jun at specific DNA response elements (AP-1 binding sites).37*38Although the MCH neurons were stimulated by the insulin-treatment as shown by ICC and ISH, they exhibited very few if any FL1 staining. This result may be related to the finding that the rat MCH gene does not contain API-binding site, 39which indicates that the MCH gene expression is regulated by other transcription factors. Several hypotheses may be put forward to explain the response of MCH neurons: (1) direct effects of insulin and/or of insulininduced acute hypoglycemia, (2) indirect effects via insulin-responsive and/or glucose-sensitive neurons, (3) indirect effect of systemic stress induced by insulin treatment. A direct action of insulin on MCH neurons seems very unlikely because insulin receptor-mRNAs were not detected in the LHT.40 Conversely direct stimulation of MCH neurons by hypoglycemia must be considered since electrophysiological studies have shown that about one third of LHT neurons are sensitive to variations of the glucose concentration. These glucose-sensitive neurons are inhibited by local application of glucose and activated by glucopenia; they also change their elecof trical activity in response to variations glycemia. 25.26However, previous in vitro studies suggested that MCH neurons are not stimulated by lowering the glucose concentration in the culture medium (N. Compagnone, unpublished results). Thus, the glucose-sensitivity of MCH neurons remains to be established. In the same area than MCH neurons, many cells showed nuclear FL1
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after insulin injection. Among them, we have previously identified PLI neurons by means of an AS to ovine prolactin.33 Other Fos-positive nuclei belong to LHT neurons whose chemical phenotypes remain to be determined. The expression of c-fos in PLI neurons early after insulin treatment as well as other unpublished results suggest that these neurons are glucose-sensitive. Since PLI and MCH neurons are closely intermingled, a response of MCH neurons to hypoglycemia, mediated by PLI neurons may be hypothetized. However, we cannot rule out a possible action of insulin via neurons expressing insulin receptors which have been reported in several brain areas40.4’ most of which are connected with LHT mainly through the medial forebrain bundle.42 Incidentally, we noticed that all the brain regions known to possess insulin receptors that we analyzed (gyrus dentatus, piriform cortex, paraventricular thalamic nucleus, ventromedial and dorsomedial nuclei and infundibular nucleus) contained abundant FL1 nuclei after insulin injection. Finally, effect of systemic stress on MCH neurons must also be considered since it has been suggested that MCH neurons are involved in the regulation of stress response in fishes,3 5 and that the rat MCH gene expression is negatively regulated by neurogenic stress.‘2 In the present study, insulin injections resulted in an activation of MCH neurons. It seems thus likely that, in our acute experimental conditions, MCH neurons responded preferentially to glucopenic and/or other homeostatic stimuli than to a stress effect of insulin-treatment. The stress response was evidenced by abundant FL1 nuclei observed in the PVN (mainly in the pars parvocellularis containing CRF neurons), but also in POMC cells of the anterior pituitary. Previous studies have shown that insulininduced hypoglycemia leads to the activation of the adrenocortical axis in various species.4346 The resulting increase in corticosteronemia may therefore stimulate MCH gene expression.22 In conclusion, the present study has revealed activation of MCH neurons following insulin injection. We are currently evaluating the hypothesis of a direct effect of glucose deprivation on these neurons. Further studies will also be required to examine to which extent MCH neurons are activated secondarily to activation of other structures by this treatment.
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References 1. Kawauchi, H., Kawazoe, I., Tsubokawa, M., Kishida, M. and Baker, B. I. Characterization of melanin-concentrating hormone in chum salmon pituitaries. Nature 1983; 305: 321323. 2. Ono. M ., Wada, C.. Oikawa, I., Kawazoe, I. and Kawauchi, H. Structures of two kinds of mRNA encoding the chum salmon melanin-concentrating hormone. Gene 1988: 7 I : 433-438. 3. Baker, B. 1. Melanin concentrating hormone: Hadley ME (ed) The Melanotropic Peptides. CRC Press, Boca Raton. 1988; ~012: 159-174. 4. Lyon, V. E. and Baker, B. 1. The effect of photoperiod on plasma levels of melanin-concentrating hormone in the trout. J. Neuroendocrinol. 1993: 5: 493499. 5. Green. J. A. and Baker. B. 1. The influence of repeated stress on the release of melanin-concentrating hormone in the rainbow trout. J. Endocrinol. 1991: 128: 261-266. 6. Fellmann. D., Bugnon, C. and Risold, P. Y. Unrelated peptide immunoreactivities coexist in neurons of the rat lateral dorsal hypothalamus: human growth hormonereleasing factor] -37. salmon melanin-concentrating hormone- and a-melanotropin-like substances. Neurosci. Lett. 1987: 74: 2755280. I. Bresson. J. L.. Clavequin. M. C.. Fellmann, D. and Bugnon, C. Human hypothalamic neuronal system revealed with a salmon melanin-concentrating hormone (MCH) antiserum. Neurosci. Lett. 1989; 102: 3943. 8. Fellmann, D.. Bugnon, C.. Verstegen, J. and Lavry. C. N. Coexpression of human GRF l-37 like and alpha MSHlike immunoreactivities in neurons of the rat lateral dorsal hypothalamus. Neurosci. Lett. 1985; 68: 122-126. 9. Naito. N.. Kawazoe. I.. Nakai. Y., Kawauchi. H. and Hirdno. T. Coexistence of immunoreactivity for melaninconcentrating hormone and r-melanocyte stimulating hormone in the hypothalamus of the rat. Neurosci. Lett. 1986; 70: 81- 85. IO. Skofitsch. G.. Jacobowitz, D. M. and Zamir, N. Immunohistochemical localization of a melanin concentrating hormone-like peptide in the rat brain. Brain Res. Bull. 1985; 15: 6355649. Il. Vaughan, J. M.. Fischer, W. H.. Rivier. J. and Vale, W. Characterization of melanin-concentrating hormone from rat hypothalamus. Endocrinol. 1989; 125: 1660-1665. 12. Fellmann. D., Bresson, J. L., Breton. C. et al. Cloning of cDNAs encoding a rat neuropeptide immunologically related to salmon melanin-concentrating hormone. Neurojci. Lett. 1989; 106: 23328. 13. Nahon. J. L, Presse. F.. Bittencourt. J. C.. Sawchenko. P. E. and Vale, W. The rat melanin-concentrating hormone messenger ribonucleic acid encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus. Endocrinol. 1989; 125: 20562065. 14. Tatemoto. K. and Mutt. V. Isolation and characterization of the intestinal peptide porcine PHI (PHI-27). a new member of the glucagon-secretin family. Proc. Nat]. Acad. Sci. USA. I98 I ; 78: 660336607. 15. Risold. P. Y ., Fellmann. D., Rivier, J.. Vale, W. and Bugnon. C. lmmunoreactivities for antisera to three putative neuropeptides of the rat melanin-concentrating hormone precursor are coexpressed in neurons of the rat lateral dorsal hypothalamus. Neurosci. Lett. 1992; 136: 145-149. 16. Bittencourt. J. C.. Presse. F.. Arias, C. et al. The melaninconcentrating hormone system of the rat brain: an immuno-
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42. Bernadis. L. L. and Bellinger, L. L. The lateral hypothalamic area revisited: Neuroanatomy, body weight regulation. neuroendocrinology and metabolism. Neurosci. Biobehav. Rev. 1993; 17: 141-193. 43. Zukoski, C. F. Mecanism of action of insulin hypoglycemia in adrenal cortical secretion. Endocrinol. 1966: 78: 1264. 44. Kraiser. J. and Logothetopoulos, J. Adrenal cortical response to insulin-induced hypoglycemia in the rat. Acta. Endocrinol. 1963; 44: 282. 45. Krey, L. C., Lu, K. H., Butler, W. R., Hotchkiss. J., Piva, F. and Knobil, E. Surgical disconnection of the medial basal hypothalamus and pituitary function in the rhesus monkey. II. GH and cortisol secretion. Endocrinol. 1975; 96: 1088. 46. Keller-Wood, M. E., Shinsako, J., Keil. L. C. and Dallman, M. F. Insulin-induced hypoglycemia in conscious dogs. I. Dose-related pituitary and adrenal responses. Endocrinol. 1981; 109: 818. 47 Boutillier, A. L., Sassone-Corsi, P. and Loeffler. J. P. The protooncogene c-fos is induced by corticotropin-releasing factor and stimulates proopiomelanocortin in pituitary cells. Mol. Endocrinol. 1991; 5: 1301-1310.
Insulin Treatment Stimulates the Rat Melaninconcentrating Hormone-producing Neurons M. BAHJAOUI-BOUHADDI,
D. FELLMANN,
B. GRIFFOND and C. BUGNON
CNRS UA 561, Laboratoire d’Histologie Embryologie Cytog&ktique, Faculty de Mhdecine, Place Saint-Jacques, 25030 Besangon Cedex, France (Reprint requests to MB-B)
Abstract-Melanin-concentrating hormone (MCH) is involved in the regulation of body colour in teleost fish. A peptide highly homologous to salmon MCH has been found in the rat brain, but its physiological functions have not yet been precisely defined. The location of MCH neurons in the lateral hypothalamus (LHT) of the rat suggests possible implication in feeding behaviour. In the present study, immunohistochemical and in situ hybridization methods were used to investigate MCH gene expression following insulin injections. Five hours after insulin injection, a significant increase in the abundance and staining intensity of MCH immunoreactive perikarya and fibres was observed. Concurrently the level of MCH mRNA significantly increased (50%). Insulin-treatment also induced a marked and progressive increase in the number and staining intensity of nuclei detected by a Fos antiserum in LHT and other brain areas. Double labelling technique demonstrated that very few if any MCH neurons exhibited Fos-like immunoreactivity. These results demonstrate that an insulin-treatment stimulates MCH neuron activity without the mediation of the proto-oncogene c-fos. The mechanisms triggering this activation remain to be elucidated.
Introduction
Melanin-concentrating hormone is a 17 amino-acid peptide, originally isolated from salmon pituitary gland (sMCH).‘,* In teleost fish sMCH plays a role in adaptive changes in skin pigmentation.3 It may also be involved in the regulation of stress response by inhibiting corticotropic activity in trouts and ee1s.j 5 In our laboratory, immunocytochemical studies
Date received 7 March 1994 Date accepted 8 April 1994
using an antiserum (AS) raised against human somatocrinin l-37 (GRF37) demonstrated an abundant population of neurons in the lateral regions of rat and human hypothalamus.6.7 It was then demonstrated that these neurons also contain epitopes related to cc-melanotropin (cr-MSH) and to salmon MCH (sMCH).*-” The sequence of the rat peptide binding sMCH antibodies (rMCH) has a high degree of homology with sMCH.” The sequence of the mRNA encoding the rat MCH-like peptide has been determined.‘2,‘3 In addition to rMCH peptide, the rat preproMCH (ppMCH) is supposed to generate two other neuropeptides, named NE1 (neuro251
252 peptide-glutamic acid-isoleucine) and NGE (neuropeptide-glycine-glutamic acid), according to Tatemoto and Mutt’s convention.‘4 The antisera (AS) raised against these peptides stained a single neuron population.” While projections of rMCH neurons are abundant throughout the brain, the sites of synthesis of rat ppMCH are confined to the lateral hypothalamus (LHT).16 This area participates in the regulation of food and water intakes as well as in the homeostatic control of various vegetative functions.‘7m20 The low amounts of immunoreactive MCH detected in the hypophysis of most vertebrates other than teleosts and the widespread distribution of MCH fibres throughout the brain suggest that the peptide would act more as a neurotransmitter or neuromodulator than as a neurohormone.21 In the rat, recent studies have shown that MCH gene expression is negatively regulated by stress and corticosteroid withdrawal.22 It has also been suggested that rat ppMCH is implicated in processes related to nocturnal regulation and drinking behavior.23 Several electrophysiological studies have shown that LHT contains glucose-sensitive neurons, whose electrical activity is altered by application of glucose or insulin.2k27 They have not been morphologically identified yet. In the present study we examined the effects of insulin treatment on the activity of rat MCH neurons for a better understanding of the roles of this neuron population and in order to verify if it could belong to the glucosesensitive neuron population of the LHT. MCH neuron activity was checked using immunocytochemical (ICC) and in situ hybridization (ISH) techniques. In addition, the expression of Fos-like-immunoreactivity (FLI), generally considered as a marker of neuronal activation,28,29 was investigated to identify the neuron populations prominently responding to insulin-treatment.
Materials and methods Animals 38 male Sprague-Dawley rats (IFFA Credo), weighing 250-300 g, were used. Animals were housed in room with controlled temperature. They were fed and watered ad libitum with a standard food for laboratory animals (UAR).
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Experiments and tissue preparation Twenty rats received a single intraperitoneal (ip) insulin injection (Actrapid HM Novo, 20 IUjkg bw diluted in isotonic saline) and were killed 1 h. 3 h or 5 h after treatment. Nine rats were injected with an equal volume of isotonic saline and 9 untreated animals served as controls. After chloral anesthesia (70 mg/200 g bw), blood was collected by cardiac puncture and centrifuged. Plasma was frozen until determination of glucose and insulin levels. Brains and pituitary glands were perfused with 1% paraformaldehyde in O.lM phosphate buffer and removed as previously described.6 They were post-fixed in the same fixative for 2 h and then soaked overnight in 15% sucrose in O.lM phosphate buffer (pH 7.2). Serial 10 pm cryostat sections were collected on gelatin-coated slides and stored at - 70°C until ICC staining and ISH were initiated. Determination of plasma glucose and insulin levels Plasma glucose and insulin levels were determined respectively by enzymatic method according to Slein et a13’and Phasedeph Insulin RIA (Pharmacia Diagnostics AB, Uppsala, Sweden). Immunocytochemistry Following rehydration, sections were incubated overnight with an antiserum (AS) to NGE (l/100) raised in rabbits and prepared in our laboratory.” The antigen-antibody complex was revealed by fluorescein-labelled goat anti-rabbit IgG (Institut Pasteur, l/SO, 1 h). Fos immunolabelling was performed using a polyclonal sheep AS against a synthetic peptide corresponding to the 2-17 sequence of Fos (OA 11823, Cambridge Research). Sections were incubated for 24 h at room temperature with the primary AS used at a final dilution of l/2000 in phosphate-buffered-saline (PBS) containing 1% bovine serum albumin (BSA). They were then washed three times (10-15 min) in PBS and incubated for 1 h with biotinylated donkey antibody to sheep IgG diluted l/l00 (Amersham), rinsed and incubated for 1 h with streptavidin-biotinylatedperoxidase complex diluted l/l00 in PBS (Amersham). Peroxidase was detected using standard 3,3’diamino-benzidine and hydrogen peroxide solution. To determine the specificity of the staining,
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random brain and pituitary sections were treated in the same manner as described above but incubated without the Fos AS. Some of the sections stained for Fos, were thoroughly rinsed and incubated overnight at room temperature with rabbit AS to ACTH or NGE at a final dilution of l/100. NGEand ACTH-immunoreactive cells were revealed using fluorescein-conjugated goat anti-rabbit IgG as described above.
In situ hybridization We used a 27 mer oligonucleotide probe complementary to the mRNA sequence coding the 9 amino-acids of the C-terminal end of the rMCH peptide with the following sequence: 5’-GCTCA GATGGCTGGGACAACCGTTCAG-3’. The probe was 5’ labelled by kination with y 32P-ATP. Hybridization steps and buffers have been previously described.3’ Slides were incubated for 1 h in prehybridization buffer at room temperature, rinsed 15 min in 4 x SSC (standard trisodium citrate buffer) and dried in ethanol. 25 ~1 hybridization buffer containing 1 ng labelled oligonucleotide was deposited on each slide, under a sealed coverslip and incubated overnight at 42°C. The coverslips were then removed and the slides were rinsed in 4 x SSC at room temperature. They were washed in solutions containing gradually decreasing concentrations of SSC and finally put in 0.1 x SSC for 30 min at 45°C. The sections were quickly dehydrated in ethanol, coated with K5 Ilford emulsion by the classical dipping procedure,3’ exposed at 4°C for lo-15 days and revealed with Kodak D19 solution.
Quantitative data Autoradiographic rMCH-mRNA signals were quantified using a Biocom 500 image analysis system by means of a grain-counting program (RAGSOO). For each animal (treated or control), the silver grains were counted at four different levels. On each section quantification was performed on 20 randomly chosen cells displaying a nuclear profile. The number of grains was determined over cell area and expressed as a density (number of grains per 100 pm’ of cell area). The results are presented as the mean+ S.E.M. Data
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between groups were compared variance analysis (ANOVA).
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using a one-way
Results
Glycemia and insulinemia Insulin level were 620-700 pU/ml in insulin treated rats and of 7-l 1 pU/ml in vehicle-treated or uninjetted rats. Plasma glucose levels dramatically decreased in insulin treated rats. Glucose concentrations were 2.45 mM 1 h after treatment, 0.21.5 mM after 3 h and O-O.2 mM after 5 h, versus 9.8-12 mM in vehicle-treated or uninjected rats. Immunocytochemical observations NGE AS revealed fibres and perikarya of the MCH system in treated as well as in control rats (Fig. 1). The abundance of fibres and the staining intensity of neurons were increased in insulin-treated animals, particularly in those sacrificed 5 h after treatment (Fig. 2). In the latter the fibre network appeared prominent in the median eminence and in the cortex but also in all other known projection zones.16 ICC using the OA 11823 AS revealed few if any Fos immunoreactive neurons in LHT of vehicletreated and control animals. In insulin-treated rats the nuclei of many LHT neurons were stained. This pattern was already detected after 1 h but the number of FL1 nuclei and the staining intensity were strongly increased 3 h and 5 h after insulin-treatment (Fig. 3). In addition, in animals sacrificed 3 h and 5 h after treatment, FL1 was observed in multiple brain areas such as piriform cortex (Fig. 5), medial portion of gyrus dentatus (Fig. 6) the paraventricular thalamic nucleus, the infundibular nucleus, ventromedial, dorsomedial and paraventricular (PVN) hypothalamic nuclei. In the latter, FL1 was observed in different cytoarchitectonic subdivisions3* including that containing the parvocellular CRF neurons (Fig. 7). Omission of the primary AS resulted in the absence of FL1 staining in all animals. Double labelling technique demonstrated that very few MCH neurons exhibited FL1 nuclei (Fig. 4). We have recently demonstrated3’ that most of the Fos positive nuclei observed in the LHT belonged to a population of neurons colocalized
254
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Figures l-10 Effect of insulin-treatment on MCH-producing neurons immunostained with NGE antiserum. The staining intensity of MCH neurons is more intense in rats sacrificed 5 h after insulin-treatment (2) than in control rats (1). Coronal sections of hypothalamus of insulin-injected rat. 5 h after this treatment FL1 increases in LHT neurons (3). Double labelling for NGE and FL1 reveals that no NGE immunoreactive neurons exhibited FL1 nuclei (4). Fos staining is also observed in piriform cortex (5) in gyrus dentatus (6), in the PVN (7) and in anterior lobe of pituitary gland (8). Double labelling for ACTH and FLI showing that majority of ACTH-immunoreactive cells in anterior lobe contain FL1 nuclei (8). (V, third ventricle; PVN, paraventicular nucleus). Hybridization histochemical localization of MCH-mRNA in LHT rat. Darkfield photomicrographs to show the effect of insulintreatment on MCH-mRNA expression (IO). Compare with control animal (9).
Figures continued.
70
60
1
So
8
40
-
induction of FL1 was detected 3 h and 5 h after insulin-treatment. Double staining with the ACTH AS showed that most of the FL1 cells were corticotrophs (Fig. 8).
71
Hybridocytochemical observations
si i
30
Ii
20
A hybridization signal was observed on MCH cell bodies of treated (Fig. 10) and control rats (Fig. 9). The autoradiographic signal was slightly higher after 3 h and significantly higher after 5 h in insulintreated rats than in vehicle-treated and control animals (Fig. 11).
10
0
C
S
lh
3h
Sh
Fig. 11 Effect of insulin-treatment on MCH gene expression. Histogram showing the number of silver grains by 100 pm*, which reflect MCH-mRNA levels in the MCH neurons of treated rat I h. 3 h or 5 h to insulin and of the control (C) and vehicletreated rats (S).
with MCH neurons and recently characterized as prolactin immunoreactivities (PLI) and dynorphincontaining neurons.34 In the anterior lobe of the pituitary a considerable
Discussion We report here that i.p. insulin injections resulted in a strong activation of rat MCH neurons as shown by a 50% increase of the MCH-mRNA and by an increase in the abundance of NGE immunoreactive material in fibres and perikarya. This activation was noticeable after 3 h but was very marked in animals
256 sacrificed 5 h after insulin-treatment. In this study, 1 h after insulin administration, some FLI-containing neurons were detected in LHT and this response became stronger after 3-5 h. This time course is very similar to that previously described for Fos protein in different experimental conditions.35.36 The AS used is directed against 2-17 sequence of the Fos protein. It is presumed to be specific for Fos since the N-terminal peptide has little homology with known Fos-related antigens (FRA).35,36 Fos has been proposed to participate in transcriptional regulation via interaction with the protein product of c-jun at specific DNA response elements (AP-1 binding sites).37*38Although the MCH neurons were stimulated by the insulin-treatment as shown by ICC and ISH, they exhibited very few if any FL1 staining. This result may be related to the finding that the rat MCH gene does not contain API-binding site, 39which indicates that the MCH gene expression is regulated by other transcription factors. Several hypotheses may be put forward to explain the response of MCH neurons: (1) direct effects of insulin and/or of insulininduced acute hypoglycemia, (2) indirect effects via insulin-responsive and/or glucose-sensitive neurons, (3) indirect effect of systemic stress induced by insulin treatment. A direct action of insulin on MCH neurons seems very unlikely because insulin receptor-mRNAs were not detected in the LHT.40 Conversely direct stimulation of MCH neurons by hypoglycemia must be considered since electrophysiological studies have shown that about one third of LHT neurons are sensitive to variations of the glucose concentration. These glucose-sensitive neurons are inhibited by local application of glucose and activated by glucopenia; they also change their elecof trical activity in response to variations glycemia. 25.26However, previous in vitro studies suggested that MCH neurons are not stimulated by lowering the glucose concentration in the culture medium (N. Compagnone, unpublished results). Thus, the glucose-sensitivity of MCH neurons remains to be established. In the same area than MCH neurons, many cells showed nuclear FL1
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after insulin injection. Among them, we have previously identified PLI neurons by means of an AS to ovine prolactin.33 Other Fos-positive nuclei belong to LHT neurons whose chemical phenotypes remain to be determined. The expression of c-fos in PLI neurons early after insulin treatment as well as other unpublished results suggest that these neurons are glucose-sensitive. Since PLI and MCH neurons are closely intermingled, a response of MCH neurons to hypoglycemia, mediated by PLI neurons may be hypothetized. However, we cannot rule out a possible action of insulin via neurons expressing insulin receptors which have been reported in several brain areas40.4’ most of which are connected with LHT mainly through the medial forebrain bundle.42 Incidentally, we noticed that all the brain regions known to possess insulin receptors that we analyzed (gyrus dentatus, piriform cortex, paraventricular thalamic nucleus, ventromedial and dorsomedial nuclei and infundibular nucleus) contained abundant FL1 nuclei after insulin injection. Finally, effect of systemic stress on MCH neurons must also be considered since it has been suggested that MCH neurons are involved in the regulation of stress response in fishes,3 5 and that the rat MCH gene expression is negatively regulated by neurogenic stress.‘2 In the present study, insulin injections resulted in an activation of MCH neurons. It seems thus likely that, in our acute experimental conditions, MCH neurons responded preferentially to glucopenic and/or other homeostatic stimuli than to a stress effect of insulin-treatment. The stress response was evidenced by abundant FL1 nuclei observed in the PVN (mainly in the pars parvocellularis containing CRF neurons), but also in POMC cells of the anterior pituitary. Previous studies have shown that insulininduced hypoglycemia leads to the activation of the adrenocortical axis in various species.4346 The resulting increase in corticosteronemia may therefore stimulate MCH gene expression.22 In conclusion, the present study has revealed activation of MCH neurons following insulin injection. We are currently evaluating the hypothesis of a direct effect of glucose deprivation on these neurons. Further studies will also be required to examine to which extent MCH neurons are activated secondarily to activation of other structures by this treatment.
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