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Non-NMDA glutamate receptor antagonist injected into the hypothalamic paraventricular nucleus blocks the suckling stimulus-induced release of prolactin
Brain Research Bulletin 65 (2005) 163–168
Non-NMDA glutamate receptor antagonist injected into the hypothalamic paraventricular nucleus blocks the suckling stimulus-induced release of prolactin Ibolya Bodn´ar, Zsuzsanna B´anky, Gy¨orgy M. Nagy, B´ela Hal´asz ∗ Neuroendocrine Research Laboratory, Department of Human Morphology & Developmental Biology, Hungarian Academy of Sciences and Semmelweis University, T˝uzolt´o u. 58, H-1094 Budapest, Hungary Received 27 October 2004; received in revised form 21 December 2004; accepted 4 January 2005 Available online 21 January 2005
Abstract The aim of the present investigations was to test the involvement of the glutamatergic innervation of the hypothalamic paraventricular nucleus in the prolactin response to the suckling stimulus. A non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-dione disodium (CNQX), or an NMDA receptor antagonist, dizocipine hydrogen malate (MK-801), was injected bilaterally into the hypothalamic paraventricular nucleus of lactating freely moving rats before the end of a 4-h separation of the dams from their pups. The litters were then returned. Blood samples for prolactin were taken at different time points. The effect of the non-NMDA receptor antagonist was also tested in animals receiving the drug bilaterally into the dorsomedial nucleus area or the arcuate nucleus. Bilateral injection of CNQX into the paraventricular nucleus blocked the elevation in plasma prolactin concentration induced by the suckling stimulus. In contrast, bilateral administration of the NMDA receptor antagonist MK-801 into the paraventricular nucleus or bilateral injection of CNQX into the dorsomedial nucleus area or the arcuate nucleus did not interfere with the prolactin response to the suckling stimulus. The findings indicate that the glutamatergic innervation of the paraventricular nucleus is involved in the mediation of the neural signal of the suckling stimulus inducing prolactin release. © 2005 Elsevier Inc. All rights reserved. Keywords: Hypothalamus; Neurotransmitter; Regulation; Hormone release; Glutamatergic innervation
1. Introduction Suckling-induced prolactin release is a widely studied neuroendocrine reflex, comprising a neural afferent and humoral efferent component [10]. The hypothalamic paraventricular nucleus region appears to be a key structure in the mediation of the suckling stimulus-induced release of prolactin. Lesion of this cell group interferes with the hormone response induced by suckling [13]. Frontal knife cut behind this nucleus, horizontal knife cut below the cell group or lesion of the medial subdivision of the nucleus blocks the prolactin response to suckling [3].
∗
Corresponding author. Tel.: +36 1 215 5847; fax: +36 1 215 3064. E-mail address: [email protected] (B. Hal´asz).
0361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.01.001
A significant amount of information has been accumulated suggesting that the excitatory amino acid glutamate is a major excitatory neurotransmitter of the hypothalamus, and besides serotonin, noradrenaline and other stimulatory or inhibitory neurotransmitters may play a critical widespread role in the control of hypothalamic neuroendocrine neurons and processes including the control of prolactin secretion [4]. Parker and Crowley [19] have reported that administration of the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX), in the third ventricle of suckling rats blocked the release of oxytocin and prolactin induced by suckling. Zelena et al. [30] have found that combined treatment with NMDA and non-NMDA receptor antagonists, giving the drugs i.v., diminished the suckling-induced prolactin elevation.
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Glutamate appears to be a prime candidate as a major excitatory signal to hypothalamic paraventricular nucleus neurons. This cell group is rich in glutamate receptor mRNA and protein expression [1,2,8,11], in glutamate/agonist binding [21] and in glutamate immunoreactive axons and synapses [7,25,27], as well as it contains glutamatergic neurons and vesicular glutamate transporter 2 protein immunoreactive fibers [5,16]. All three subtypes of ionotropic glutamate receptors and certain subtypes of metabotropic glutamate receptors are present in the cell group [4,26]. Wuarin and Dudek [29] have reported that excitatory amino acid antagonists inhibit almost completely synaptic responses in the magnocellular and parvocellular division of the paraventricular nucleus. van den Pol et al. [27] have found that spontaneous excitatory postsynaptic potentials in the paraventricular nucleus were markedly attenuated by application of the non-NMDA receptor antagonist CNQX. The aim of the present investigations was to test the involvement of the glutamatergic innervation of the paraventricular nucleus in the prolactin response to the suckling stimulus. Non-NMDA or NMDA receptor antagonist was injected bilaterally into the paraventricular nucleus of lactating rats 15 min before the end of a 4-h separation of the dams from their pups. The litters were returned and the mothers exposed to the suckling stimulus. Blood samples for prolactin were taken at different time points. For testing the selectivity of the effect of the non-NMDA receptor antagonist given into the paraventricular nucleus, the non-NMDA receptor antagonist was also injected into the area of the dorsomedial nucleus or the arcuate nucleus.
2.2. Implantation of bilateral guide cannulae into the hypothalamus Dams were anaesthesized (chloral hydrate, 400 mg/kg, i.p.) and stereotaxically implanted with double guide (26 gauge) cannulae (Plastics One Inc., Roanoke, VA, USA). In the case of microinjections into the paraventricular and dorsomedial nuclei the tubing length below pedestal was 7.0 mm and center to center distance between the two tubings was in all three cases 1.0 mm. Double dummy cannulae (obturator) sealed the top of the double guide cannulae. The obturator had two stainless steel wires, which were inserted into the double guide cannula tubes. A dust cap secured the unit to the double guide cannulae. The cannulae were fixed by screws and dental cement to the top of the skull. The stereotaxic coordinates taking the bregma suture as zero reference point [20] used for the implantations were: (i) Paraventricular nucleus: AP, bregma −1.7 mm; V, 7.1 mm. (ii) Dorsomedial nucleus: AP, bregma −3.1 mm; V, 7.1 mm. In this case the double cannulae were placed in a similar position than those for the paraventricular nuclei except they were 1.4 mm more caudal and were considered as a control to test the selectivity of the effect of the paraventricular microinjections. (iii) Arcuate nucleus: AP, bregma −3.0; V, 9.0 mm.
2. Materials and methods
In all three cases the two guide cannulae were in the coronal plane and 0.5 mm laterally from the midline (interhemispheric fissure). Implantation of bilateral guide cannulae was made on postpartum days 3–4. Rats were given a 5–7 day recovery period, during which they were handled for the microinjection procedure.
2.1. Animals
2.3. Microinjections
Primiparous lactating female rats were used. They were bred in our animal facilities from Sprague–Dawley stock. The rats were individually housed with their litters; litter size was reduced to eight. The rats were maintained in a climateand illumination-controlled room (14:10-h light/14:10-h dark cycle; lights on at 06:00 h). Water and standard rat chow were available ad libitum. All animal experiments were conducted in accordance with the Guide of the University for the Care and Use of Laboratory Animals. Microinjections were given into the hypothalamic:
Microinjections were made in non-anaesthesized rats. The inserted double internal cannulae (gauge 33) (Plastics One Inc., Roanoke, VA, USA) extended 1.0 mm below the tip of the guide cannulae. They were attached to Teflon tubing and supplied by a 0.5 l Hamilton syringe. Animals received through the internal cannulae 0.1 l distilled water or one of the glutamate receptor antagonists: 6-cyano-7-nitroquinoxaline2,3-dione disodium, a competitive non-NMDA glutamate receptor antagonist (Sigma–Aldrich) (20 mM solved in 0.1 ml distilled water) or dizocipine hydrogen malate (MK-801) (20 mM solved in 0.1 ml distilled water), a selective non-competitive NMDA receptor antagonist (Sigma–Aldrich). Water was injected into the paraventricular and the arcuate nucleus, CNQX into the paraventricular nucleus, the dorsomedial nucleus area and the arcuate nucleus. MK-801 was given into the paraventricular nucleus.
• paraventricular nucleus; • dorsomedial nucleus; and • arcuate nucleus. Cannulae for injections were inserted through bilateral guide cannulae.
I. Bodn´ar et al. / Brain Research Bulletin 65 (2005) 163–168
Microinjections were made on the day of testing the suckling-induced prolactin release 15 min before the reunion of the dams with their pups. 2.4. Testing the suckling-induced prolactin release: blood sampling The suckling-induced prolactin releasing effect was examined on postpartum days 8–10. Prior to exposing the mothers to the suckling stimulus, the dams were separated from their pups for 4 h. Two days prior to taking blood samples, a permanent cannula (silicon tubing inner diameter 0.50 mm, external diameter 0.90 mm; Dow Corning Corp., Midland, MI, USA and Becton-Dickinson, Parsipparny, NY, USA) was implanted into the jugular vein under chloral hydrate anaesthesia, allowing frequent blood sampling from freely moving rats. The rats were habituated to the procedure of blood sampling by connecting them to a polyethylene tube every day. On the day of experiment the first blood sample was taken before separation. Fifteen minutes before the end of the separation period a second blood sample was taken and the animals received the intrahypothalamic microinjection. Fifteen minutes later another blood sample was taken and the pups were returned to the dams. Full pup attachment was achieved within 5 min after reunion. Further blood samples were taken 15, 30 and 60 min after reunion (using only the rats showing full pup attachment, which was true for approximately 90% of each group, and was not influenced by the type of microinjection). At each time point 200 l blood was obtained, plasma was separated and stored at −20 ◦ C until assayed for prolactin. 2.5. Hormone analysis Prolactin was measured by radioimmunoassay (RIA), with kits kindly provided by NHPP, NIDDK and Dr. A.F. Parlow. The RIA procedure was similar to the instructions supplied with the kit, with modifications as described previously [12]. We used the Chloramine-T method for iodination and protein A (BactASorb, Human Rt, G¨od¨oll˝o, Hungary) for separation of bound and free hormone. Data collection and calculations for curve fitting were made using LKB Clinigamma software. The data were expressed in terms of NIAMDD-Rat-RP-3. The within- and between-assay variances were 10 and 14%, respectively. The sensitivity of the prolactin assay was 0.5 ng/ml rat plasma, expressed in terms of the rat prolactin RP-3 standard (or 25 pg PRL). We used 50 l plasma per samples. All samples were measured in duplicate. 2.6. Brain histology After the last blood sample was taken, rats were perfused under deep hexobarbital anaesthesia with 200 ml saline followed by 250 ml 4% paraformaldehyde via transcardiac
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puncture. The cannulae were taken out from the brains and the brains were removed and placed in the same fixative at 4 ◦ C for 24 h. Subsequently, they were transferred to a buffered sucrose solution (25% sucrose in phosphate buffer saturated) for 24 h. For histological localization of the microinjections, 20 m thick coronal sections were made by means of a cryostat of all brains. The sections were stained with haematoxylin and eosin and the localization of each microinjection determined. Only those rats in which the tips of the internal cannulae were verified to be in the right place were included in the study. 2.7. Statistical analysis Statistical analysis of the data (amplitude of the response) was performed using two-way ANOVA followed by Dunnett’s test for multiple comparisons. Values are considered significantly different at p < 0.05.
3. Results Animals were grouped according to the histological assessment of cannulae placement. Only injections that appeared to be centered within the paraventricular nuclei were designated as paraventricular microinjections (Fig. 1A), those centered within the dorsomedial nuclei area as dorsomedial microinjections (Fig. 1B) and those centered within the arcuate nuclei as arcuate microinjections (Fig. 1C). The proportion of the successful placements of the cannulae was about 50%. Animals in which the microinjections were misplaced had been discarded. Microinjections of the solvent into the paraventricular or into the arcuate nuclei did not interfere with the rise in plasma prolactin concentration induced by the suckling stimulus (Figs. 2 and 3). In animals receiving bilateral microinjections of the nonNMDA receptor antagonist CNQX into the paraventricular nucleus, there was no change in hormone concentration after replacement of the pups, suckling did not induce any elevation in plasma prolactin concentration (Fig. 2). The blockade of the prolactin response compared to the response of all other groups is statistically significantly different (p < 0.01). Dams, which received microinjections of the NMDA receptor antagonist MK-801 into the paraventricular nuclei, did respond to the suckling stimulus (Fig. 2). Microinjections of the non-NMDA receptor antagonist CNQX into the area of the dorsomedial nucleus or into the arcuate nucleus did not affect the prolactin response. In these two groups, in contrast to those animals receiving the same receptor antagonist into the paraventricular nuclei, suckling induced a significant rise in plasma prolactin concentration (Fig. 3). The elevations in plasma prolactin concentrations induced by the suckling stimulus are statistically significant in all groups in which there was a rise in hormone levels (p < 0.05).
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Fig. 1. Light micrographs show the location of the tracks of internal cannulae (TIC) for the microinjections into the paraventricular nucleus (PVN) (A), dorsomedial nucleus (DMN) (B) and arcuate nucleus (AN) (C). 3V: third ventricle; TGC: track of the guide cannula. Scale bars: 100 m.
4. Discussion Our present findings indicate that a non-NMDA receptor antagonist injected bilaterally into the hypothalamic paraventricular nucleus interferes with the suckling stimulus-induced release of prolactin. The observations are in line with previous investigations showing that injection of the non-NMDA receptor antagonist, CNQX into the third ventricle [19] or combined treatment with non-NMDA and NMDA receptor antagonists giving the drugs i.v. [30], blocked the suckling stimulus-induced prolactin elevation. In addition, our results provide for the first time direct information about the site of action of the receptor antagonist in blocking the prolactin response. They demonstrate that this action of the drug was exerted on the paraventricular nucleus. A similar effect was not observed if the same receptor antagonist was administered into the area of the dorsomedial nucleus or the arcuate nucleus. The fact that the non-NMDA receptor antagonist was effective in blocking the prolactin response only in that case when it was injected into the paraventricular nucleus, strongly suggests that the glutamatergic innervation of the cell group is involved in the mediation of the suckling stimulus. The glutamatergic fibers terminating in the paraventricular nuclei originate partly from glutamatergic neurons of several diencephalic and telencephalic structures and partly belong to glutamatergic interneurons of the paraventricular nucleus [5]. It should be pointed out that glutamatergic neurons in the brain stem projecting to the paraventricular nuclei were not detected [5]. When taking into account that the signal of the suckling stimulus is mediated through the spinal cord and brain stem up to the hypothalamus and further, that glutamatergic neurons in the brain stem do not terminate in the paraventricular nuclei, it is close at hand to assume that the glutamatergic innervation of the paraventricular nucleus to be involved in the mediation of the suckling stimulus, most probably belongs to the glutamatergic interneurons of the cell group. We postulate that these interneurons take part in forwarding the stimulatory influ-
ence of the suckling stimulus reaching the paraventricular nuclei. A large amount of information has been accumulated suggesting that serotonergic elements arising from the raphe nuclei [23] and terminating in the hypothalamic paraventricular nucleus are primarily involved in leading the suckling stimulus to the cell group [3,14,15,17,18,28]. Our present findings suggest that the glutamatergic interneurons in the paraventricular nucleus are participating in the mediation of the stimulatory effect of the serotonergic afferents terminating in the paraventricular nucleus. In a recent publication Feldman and Weidenfeld [9] have formulated an almost identical concept. They postulated that glutamatergic interneurons in the paraventricular nucleus acting via non-NMDA and NMDA receptors might act as an excitatory mechanism in the norepinephrine and serotonin control of hypothalamic adrenocorticotropin secretagogues. Daftary et al. [6] have provided data suggesting that glutamatergic interneurons in the paraventricular nucleus serve as an excitatory relay in the afferent noradrenergic control of oxytocin and vasopressin release under certain physiological conditions. Pirnik et al. [22] have provided evidence that oxytocin and vasopressin neurons in the paraventricular nucleus play important role in the mediation of signals induced by hypertonic saline. In the present investigations only CNQX, the non-NMDA receptor antagonist was effective in interfering with the prolactin response to the suckling stimulus. The NMDA receptor antagonist MK-801 administered into the paraventricular nucleus did not cause any significant alteration in the plasma prolactin concentration of dams exposed to the suckling stimulus. van den Pol et al. [27] have reported that spontaneous excitatory postsynaptic potentials in the paraventricular nucleus were markedly attenuated by application of the non-NMDA receptor antagonist, CNQX. NMDA antagonists only slightly suppressed excitatory postsynaptic potentials. These findings suggest that in the paraventricular nucleus the excitatory amino acid acts primarily on non-NMDA receptors.
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Fig. 2. Plasma prolactin concentrations of dams separated for 4 h from their litters, receiving microinjection of the solvent (water), the non-NMDA antagonist CNQX or the NMDA antagonist MK-801 into the paraventricular nucleus (PVN) and 15 min later exposed to the suckling stimulus. Separation of the mothers caused the well-known marked reduction in hormone concentrations. Suckling induced a statistically significant elevation in plasma prolactin of rats getting water or MK-801 into the paraventricular nucleus, but it did not result in any increase in hormone concentrations in rats receiving microinjection of CNQX into this cell group, n: number of animals.
The present findings do not provide any information on where the neural afferent component of the suckling stimulus-induced release of prolactin is transformed into a humoral efferent reflex answer expressed via changes in the secretion of prolactin releasing and/or release-inhibiting factors (vasoactive intestinal peptide, oxytocin, vasopressin, salsolinol, dopamine) produced by hypothalamic neurons [10,24]. Further investigations are needed to study this question.
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Fig. 3. Plasma prolactin concentrations of dams separated for 4 h from their litters receiving microinjection of the solvent (water) into the arcuate nucleus (AN), CNQX into the dorsomedial nucleus area (DMN) or into the arcuate nucleus (AN) and 15 min later exposed to the suckling stimulus. Rats getting CNQX into the dorsomedial nucleus area or into the arcuate nucleus responded to the suckling stimulus, n: number of animals.
Acknowledgements The authors are grateful to Mrs. M. M´esz´aros and Mrs. M. Bal´azs for their excellent technical assistance, to Mrs. I. Salamon for excellent histotechnical work and Mrs. E. Pirity for typing the manuscript. This work was supported by the Ministry of Health (ETT 054/2003 to B.H.), National Scientific Research Fund (OTKA T-042516 to B.H.) and the Hungarian Academy of Sciences. References [1] W.M. Al-Ghoul, R.B. Meeker, R.S. Greenwood, Differential expression of five N-methyl-d-aspartate receptor subunit mRNAs in vaso-
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[16] W. Lin, K. McKinney, L. Liu, S. Lakhlani, L. Jennes, Distribution of vesicular glutamate transporter 2 messenger ribonucleic acid and protein in the septum-hypothalamus of the rat, Endocrinology 144 (2003) 662–670. [17] K.H. Lu, J. Meites, Effects of serotonin precursors and melatonin on serum prolactin release in rats, Endocrinology 93 (1973) 152–155. [18] F. Mena, A. Enjalbert, L. Carbonell, M. Priam, C. Kordon, Effects of suckling on plasma and hypothalamic monoamine levels in the rat, Endocrinology 99 (1976) 445–451. [19] S.L. Parker, W.R. Crowley, Stimulation of oxytocin release in the lactating rat by central excitatory amino acid mechanisms: evidence for specific involvement of R,S-␣-amino-3-hydroxy-5-methylisoxazole4-propionic acid-sensitive glutamate receptors, Endocrinology 133 (1993) 2847–2854. [20] G. Paxinos, C. Watson (Eds.), The Rat Brain in Stereotaxic Coordinates, third ed., Academic Press, San Diego, 1997. [21] R.S. Petralia, R.J. Wenthold, Types of excitatory amino acid receptors and their localization in the nervous system and hypothalamus, in: D.W. Brann, V.B. Mahesh (Eds.), Excitatory Amino Acid Receptors: Their Role in Neuroendocrine Function, CRC Press, New York, 1996, pp. 55–101. [22] Z. Pirnik, B. Mravec, A. Kiss, Fos protein expression in mouse hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei upon osmotic stimulus: colocalization with vasopressin, oxytocin and tyrosine hydroxylase, Neurochem. Int. 45 (2004) 597–607. [23] P.E. Sawchenko, L.W. Swanson, H.W.M. Steinbusch, A.A.J. Verhofstad, The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat, Brain Res. 277 (1983) 355–360. [24] B.E. T´oth, K. Homicsk´o, B. Radnai, W. Maruyama, J.E. DeMaria, M. Vecserny´es, M.I.K. Fekete, F. F¨ul¨op, M. Naoi, M.E. Freeman, G.M. Nagy, Salsolinol is a putative endogenous neuro-intermediate lobe prolactin-releasing factor, J. Neuroendocrinol. 13 (2001) 1042–1050. [25] A.N. van den Pol, Glutamate and aspartate immunoreactivity in hypothalamic presynaptic axons, J. Neurosci. 11 (1991) 2087–2101. [26] A.N. van den Pol, I.R.M. Hermans-Borgmeyer, M. Hofer, P. Ghosh, S. Heinemann, Ionotropic glutamate-receptor gene expression in hypothalamus: localization of AMPA, kainate and NMDA receptor RNA with in situ hybridization, J. Comp. Neurol. 343 (1994) 428–444. [27] A.N. van den Pol, J.P. Wuarin, F.E. Dudek, Glutamate, the dominant excitatory transmitter in neuroendocrine regulation, Science 250 (1990) 1276–1278. [28] J.O. Willoughby, M. Menadue, P. Jervois, Function of serotonin in physiologic secretion of growth hormone and prolactin. Action of 5,7-dihydroxytryptamine, fenfluramine and p-chlorophenylalanine, Brain Res. 249 (1982) 291–299. [29] J.P. Wuarin, F.E. Dudek, Excitatory amino acid antagonists inhibit synaptic responses in the guinea pig hypothalamic paraventricular nucleus, J. Neurophysiol. 65 (1991) 946–951. [30] D. Zelena, B.G. Makara, M.G. Nagy, Effect of glutamate receptor antagonists on suckling-induced prolactin release in rats, Endocrine 21 (2003) 147–152.
Non-NMDA glutamate receptor antagonist injected into the hypothalamic paraventricular nucleus blocks the suckling stimulus-induced release of prolactin Ibolya Bodn´ar, Zsuzsanna B´anky, Gy¨orgy M. Nagy, B´ela Hal´asz ∗ Neuroendocrine Research Laboratory, Department of Human Morphology & Developmental Biology, Hungarian Academy of Sciences and Semmelweis University, T˝uzolt´o u. 58, H-1094 Budapest, Hungary Received 27 October 2004; received in revised form 21 December 2004; accepted 4 January 2005 Available online 21 January 2005
Abstract The aim of the present investigations was to test the involvement of the glutamatergic innervation of the hypothalamic paraventricular nucleus in the prolactin response to the suckling stimulus. A non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-dione disodium (CNQX), or an NMDA receptor antagonist, dizocipine hydrogen malate (MK-801), was injected bilaterally into the hypothalamic paraventricular nucleus of lactating freely moving rats before the end of a 4-h separation of the dams from their pups. The litters were then returned. Blood samples for prolactin were taken at different time points. The effect of the non-NMDA receptor antagonist was also tested in animals receiving the drug bilaterally into the dorsomedial nucleus area or the arcuate nucleus. Bilateral injection of CNQX into the paraventricular nucleus blocked the elevation in plasma prolactin concentration induced by the suckling stimulus. In contrast, bilateral administration of the NMDA receptor antagonist MK-801 into the paraventricular nucleus or bilateral injection of CNQX into the dorsomedial nucleus area or the arcuate nucleus did not interfere with the prolactin response to the suckling stimulus. The findings indicate that the glutamatergic innervation of the paraventricular nucleus is involved in the mediation of the neural signal of the suckling stimulus inducing prolactin release. © 2005 Elsevier Inc. All rights reserved. Keywords: Hypothalamus; Neurotransmitter; Regulation; Hormone release; Glutamatergic innervation
1. Introduction Suckling-induced prolactin release is a widely studied neuroendocrine reflex, comprising a neural afferent and humoral efferent component [10]. The hypothalamic paraventricular nucleus region appears to be a key structure in the mediation of the suckling stimulus-induced release of prolactin. Lesion of this cell group interferes with the hormone response induced by suckling [13]. Frontal knife cut behind this nucleus, horizontal knife cut below the cell group or lesion of the medial subdivision of the nucleus blocks the prolactin response to suckling [3].
∗
Corresponding author. Tel.: +36 1 215 5847; fax: +36 1 215 3064. E-mail address: [email protected] (B. Hal´asz).
0361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.01.001
A significant amount of information has been accumulated suggesting that the excitatory amino acid glutamate is a major excitatory neurotransmitter of the hypothalamus, and besides serotonin, noradrenaline and other stimulatory or inhibitory neurotransmitters may play a critical widespread role in the control of hypothalamic neuroendocrine neurons and processes including the control of prolactin secretion [4]. Parker and Crowley [19] have reported that administration of the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX), in the third ventricle of suckling rats blocked the release of oxytocin and prolactin induced by suckling. Zelena et al. [30] have found that combined treatment with NMDA and non-NMDA receptor antagonists, giving the drugs i.v., diminished the suckling-induced prolactin elevation.
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Glutamate appears to be a prime candidate as a major excitatory signal to hypothalamic paraventricular nucleus neurons. This cell group is rich in glutamate receptor mRNA and protein expression [1,2,8,11], in glutamate/agonist binding [21] and in glutamate immunoreactive axons and synapses [7,25,27], as well as it contains glutamatergic neurons and vesicular glutamate transporter 2 protein immunoreactive fibers [5,16]. All three subtypes of ionotropic glutamate receptors and certain subtypes of metabotropic glutamate receptors are present in the cell group [4,26]. Wuarin and Dudek [29] have reported that excitatory amino acid antagonists inhibit almost completely synaptic responses in the magnocellular and parvocellular division of the paraventricular nucleus. van den Pol et al. [27] have found that spontaneous excitatory postsynaptic potentials in the paraventricular nucleus were markedly attenuated by application of the non-NMDA receptor antagonist CNQX. The aim of the present investigations was to test the involvement of the glutamatergic innervation of the paraventricular nucleus in the prolactin response to the suckling stimulus. Non-NMDA or NMDA receptor antagonist was injected bilaterally into the paraventricular nucleus of lactating rats 15 min before the end of a 4-h separation of the dams from their pups. The litters were returned and the mothers exposed to the suckling stimulus. Blood samples for prolactin were taken at different time points. For testing the selectivity of the effect of the non-NMDA receptor antagonist given into the paraventricular nucleus, the non-NMDA receptor antagonist was also injected into the area of the dorsomedial nucleus or the arcuate nucleus.
2.2. Implantation of bilateral guide cannulae into the hypothalamus Dams were anaesthesized (chloral hydrate, 400 mg/kg, i.p.) and stereotaxically implanted with double guide (26 gauge) cannulae (Plastics One Inc., Roanoke, VA, USA). In the case of microinjections into the paraventricular and dorsomedial nuclei the tubing length below pedestal was 7.0 mm and center to center distance between the two tubings was in all three cases 1.0 mm. Double dummy cannulae (obturator) sealed the top of the double guide cannulae. The obturator had two stainless steel wires, which were inserted into the double guide cannula tubes. A dust cap secured the unit to the double guide cannulae. The cannulae were fixed by screws and dental cement to the top of the skull. The stereotaxic coordinates taking the bregma suture as zero reference point [20] used for the implantations were: (i) Paraventricular nucleus: AP, bregma −1.7 mm; V, 7.1 mm. (ii) Dorsomedial nucleus: AP, bregma −3.1 mm; V, 7.1 mm. In this case the double cannulae were placed in a similar position than those for the paraventricular nuclei except they were 1.4 mm more caudal and were considered as a control to test the selectivity of the effect of the paraventricular microinjections. (iii) Arcuate nucleus: AP, bregma −3.0; V, 9.0 mm.
2. Materials and methods
In all three cases the two guide cannulae were in the coronal plane and 0.5 mm laterally from the midline (interhemispheric fissure). Implantation of bilateral guide cannulae was made on postpartum days 3–4. Rats were given a 5–7 day recovery period, during which they were handled for the microinjection procedure.
2.1. Animals
2.3. Microinjections
Primiparous lactating female rats were used. They were bred in our animal facilities from Sprague–Dawley stock. The rats were individually housed with their litters; litter size was reduced to eight. The rats were maintained in a climateand illumination-controlled room (14:10-h light/14:10-h dark cycle; lights on at 06:00 h). Water and standard rat chow were available ad libitum. All animal experiments were conducted in accordance with the Guide of the University for the Care and Use of Laboratory Animals. Microinjections were given into the hypothalamic:
Microinjections were made in non-anaesthesized rats. The inserted double internal cannulae (gauge 33) (Plastics One Inc., Roanoke, VA, USA) extended 1.0 mm below the tip of the guide cannulae. They were attached to Teflon tubing and supplied by a 0.5 l Hamilton syringe. Animals received through the internal cannulae 0.1 l distilled water or one of the glutamate receptor antagonists: 6-cyano-7-nitroquinoxaline2,3-dione disodium, a competitive non-NMDA glutamate receptor antagonist (Sigma–Aldrich) (20 mM solved in 0.1 ml distilled water) or dizocipine hydrogen malate (MK-801) (20 mM solved in 0.1 ml distilled water), a selective non-competitive NMDA receptor antagonist (Sigma–Aldrich). Water was injected into the paraventricular and the arcuate nucleus, CNQX into the paraventricular nucleus, the dorsomedial nucleus area and the arcuate nucleus. MK-801 was given into the paraventricular nucleus.
• paraventricular nucleus; • dorsomedial nucleus; and • arcuate nucleus. Cannulae for injections were inserted through bilateral guide cannulae.
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Microinjections were made on the day of testing the suckling-induced prolactin release 15 min before the reunion of the dams with their pups. 2.4. Testing the suckling-induced prolactin release: blood sampling The suckling-induced prolactin releasing effect was examined on postpartum days 8–10. Prior to exposing the mothers to the suckling stimulus, the dams were separated from their pups for 4 h. Two days prior to taking blood samples, a permanent cannula (silicon tubing inner diameter 0.50 mm, external diameter 0.90 mm; Dow Corning Corp., Midland, MI, USA and Becton-Dickinson, Parsipparny, NY, USA) was implanted into the jugular vein under chloral hydrate anaesthesia, allowing frequent blood sampling from freely moving rats. The rats were habituated to the procedure of blood sampling by connecting them to a polyethylene tube every day. On the day of experiment the first blood sample was taken before separation. Fifteen minutes before the end of the separation period a second blood sample was taken and the animals received the intrahypothalamic microinjection. Fifteen minutes later another blood sample was taken and the pups were returned to the dams. Full pup attachment was achieved within 5 min after reunion. Further blood samples were taken 15, 30 and 60 min after reunion (using only the rats showing full pup attachment, which was true for approximately 90% of each group, and was not influenced by the type of microinjection). At each time point 200 l blood was obtained, plasma was separated and stored at −20 ◦ C until assayed for prolactin. 2.5. Hormone analysis Prolactin was measured by radioimmunoassay (RIA), with kits kindly provided by NHPP, NIDDK and Dr. A.F. Parlow. The RIA procedure was similar to the instructions supplied with the kit, with modifications as described previously [12]. We used the Chloramine-T method for iodination and protein A (BactASorb, Human Rt, G¨od¨oll˝o, Hungary) for separation of bound and free hormone. Data collection and calculations for curve fitting were made using LKB Clinigamma software. The data were expressed in terms of NIAMDD-Rat-RP-3. The within- and between-assay variances were 10 and 14%, respectively. The sensitivity of the prolactin assay was 0.5 ng/ml rat plasma, expressed in terms of the rat prolactin RP-3 standard (or 25 pg PRL). We used 50 l plasma per samples. All samples were measured in duplicate. 2.6. Brain histology After the last blood sample was taken, rats were perfused under deep hexobarbital anaesthesia with 200 ml saline followed by 250 ml 4% paraformaldehyde via transcardiac
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puncture. The cannulae were taken out from the brains and the brains were removed and placed in the same fixative at 4 ◦ C for 24 h. Subsequently, they were transferred to a buffered sucrose solution (25% sucrose in phosphate buffer saturated) for 24 h. For histological localization of the microinjections, 20 m thick coronal sections were made by means of a cryostat of all brains. The sections were stained with haematoxylin and eosin and the localization of each microinjection determined. Only those rats in which the tips of the internal cannulae were verified to be in the right place were included in the study. 2.7. Statistical analysis Statistical analysis of the data (amplitude of the response) was performed using two-way ANOVA followed by Dunnett’s test for multiple comparisons. Values are considered significantly different at p < 0.05.
3. Results Animals were grouped according to the histological assessment of cannulae placement. Only injections that appeared to be centered within the paraventricular nuclei were designated as paraventricular microinjections (Fig. 1A), those centered within the dorsomedial nuclei area as dorsomedial microinjections (Fig. 1B) and those centered within the arcuate nuclei as arcuate microinjections (Fig. 1C). The proportion of the successful placements of the cannulae was about 50%. Animals in which the microinjections were misplaced had been discarded. Microinjections of the solvent into the paraventricular or into the arcuate nuclei did not interfere with the rise in plasma prolactin concentration induced by the suckling stimulus (Figs. 2 and 3). In animals receiving bilateral microinjections of the nonNMDA receptor antagonist CNQX into the paraventricular nucleus, there was no change in hormone concentration after replacement of the pups, suckling did not induce any elevation in plasma prolactin concentration (Fig. 2). The blockade of the prolactin response compared to the response of all other groups is statistically significantly different (p < 0.01). Dams, which received microinjections of the NMDA receptor antagonist MK-801 into the paraventricular nuclei, did respond to the suckling stimulus (Fig. 2). Microinjections of the non-NMDA receptor antagonist CNQX into the area of the dorsomedial nucleus or into the arcuate nucleus did not affect the prolactin response. In these two groups, in contrast to those animals receiving the same receptor antagonist into the paraventricular nuclei, suckling induced a significant rise in plasma prolactin concentration (Fig. 3). The elevations in plasma prolactin concentrations induced by the suckling stimulus are statistically significant in all groups in which there was a rise in hormone levels (p < 0.05).
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Fig. 1. Light micrographs show the location of the tracks of internal cannulae (TIC) for the microinjections into the paraventricular nucleus (PVN) (A), dorsomedial nucleus (DMN) (B) and arcuate nucleus (AN) (C). 3V: third ventricle; TGC: track of the guide cannula. Scale bars: 100 m.
4. Discussion Our present findings indicate that a non-NMDA receptor antagonist injected bilaterally into the hypothalamic paraventricular nucleus interferes with the suckling stimulus-induced release of prolactin. The observations are in line with previous investigations showing that injection of the non-NMDA receptor antagonist, CNQX into the third ventricle [19] or combined treatment with non-NMDA and NMDA receptor antagonists giving the drugs i.v. [30], blocked the suckling stimulus-induced prolactin elevation. In addition, our results provide for the first time direct information about the site of action of the receptor antagonist in blocking the prolactin response. They demonstrate that this action of the drug was exerted on the paraventricular nucleus. A similar effect was not observed if the same receptor antagonist was administered into the area of the dorsomedial nucleus or the arcuate nucleus. The fact that the non-NMDA receptor antagonist was effective in blocking the prolactin response only in that case when it was injected into the paraventricular nucleus, strongly suggests that the glutamatergic innervation of the cell group is involved in the mediation of the suckling stimulus. The glutamatergic fibers terminating in the paraventricular nuclei originate partly from glutamatergic neurons of several diencephalic and telencephalic structures and partly belong to glutamatergic interneurons of the paraventricular nucleus [5]. It should be pointed out that glutamatergic neurons in the brain stem projecting to the paraventricular nuclei were not detected [5]. When taking into account that the signal of the suckling stimulus is mediated through the spinal cord and brain stem up to the hypothalamus and further, that glutamatergic neurons in the brain stem do not terminate in the paraventricular nuclei, it is close at hand to assume that the glutamatergic innervation of the paraventricular nucleus to be involved in the mediation of the suckling stimulus, most probably belongs to the glutamatergic interneurons of the cell group. We postulate that these interneurons take part in forwarding the stimulatory influ-
ence of the suckling stimulus reaching the paraventricular nuclei. A large amount of information has been accumulated suggesting that serotonergic elements arising from the raphe nuclei [23] and terminating in the hypothalamic paraventricular nucleus are primarily involved in leading the suckling stimulus to the cell group [3,14,15,17,18,28]. Our present findings suggest that the glutamatergic interneurons in the paraventricular nucleus are participating in the mediation of the stimulatory effect of the serotonergic afferents terminating in the paraventricular nucleus. In a recent publication Feldman and Weidenfeld [9] have formulated an almost identical concept. They postulated that glutamatergic interneurons in the paraventricular nucleus acting via non-NMDA and NMDA receptors might act as an excitatory mechanism in the norepinephrine and serotonin control of hypothalamic adrenocorticotropin secretagogues. Daftary et al. [6] have provided data suggesting that glutamatergic interneurons in the paraventricular nucleus serve as an excitatory relay in the afferent noradrenergic control of oxytocin and vasopressin release under certain physiological conditions. Pirnik et al. [22] have provided evidence that oxytocin and vasopressin neurons in the paraventricular nucleus play important role in the mediation of signals induced by hypertonic saline. In the present investigations only CNQX, the non-NMDA receptor antagonist was effective in interfering with the prolactin response to the suckling stimulus. The NMDA receptor antagonist MK-801 administered into the paraventricular nucleus did not cause any significant alteration in the plasma prolactin concentration of dams exposed to the suckling stimulus. van den Pol et al. [27] have reported that spontaneous excitatory postsynaptic potentials in the paraventricular nucleus were markedly attenuated by application of the non-NMDA receptor antagonist, CNQX. NMDA antagonists only slightly suppressed excitatory postsynaptic potentials. These findings suggest that in the paraventricular nucleus the excitatory amino acid acts primarily on non-NMDA receptors.
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Fig. 2. Plasma prolactin concentrations of dams separated for 4 h from their litters, receiving microinjection of the solvent (water), the non-NMDA antagonist CNQX or the NMDA antagonist MK-801 into the paraventricular nucleus (PVN) and 15 min later exposed to the suckling stimulus. Separation of the mothers caused the well-known marked reduction in hormone concentrations. Suckling induced a statistically significant elevation in plasma prolactin of rats getting water or MK-801 into the paraventricular nucleus, but it did not result in any increase in hormone concentrations in rats receiving microinjection of CNQX into this cell group, n: number of animals.
The present findings do not provide any information on where the neural afferent component of the suckling stimulus-induced release of prolactin is transformed into a humoral efferent reflex answer expressed via changes in the secretion of prolactin releasing and/or release-inhibiting factors (vasoactive intestinal peptide, oxytocin, vasopressin, salsolinol, dopamine) produced by hypothalamic neurons [10,24]. Further investigations are needed to study this question.
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Fig. 3. Plasma prolactin concentrations of dams separated for 4 h from their litters receiving microinjection of the solvent (water) into the arcuate nucleus (AN), CNQX into the dorsomedial nucleus area (DMN) or into the arcuate nucleus (AN) and 15 min later exposed to the suckling stimulus. Rats getting CNQX into the dorsomedial nucleus area or into the arcuate nucleus responded to the suckling stimulus, n: number of animals.
Acknowledgements The authors are grateful to Mrs. M. M´esz´aros and Mrs. M. Bal´azs for their excellent technical assistance, to Mrs. I. Salamon for excellent histotechnical work and Mrs. E. Pirity for typing the manuscript. This work was supported by the Ministry of Health (ETT 054/2003 to B.H.), National Scientific Research Fund (OTKA T-042516 to B.H.) and the Hungarian Academy of Sciences. References [1] W.M. Al-Ghoul, R.B. Meeker, R.S. Greenwood, Differential expression of five N-methyl-d-aspartate receptor subunit mRNAs in vaso-
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