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Neuropeptide Y and [Leu31,Pro34]neuropeptide Y potentiate potassium-induced noradrenaline release in the paraventricular nucleus of the aged rat
Brain Research 750 Ž1997. 301–304
Short communication
Neuropeptide Y and wLeu31,Pro 34 xneuropeptide Y potentiate potassium-induced noradrenaline release in the paraventricular nucleus of the aged rat J.A. Hastings a , J.M. Pavia a , M.J. Morris b
b, )
a School of Biological and Chemical Sciences, Deakin UniÕersity, Victoria 3217, Australia Department of Pharmacology, The UniÕersity of Melbourne, ParkÕille, Victoria 3052, Australia
Accepted 10 December 1996
Abstract This microdialysis study investigated the effects of NPY and the Y1 selective agonist wLeu31,Pro 34 xNPY on basal and potassiumstimulated noradrenaline release in the PVN of 18-month-old anaesthetised male Sprague–Dawley rats. Microdialysate noradrenaline, DOPAC and HVA concentrations were measured by HPLC after i.c.v. administration of 2 nmol NPY, wLeu31,Pro 34 xNPY or vehicle. wLeu31,Pro 34 xNPY produced a significant 40% reduction in basal noradrenaline concentration Ž P - 0.05.. Aged rats had blunted noradrenaline responses to potassium stimulation, however stimulated noradrenaline release was similar in 18-month-old NPY-treated animals and 3-month-old saline treated age controls Ž2.8 and 3.2 times resting, respectively.. wLeu31,Pro 34 xNPY induced a significantly greater release of noradrenaline in response to KCl Ž5.0 times resting, P - 0.05.. Thus, in 18-month-old animals with reduced endogenous hypothalamic NPY content, administration of NPY or wLeu31,Pro 34 xNPY increased potassium-induced noradrenaline release to levels seen in 3-month-old rats. This effect may be mediated by an NPY Y1 receptor. Keywords: Ageing; Hypothalamic paraventricular nucleus; Microdialysis, in vivo; wLeu31 ,Pro 34 xNPY; Neuropeptide Y; Noradrenaline
Neuropeptide Y ŽNPY., a 36 amino acid peptide, is extensively co-localised with noradrenaline in nerves supplying the cardiovascular system w15x. NPY is co-released with noradrenaline from sympathetic nerves w10,15x. In the central nervous system ŽCNS., NPY is co-localised with medullary catecholamines in the A1, A2 and A6 cell groups which send afferent fibres to the paraventricular nucleus ŽPVN. of the hypothalamus w1,17x. NPY regulates a number of functions in the CNS, including blood pressure, feeding, drinking, temperature, memory, and exerts neuroendocrine effects on luteinizing hormone and the hypothalamo-pituitary-adrenal axis w5,15,19x. Recent evidence supports important co-modulatory actions of NPY and noradrenaline, with reciprocal inhibitory effects of each transmitter on the release of its co-transmitter described in the periphery w15x. Work to date has provided evidence for an interaction between NPY and a 2-adrenoceptors in various brain regions w20x, however
) Corresponding author. Fax: q61 Ž3. 9347-1452; E-mail: [email protected]
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 4 7 5 - 8
the physiological role of NPY in the modulation of noradrenergic transmission in the CNS remains unclear. NPY receptors have been classified into various types according to the potency order of analogues, and multiple NPY receptor subtypes are present in the brain w2,8,19x. Few studies have investigated the effects of NPY on noradrenaline release in the brain in vivo. We reported a stimulatory effect of intracerebroventricular Ži.c.v.. NPY administration on potassium-induced noradrenaline release in the PVN of 3-month-old rats w14x. Furthermore, we observed reduced noradrenaline release to maximal stimulation with potassium in 18-month-old animals w3x, in whom endogenous NPY concentrations are markedly reduced w13,16x. Therefore, the present in vivo microdialysis study examined the effect of exogenous NPY and the selective NPY Y1 agonist wLeu31 ,Pro 34 xNPY on the release of noradrenaline and associated dopamine metabolites, dihydroxyphenylacetic acid ŽDOPAC. and homovanillic acid ŽHVA., from the PVN of the 18-month-old rat. Nineteen aged Ž18.5 " 0.7 months; 614 " 14 g. and five adult Ž3.5 " 0.2 months; 411 " 30 g. male Sprague–Dawley rats were housed at 218C under a 12 h lightrdark cycle
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with free access to standard rat chow and water. All procedures were approved by the Deakin University Animal Experimentation Ethics Committee. Animals were anaesthetised with 10% urethane Ž140 mgr100 g b.wt, i.p.. supplemented two hourly, and ventilated with room air through a Harvard rodent respirator ŽHarvard Apparatus Inc, MA, USA.. A femoral artery was cannulated to allow continuous monitoring of mean arterial pressure ŽMAP. and heart rate ŽHR. via a pressure transducer ŽBentley Trantec, Model 800, CA, USA.. Animals were placed in a stereotaxic frame ŽDavid Kopf Instruments, CA, USA. and an i.c.v. cannula Ž0.4 mm O.D.. was placed in the lateral ventricle Žanterior y0.8 mm; lateral 1.5 mm; ventral y3.7 mm relative to bregma.. A microdialysis probe ŽCMAr12; CMArMicrodialysis, Sweden. was inserted into the hypothalamic PVN Žanterior y1.6 mm; lateral 0.6 mm; ventral y8.5 mm. w11x. Modified physiological Ringer solution; Žin mM. NaCl 132; KCl 3; CaCl 2 1.26; MgCl 2 1.18; NaH 2 PO4 0.3; Na 2 HPO4 1.2; pH 7.12, was continuously perfused through the probe at 1.1 mlrmin by an Ismatec peristaltic pump ŽCole Palmer Instruments, IL, USA.. After 90 min equilibration, dialysate collections were made every 30 min on ice into hydrochloric acid Žfinal concentration 0.1 M.. Following three basal collections, either 2 nmol NPY Ž n s 8., 2 nmol wLeu31 ,Pro 34 xNPY Ž n s 5. in 4 ml saline, or 4 ml saline vehicle Ž n s 6. was injected i.c.v. over 90 s in separate groups of 18-month-old rats. Saline vehicle was injected into five 3-month-old rats serving as an age control. Sixty minutes later noradrenaline release was stimulated during one collection using modified Ringer solution containing 100 mM KCl and 32 mM NaCl. Normal modified Ringer solution was returned in the final dialysate collection to assess recovery. At the end of the experiment the i.c.v. cannula location was checked using toluidine blue dye and the probe implantation site was verified histologically. Each dialysate collection was analysed by reverse-phase HPLC with electrochemical detection using a Rheodyne 7125 syringe-loading injector, a phase-II narrow bore column Ž100 = 3.2 mm I.D., 3 mm ODS, Bioanalytical Systems, IA, USA. and a dual glassy-carbon working electrode Žoxidation potential q775 mV. w11x. Mobile phase was recirculated at 0.6 mlrmin. All chemicals were of analytical quality. The detection limit for noradrenaline was 1–2 pg w11x. Under these conditions, the noradrenaline metabolites 3-methoxy-4-hydroxyphenylethylene glycol ŽMOPEG . and 3,4-dihydroxyphenylethylene glycol ŽDOPEG. were not observed, and levels of adrenaline and dopamine in the rat microdialysates were below detection. Resting dialysate concentrations of noradrenaline were similar in all groups of 18-month-old rats Žaverage 87.6 " 6.1 pgrml, n s 19, mean " S.E.M... To determine overall time- and treatment-related differences, noradrenaline, DOPAC and HVA concentrations, expressed as a percentage of each animal’s basal concentration, were analysed by
repeated measures ANOVA followed by least significant difference tests ŽLSD.. The responses to potassium-induced depolarisation were also compared by one-factor ANOVA of the fold changes induced ŽFig. 1.. A significant reduction in microdialysate noradrenaline concentration was observed following i.c.v. administration of 2 nmol wLeu31 ,Pro 34 xNPY but not NPY ŽFig. 1, P 0.05.. Perfusion of a depolarising concentration of 100 mM KCl through the microdialysis probe produced an increase in noradrenaline release in 18-month-old rats although this was blunted in the saline treated group ŽFig. 1.. Prior administration of NPY or wLeu31 ,Pro 34 xNPY resulted in enhanced responses to KCl compared to saline pretreatment Ž P - 0.01, Fig. 1.. When the fold increases in noradrenaline concentrations in response to potassium were compared, 3-month-old rats Žage control. treated with saline showed a similar increase Ž3.2 " 0.5 times resting. to that observed in 18-month-old rats treated with NPY Ž2.8 " 0.7 times., while after wLeu31 ,Pro 34 xNPY, potassium produced a significantly greater increase Ž5.0 " 0.9 times, P - 0.05 compared to NPY or saline pretreatment.. Administration of NPY and wLeu31 ,Pro 34 xNPY produced cardiovascular effects, with increases in MAP and HR of 25 " 4 mmHg and 35 " 9 b.p.m. Ž n s 8. and 15 " 2 mmHg and 62 " 12 b.p.m. Ž n s 5., respectively, significantly greater than the changes observed after saline Ž P - 0.05.. Basal microdialysate concentrations of the dopamine metabolites DOPAC and HVA were similar across the three groups of 18-month-old rats Žaverage 3.2 " 0.5 and 2.9 " 0.6 ngrml, respectively. and were not different to levels in 3-month-old rats. Administration of NPY or wLeu31 ,Pro 34 xNPY had no effect on basal DOPAC and HVA levels ŽTable 1.. Potassium depolarisation significantly reduced DOPAC and HVA levels in all 18-monthold rats ŽTable 1, P - 0.01. to a similar extent to that
Fig. 1. Noradrenaline, expressed as % basal concentration, in 30 min dialysate collections from the PVN of 18-month-old rats, before and after i.c.v. injection Žarrow. of saline vehicle ŽI, ns6., NPY ŽB, ns8. and wLeu31 ,Pro 34 xNPY Ž Ø , ns 5.. Potassium depolarisation ŽKq . was evoked by perfusing KCl through the probe as indicated. Data were analysed by one-way ANOVA with repeated measures and LSD tests. ), Significantly different to preceding collection, Ž P - 0.05.; †, significantly different to NPY and saline-treated rats, Ž P - 0.05..
J.A. Hastings et al.r Brain Research 750 (1997) 301–304
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Table 1 PVN microdialysate dihydroxyphenylacetic acid ŽDOPAC. and homovanillic acid ŽHVA. levels, expressed as % basal concentration, in 30 min dialysate collections in 18-month-old rats treated with i.c.v. saline, NPY or wLeu31 ,Pro 34 xNPY
DOPAC Saline NPY wLeu31 ,Pro 34 xNPY HVA Saline NPY wLeu31 ,Pro 34 xNPY a
Resting
Post i.c.v. drug
Pre Kq
Kq
104.0 " 5.8 105.7 " 6.5 111.2 " 7.5
100.8 " 7.7 116.7 " 5.9 107.5 " 6.5
120.9 " 16.8 110.0 " 9.0 102.7 " 8.2
87.0 " 12.4 a 58.8 " 7.8 a 69.3 " 6.2 a
96.9 " 6.8 111.3 " 4.4 105.5 " 5.2
103.4 " 7.6 127.8 " 7.6 108.2 " 5.5
104.8 " 15.1 118.9 " 13.4 111.5 " 6.8
60.0 " 7.6 49.3 " 9.7 56.1 " 9.5
a a a
Significantly different to preceding collection, P - 0.01; n s 6, 8, 5.
observed in 3-month-old animals Ž64.9 " 6.1 and 56.9 " 8.9% basal, respectively.. The major finding of this study is that in the hypothalamic PVN of 18-month-old rats, exogenous NPY and wLeu31 ,Pro 34 xNPY potentiated the potassium-induced increase in noradrenaline release. The blunted depolarisation response seen in 18-month-old rats was increased by pretreatment with NPY to levels observed in younger animals in the absence of NPY. At equimolar doses the potent selective NPY Y1 agonist wLeu31 ,Pro 34 xNPY produced a significantly greater potentiation of potassium-stimulated noradrenaline release than NPY. We have previously reported a facilitatory effect of NPY on potassium-induced noradrenaline release in 3–4-month-old rats w14x; results of the present study suggest that a Y1 NPY receptor may be responsible for this effect. wLeu31 ,Pro 34 xNPY caused a 40% reduction in basal PVN noradrenaline release. Several NPY receptor subtypes are present in rat brain ŽY1 , Y2 , Y3 , Y4 and Y5 . with regional and species differences in receptor density w2,7,8,19x. Several of these receptor subtypes have been localised to the hypothalamus, and Y1 and Y2 receptors are expressed in the PVN w7x. Whether wLeu31 ,Pro 34 xNPY and NPY affect noradrenaline release by stimulating Y1 NPY receptors located pre-synaptically or post-synaptically in the hypothalamus requires further investigation. We cannot rule out the possibility that NPY receptors in other brain regions are being activated or that the effect is being mediated by another NPY receptor subtype. For example, wLeu31 ,Pro 34 xNPY was recently shown to bind to rat Y5 receptors with an EC 50 in the nM range w2x. NPY may also be mediating effects via a 2adrenoceptors as several lines of evidence suggest an interaction between NPY receptors and a 2-adrenoceptors in the brain w20x. Similar basal concentrations of noradrenaline were detected in the PVN of both age groups, thus the functional pool of noradrenergic neurons in the PVN which we were examining was apparently largely preserved with ageing w3x. However, the 18-month-old rats had a reduced ability to release noradrenaline upon potassium depolarisation. As aged animals have markedly reduced hypothalamic NPY concentrations w13,16x we reasoned that physiological
changes in endogenous NPY with ageing may affect the ability to release noradrenaline in response to a stimulus. Exogenous NPY and wLeu31 ,Pro 34 xNPY potentiated the noradrenaline response induced by potassium in 18-monthold rats. Clearly factors other than changes in NPY or NPY receptors may contribute to the reduced responsiveness seen in 18-month-old rats. Decreased noradrenaline responsiveness to potassium in 18-month-old saline-treated animals may be related to changes in the density of a 2-adrenoceptors, alterations in neuronal uptake or vesicular monoamine transport with ageing w3x. Age-related changes in other neurotransmitters which modulate noradrenaline may also affect the ability to release noradrenaline. The nature of the NPY-noradrenaline interaction, and indeed the NPY receptor subtypes involved in the regulation of noradrenaline release in the CNS in vivo remains to be elucidated. The mechanism by which NPY and its analogues might reduce basal release but increase stimulated release is unclear. Results of other studies examining the effect of NPY on hypothalamic noradrenaline release in vivo reveal site-specific effects, with a reduction in noradrenaline release in the ventro-medial hypothalamus and an increase in the lateral hypothalamus w18x and the perifornical region w12x. Food availability also influenced the NPY-induced changes in extracellular noradrenaline concentrations w6x. These results probably reflect regional differences in NPY receptor distribution, effective NPY dose, and the presence of anaesthesia. Any alteration in CNS noradrenaline-NPY interactions with ageing may have implications for the regulation of cardiovascular and ingestive functions subserved by these transmitters in the hypothalamus w2,11,15,19x. The PVN receives a high density of noradrenergic neurons, therefore it is likely that some of the observed DOPAC and HVA originated in noradrenergic neurons. No significant alterations in microdialysate DOPAC or HVA concentrations were observed following i.c.v. NPY or wLeu31 ,Pro 34 xNPY, thus the effect appears selective for noradrenaline. While a dose-related increase in hypothalamic dopamine and DOPAC content has been reported following treatment with NPY w4x, few studies have exam-
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ined the effect of NPY on the release of dopamine metabolites in vivo. No age-related differences in potassiumstimulated reductions in DOPAC and HVA concentrations were observed. Reductions in DOPAC and HVA possibly reflect a shift in catecholaminergic metabolism during depolarisation towards an increasing production of noradrenaline, therefore decreasing dopamine availability for metabolism into DOPAC and HVA in noradrenergic neurons w11x. The observed reductions in DOPAC and HVA concentrations with potassium stimulation may also be attributed to a reduced efflux via a membrane potential-dependent process w9x. Few studies have investigated the relationship between dopamine metabolism and NPY and clearly more work needs to be carried out regarding the interaction between NPY and catecholamine metabolism in general. In conclusion, administration of wLeu31 ,Pro 34 xNPY to 18-month-old rats caused a reduction in basal noradrenaline release in the PVN of the hypothalamus. In 18-monthold rats with marked reductions in endogenous hypothalamic NPY, exogenous NPY and the NPY Y1 agonist wLeu31 ,Pro 34 xNPY potentiated potassium-stimulated noradrenaline release in the PVN, suggesting the involvement of a Y1 or Y5 receptor in mediating this effect.
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Acknowledgements This work was supported by the National Health and Medical Research Council of Australia. We are indebted to the William Buckland Foundation for equipment support. J.A.H. was supported by a Deakin University Postgraduate Scholarship.
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References w1x Everitt, B.J., Hokfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and ¨ Goldstein, M., Differential co-existence of neuropeptide Y ŽNPY.like immunoreactivity with catecholamines in the central nervous system of the rat, Neuroscience, 11 Ž1984. 443–462. w2x Gerald, C., Walker, M.W., Criscione, L., Gustafson, E.L., BatziHartmann, C., Smith, K.E., Vaysse, P., Durkin, M.M., Laz, T.M., Linemeyer, D.L., Schaffhauser, A.O., Whitebread, S., Hofbauer, K.G., Taber, R.I., Branchek, T.A. and Weinshank, R.L., A receptor subtype involved in neuropeptide-Y-induced food intake, Nature, 382 Ž1996. 168–171. w3x Hastings, J.A., Pavia, J.M. and Morris, M.J., Altered in vivo catecholamine release in the hypothalamic paraventricular nucleus of the aged rat, Naunyn-Schmiedeberg’s Arch. Pharmacol., 353 Ž1996. 161–167. w4x Heilig, M., Vecsei, ` ´ L., Wahlestedt, C., Alling, Ch. and Widerlov, ¨ E., Effects of centrally administered neuropeptide Y ŽNPY. and NPY13-36
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on the brain monoaminergic systems of the rat, J. Neural Transm., 79 Ž1990. 193–208. Kalra, S.P. and Crowley W. R., Neuropeptide Y: a novel neuroendocrine peptide in the control of pituitary hormone secretion, and its relation to luteinizing hormone, Front. Neuroendocrinol., 13 Ž1992. 1–46. Kyrkouli, S.E., Stanley, B.G. and Leibowitz, S.F., Differential effects of galanin and neuropeptide Y on extracellular norepinephrine levels in the paraventricular hypothalamic nucleus of the rat: a microdialysis study, Life Sci., 51 Ž1992. 203–210. Larsen, P.J., Sheikh, S.P., Jakobsen, C.R., Schwartz, T.W. and Mikkelsen, J.D., Regional distribution of putative NPY Y1 receptors and neurons expressing Y1 mRNA in forebrain areas of the rat central nervous system, Eur. J. Neurosci., 5 Ž1993. 1622–1637. Lundell, I., Statnick, M.A., Johnson, D., Schober, D.A., Starback, ¨ P., Gehlert, D.R. and Larhammar, D., The cloned rat pancreatic polypeptide receptor exhibits profound differences to the orthologous human receptor, Proc. Natl. Acad. Sci. USA, 93 Ž1996. 5111– 5115. Miyamoto, J.K., Uezu, E. and Terashima, S.-I., Active transport pumps of HVA and DOPAC in dopaminergic nerve terminals, Physiol. BehaÕ., 49 Ž1991. 141–147. Morris, M.J., Russell, A.E., Kapoor, V., Cain, M.D., Elliott, J.M., West, M.J., Wing, L.M.H. and Chalmers, J.P., Increases in plasma neuropeptide Y concentrations during sympathetic activation in man, J. Auton. NerÕ. Syst., 17 Ž1986. 143–149. Morris, M.J., Hastings, J.A. and Pavia, J.M., Catecholamine release in the rat hypothalamic paraventricular nucleus in response to haemorrhage, desipramine and potassium, Brain Res., 665 Ž1994. 5–12. Myers, R.D., Lankford, M.F. and Paez, X., Norepinephrine, dopamine, and 5-HT release from perfused hypothalamus of the rat during feeding induced by neuropeptide Y, Neurochem. Res., 17 Ž1992. 1123–1132. Pavia, J.M. and Morris, M.J., Age-related changes in neuropeptide Y content in brain and peripheral tissues of spontaneously hypertensive rats, Clin. Exp. Pharmacol. Physiol., 21 Ž1994. 335–338. Pavia, J.M., Hastings, J.A. and Morris, M.J., Neuropeptide Y potentiation of potassium-induced noradrenaline release in the hypothalamic paraventricular nucleus of the rat in vivo, Brain Res., 690 Ž1995. 108–111. Potter, E.K., Neuropeptide Y as an autonomic neurotransmitter, Pharmacol. Ther., 37 Ž1988. 251–273. Sahu, A., Kalra, P.S., Crowley, W.R. and Kalra, S.P., Evidence that hypothalamic neuropeptide Y secretion decreases in aged male rats: Implications for reproductive aging, Endocrinology, 122 Ž1988. 2199–2203. Sawchenko, P.E., Swanson, L.W., Grzanna, R., Howe, P.R.C., Bloom, S.R. and Polak, J.M., Colocalization of neuropeptide Y immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus, J. Comp. Neurol., 241 Ž1985. 138–153. Shimizu, H. and Bray, G.A., Effects of neuropeptide Y on norepinephrine and serotonin metabolism in rat hypothalamus in vivo, Brain Res. Bull., 22 Ž1989. 945–950. Wahlestedt, C. and Reis, D.J., Neuropeptide Y-related peptides and their receptors-are the receptors potential therapeutic drug targets? Annu. ReÕ. Pharmacol. Toxicol., 32 Ž1993. 309–352. Widdowson, P.S., Masten, T. and Halaris, A.E., Interactions between neuropeptide Y and alpha2-adrenoceptors in selective rat brain regions, Peptides, 12 Ž1991. 71–75.
Short communication
Neuropeptide Y and wLeu31,Pro 34 xneuropeptide Y potentiate potassium-induced noradrenaline release in the paraventricular nucleus of the aged rat J.A. Hastings a , J.M. Pavia a , M.J. Morris b
b, )
a School of Biological and Chemical Sciences, Deakin UniÕersity, Victoria 3217, Australia Department of Pharmacology, The UniÕersity of Melbourne, ParkÕille, Victoria 3052, Australia
Accepted 10 December 1996
Abstract This microdialysis study investigated the effects of NPY and the Y1 selective agonist wLeu31,Pro 34 xNPY on basal and potassiumstimulated noradrenaline release in the PVN of 18-month-old anaesthetised male Sprague–Dawley rats. Microdialysate noradrenaline, DOPAC and HVA concentrations were measured by HPLC after i.c.v. administration of 2 nmol NPY, wLeu31,Pro 34 xNPY or vehicle. wLeu31,Pro 34 xNPY produced a significant 40% reduction in basal noradrenaline concentration Ž P - 0.05.. Aged rats had blunted noradrenaline responses to potassium stimulation, however stimulated noradrenaline release was similar in 18-month-old NPY-treated animals and 3-month-old saline treated age controls Ž2.8 and 3.2 times resting, respectively.. wLeu31,Pro 34 xNPY induced a significantly greater release of noradrenaline in response to KCl Ž5.0 times resting, P - 0.05.. Thus, in 18-month-old animals with reduced endogenous hypothalamic NPY content, administration of NPY or wLeu31,Pro 34 xNPY increased potassium-induced noradrenaline release to levels seen in 3-month-old rats. This effect may be mediated by an NPY Y1 receptor. Keywords: Ageing; Hypothalamic paraventricular nucleus; Microdialysis, in vivo; wLeu31 ,Pro 34 xNPY; Neuropeptide Y; Noradrenaline
Neuropeptide Y ŽNPY., a 36 amino acid peptide, is extensively co-localised with noradrenaline in nerves supplying the cardiovascular system w15x. NPY is co-released with noradrenaline from sympathetic nerves w10,15x. In the central nervous system ŽCNS., NPY is co-localised with medullary catecholamines in the A1, A2 and A6 cell groups which send afferent fibres to the paraventricular nucleus ŽPVN. of the hypothalamus w1,17x. NPY regulates a number of functions in the CNS, including blood pressure, feeding, drinking, temperature, memory, and exerts neuroendocrine effects on luteinizing hormone and the hypothalamo-pituitary-adrenal axis w5,15,19x. Recent evidence supports important co-modulatory actions of NPY and noradrenaline, with reciprocal inhibitory effects of each transmitter on the release of its co-transmitter described in the periphery w15x. Work to date has provided evidence for an interaction between NPY and a 2-adrenoceptors in various brain regions w20x, however
) Corresponding author. Fax: q61 Ž3. 9347-1452; E-mail: [email protected]
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 4 7 5 - 8
the physiological role of NPY in the modulation of noradrenergic transmission in the CNS remains unclear. NPY receptors have been classified into various types according to the potency order of analogues, and multiple NPY receptor subtypes are present in the brain w2,8,19x. Few studies have investigated the effects of NPY on noradrenaline release in the brain in vivo. We reported a stimulatory effect of intracerebroventricular Ži.c.v.. NPY administration on potassium-induced noradrenaline release in the PVN of 3-month-old rats w14x. Furthermore, we observed reduced noradrenaline release to maximal stimulation with potassium in 18-month-old animals w3x, in whom endogenous NPY concentrations are markedly reduced w13,16x. Therefore, the present in vivo microdialysis study examined the effect of exogenous NPY and the selective NPY Y1 agonist wLeu31 ,Pro 34 xNPY on the release of noradrenaline and associated dopamine metabolites, dihydroxyphenylacetic acid ŽDOPAC. and homovanillic acid ŽHVA., from the PVN of the 18-month-old rat. Nineteen aged Ž18.5 " 0.7 months; 614 " 14 g. and five adult Ž3.5 " 0.2 months; 411 " 30 g. male Sprague–Dawley rats were housed at 218C under a 12 h lightrdark cycle
302
J.A. Hastings et al.r Brain Research 750 (1997) 301–304
with free access to standard rat chow and water. All procedures were approved by the Deakin University Animal Experimentation Ethics Committee. Animals were anaesthetised with 10% urethane Ž140 mgr100 g b.wt, i.p.. supplemented two hourly, and ventilated with room air through a Harvard rodent respirator ŽHarvard Apparatus Inc, MA, USA.. A femoral artery was cannulated to allow continuous monitoring of mean arterial pressure ŽMAP. and heart rate ŽHR. via a pressure transducer ŽBentley Trantec, Model 800, CA, USA.. Animals were placed in a stereotaxic frame ŽDavid Kopf Instruments, CA, USA. and an i.c.v. cannula Ž0.4 mm O.D.. was placed in the lateral ventricle Žanterior y0.8 mm; lateral 1.5 mm; ventral y3.7 mm relative to bregma.. A microdialysis probe ŽCMAr12; CMArMicrodialysis, Sweden. was inserted into the hypothalamic PVN Žanterior y1.6 mm; lateral 0.6 mm; ventral y8.5 mm. w11x. Modified physiological Ringer solution; Žin mM. NaCl 132; KCl 3; CaCl 2 1.26; MgCl 2 1.18; NaH 2 PO4 0.3; Na 2 HPO4 1.2; pH 7.12, was continuously perfused through the probe at 1.1 mlrmin by an Ismatec peristaltic pump ŽCole Palmer Instruments, IL, USA.. After 90 min equilibration, dialysate collections were made every 30 min on ice into hydrochloric acid Žfinal concentration 0.1 M.. Following three basal collections, either 2 nmol NPY Ž n s 8., 2 nmol wLeu31 ,Pro 34 xNPY Ž n s 5. in 4 ml saline, or 4 ml saline vehicle Ž n s 6. was injected i.c.v. over 90 s in separate groups of 18-month-old rats. Saline vehicle was injected into five 3-month-old rats serving as an age control. Sixty minutes later noradrenaline release was stimulated during one collection using modified Ringer solution containing 100 mM KCl and 32 mM NaCl. Normal modified Ringer solution was returned in the final dialysate collection to assess recovery. At the end of the experiment the i.c.v. cannula location was checked using toluidine blue dye and the probe implantation site was verified histologically. Each dialysate collection was analysed by reverse-phase HPLC with electrochemical detection using a Rheodyne 7125 syringe-loading injector, a phase-II narrow bore column Ž100 = 3.2 mm I.D., 3 mm ODS, Bioanalytical Systems, IA, USA. and a dual glassy-carbon working electrode Žoxidation potential q775 mV. w11x. Mobile phase was recirculated at 0.6 mlrmin. All chemicals were of analytical quality. The detection limit for noradrenaline was 1–2 pg w11x. Under these conditions, the noradrenaline metabolites 3-methoxy-4-hydroxyphenylethylene glycol ŽMOPEG . and 3,4-dihydroxyphenylethylene glycol ŽDOPEG. were not observed, and levels of adrenaline and dopamine in the rat microdialysates were below detection. Resting dialysate concentrations of noradrenaline were similar in all groups of 18-month-old rats Žaverage 87.6 " 6.1 pgrml, n s 19, mean " S.E.M... To determine overall time- and treatment-related differences, noradrenaline, DOPAC and HVA concentrations, expressed as a percentage of each animal’s basal concentration, were analysed by
repeated measures ANOVA followed by least significant difference tests ŽLSD.. The responses to potassium-induced depolarisation were also compared by one-factor ANOVA of the fold changes induced ŽFig. 1.. A significant reduction in microdialysate noradrenaline concentration was observed following i.c.v. administration of 2 nmol wLeu31 ,Pro 34 xNPY but not NPY ŽFig. 1, P 0.05.. Perfusion of a depolarising concentration of 100 mM KCl through the microdialysis probe produced an increase in noradrenaline release in 18-month-old rats although this was blunted in the saline treated group ŽFig. 1.. Prior administration of NPY or wLeu31 ,Pro 34 xNPY resulted in enhanced responses to KCl compared to saline pretreatment Ž P - 0.01, Fig. 1.. When the fold increases in noradrenaline concentrations in response to potassium were compared, 3-month-old rats Žage control. treated with saline showed a similar increase Ž3.2 " 0.5 times resting. to that observed in 18-month-old rats treated with NPY Ž2.8 " 0.7 times., while after wLeu31 ,Pro 34 xNPY, potassium produced a significantly greater increase Ž5.0 " 0.9 times, P - 0.05 compared to NPY or saline pretreatment.. Administration of NPY and wLeu31 ,Pro 34 xNPY produced cardiovascular effects, with increases in MAP and HR of 25 " 4 mmHg and 35 " 9 b.p.m. Ž n s 8. and 15 " 2 mmHg and 62 " 12 b.p.m. Ž n s 5., respectively, significantly greater than the changes observed after saline Ž P - 0.05.. Basal microdialysate concentrations of the dopamine metabolites DOPAC and HVA were similar across the three groups of 18-month-old rats Žaverage 3.2 " 0.5 and 2.9 " 0.6 ngrml, respectively. and were not different to levels in 3-month-old rats. Administration of NPY or wLeu31 ,Pro 34 xNPY had no effect on basal DOPAC and HVA levels ŽTable 1.. Potassium depolarisation significantly reduced DOPAC and HVA levels in all 18-monthold rats ŽTable 1, P - 0.01. to a similar extent to that
Fig. 1. Noradrenaline, expressed as % basal concentration, in 30 min dialysate collections from the PVN of 18-month-old rats, before and after i.c.v. injection Žarrow. of saline vehicle ŽI, ns6., NPY ŽB, ns8. and wLeu31 ,Pro 34 xNPY Ž Ø , ns 5.. Potassium depolarisation ŽKq . was evoked by perfusing KCl through the probe as indicated. Data were analysed by one-way ANOVA with repeated measures and LSD tests. ), Significantly different to preceding collection, Ž P - 0.05.; †, significantly different to NPY and saline-treated rats, Ž P - 0.05..
J.A. Hastings et al.r Brain Research 750 (1997) 301–304
303
Table 1 PVN microdialysate dihydroxyphenylacetic acid ŽDOPAC. and homovanillic acid ŽHVA. levels, expressed as % basal concentration, in 30 min dialysate collections in 18-month-old rats treated with i.c.v. saline, NPY or wLeu31 ,Pro 34 xNPY
DOPAC Saline NPY wLeu31 ,Pro 34 xNPY HVA Saline NPY wLeu31 ,Pro 34 xNPY a
Resting
Post i.c.v. drug
Pre Kq
Kq
104.0 " 5.8 105.7 " 6.5 111.2 " 7.5
100.8 " 7.7 116.7 " 5.9 107.5 " 6.5
120.9 " 16.8 110.0 " 9.0 102.7 " 8.2
87.0 " 12.4 a 58.8 " 7.8 a 69.3 " 6.2 a
96.9 " 6.8 111.3 " 4.4 105.5 " 5.2
103.4 " 7.6 127.8 " 7.6 108.2 " 5.5
104.8 " 15.1 118.9 " 13.4 111.5 " 6.8
60.0 " 7.6 49.3 " 9.7 56.1 " 9.5
a a a
Significantly different to preceding collection, P - 0.01; n s 6, 8, 5.
observed in 3-month-old animals Ž64.9 " 6.1 and 56.9 " 8.9% basal, respectively.. The major finding of this study is that in the hypothalamic PVN of 18-month-old rats, exogenous NPY and wLeu31 ,Pro 34 xNPY potentiated the potassium-induced increase in noradrenaline release. The blunted depolarisation response seen in 18-month-old rats was increased by pretreatment with NPY to levels observed in younger animals in the absence of NPY. At equimolar doses the potent selective NPY Y1 agonist wLeu31 ,Pro 34 xNPY produced a significantly greater potentiation of potassium-stimulated noradrenaline release than NPY. We have previously reported a facilitatory effect of NPY on potassium-induced noradrenaline release in 3–4-month-old rats w14x; results of the present study suggest that a Y1 NPY receptor may be responsible for this effect. wLeu31 ,Pro 34 xNPY caused a 40% reduction in basal PVN noradrenaline release. Several NPY receptor subtypes are present in rat brain ŽY1 , Y2 , Y3 , Y4 and Y5 . with regional and species differences in receptor density w2,7,8,19x. Several of these receptor subtypes have been localised to the hypothalamus, and Y1 and Y2 receptors are expressed in the PVN w7x. Whether wLeu31 ,Pro 34 xNPY and NPY affect noradrenaline release by stimulating Y1 NPY receptors located pre-synaptically or post-synaptically in the hypothalamus requires further investigation. We cannot rule out the possibility that NPY receptors in other brain regions are being activated or that the effect is being mediated by another NPY receptor subtype. For example, wLeu31 ,Pro 34 xNPY was recently shown to bind to rat Y5 receptors with an EC 50 in the nM range w2x. NPY may also be mediating effects via a 2adrenoceptors as several lines of evidence suggest an interaction between NPY receptors and a 2-adrenoceptors in the brain w20x. Similar basal concentrations of noradrenaline were detected in the PVN of both age groups, thus the functional pool of noradrenergic neurons in the PVN which we were examining was apparently largely preserved with ageing w3x. However, the 18-month-old rats had a reduced ability to release noradrenaline upon potassium depolarisation. As aged animals have markedly reduced hypothalamic NPY concentrations w13,16x we reasoned that physiological
changes in endogenous NPY with ageing may affect the ability to release noradrenaline in response to a stimulus. Exogenous NPY and wLeu31 ,Pro 34 xNPY potentiated the noradrenaline response induced by potassium in 18-monthold rats. Clearly factors other than changes in NPY or NPY receptors may contribute to the reduced responsiveness seen in 18-month-old rats. Decreased noradrenaline responsiveness to potassium in 18-month-old saline-treated animals may be related to changes in the density of a 2-adrenoceptors, alterations in neuronal uptake or vesicular monoamine transport with ageing w3x. Age-related changes in other neurotransmitters which modulate noradrenaline may also affect the ability to release noradrenaline. The nature of the NPY-noradrenaline interaction, and indeed the NPY receptor subtypes involved in the regulation of noradrenaline release in the CNS in vivo remains to be elucidated. The mechanism by which NPY and its analogues might reduce basal release but increase stimulated release is unclear. Results of other studies examining the effect of NPY on hypothalamic noradrenaline release in vivo reveal site-specific effects, with a reduction in noradrenaline release in the ventro-medial hypothalamus and an increase in the lateral hypothalamus w18x and the perifornical region w12x. Food availability also influenced the NPY-induced changes in extracellular noradrenaline concentrations w6x. These results probably reflect regional differences in NPY receptor distribution, effective NPY dose, and the presence of anaesthesia. Any alteration in CNS noradrenaline-NPY interactions with ageing may have implications for the regulation of cardiovascular and ingestive functions subserved by these transmitters in the hypothalamus w2,11,15,19x. The PVN receives a high density of noradrenergic neurons, therefore it is likely that some of the observed DOPAC and HVA originated in noradrenergic neurons. No significant alterations in microdialysate DOPAC or HVA concentrations were observed following i.c.v. NPY or wLeu31 ,Pro 34 xNPY, thus the effect appears selective for noradrenaline. While a dose-related increase in hypothalamic dopamine and DOPAC content has been reported following treatment with NPY w4x, few studies have exam-
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ined the effect of NPY on the release of dopamine metabolites in vivo. No age-related differences in potassiumstimulated reductions in DOPAC and HVA concentrations were observed. Reductions in DOPAC and HVA possibly reflect a shift in catecholaminergic metabolism during depolarisation towards an increasing production of noradrenaline, therefore decreasing dopamine availability for metabolism into DOPAC and HVA in noradrenergic neurons w11x. The observed reductions in DOPAC and HVA concentrations with potassium stimulation may also be attributed to a reduced efflux via a membrane potential-dependent process w9x. Few studies have investigated the relationship between dopamine metabolism and NPY and clearly more work needs to be carried out regarding the interaction between NPY and catecholamine metabolism in general. In conclusion, administration of wLeu31 ,Pro 34 xNPY to 18-month-old rats caused a reduction in basal noradrenaline release in the PVN of the hypothalamus. In 18-monthold rats with marked reductions in endogenous hypothalamic NPY, exogenous NPY and the NPY Y1 agonist wLeu31 ,Pro 34 xNPY potentiated potassium-stimulated noradrenaline release in the PVN, suggesting the involvement of a Y1 or Y5 receptor in mediating this effect.
w5x
w6x
w7x
w8x
w9x
w10x
w11x
w12x
Acknowledgements This work was supported by the National Health and Medical Research Council of Australia. We are indebted to the William Buckland Foundation for equipment support. J.A.H. was supported by a Deakin University Postgraduate Scholarship.
w13x
w14x
w15x
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