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Chronic desipramine treatment reduces regional neuropeptide Y binding to Y2-type receptors in rat brain
196
Brain Research. 539 (1991) 196-202 Elsevier
BRES 16254
Chronic desipramine treatment reduces regional neuropeptide Y binding to Ye-type receptors in rat brain P e t e r S. W i d d o w s o n 1"2 a n d A . E . Halaris 1"3 Departments of 1Psychiatry, 2Neuroscience and ~Pharmacology, Case Western Reserve University and MetroHealth Medical Center, Cleveland, OH 44109 (U.S.A.)
(Accepted 14 August 1990) Key words: Neuropeptide Y; Antidepressant drug; Yl-type receptor; Y2-type receptor; Second messenger
Chronic treatment of rats with desipramine and imipramine (5 mg/kg/twice daily/i.p.) for 14 days caused a significant reduction in the binding of [3H]proprionyl NPY to membranes prepared from frontal cortex, nucleus accumbens, hypothalamus and hippocampus. There was no change in binding of [3H]proprionyl NPY in the parieto-occipital cortex, striatum or amygdala. Scatchard analysis of binding data from frontal cortical and hippocampal membranes showed that [3H]proprionyl NPY bound to a single site with a Kd of approximately 0.3 nM. The loss of [3H]proprionyl NPY binding in hippocampal and frontal cortical membranes revealed that chronic tricyclic antidepressant treatment produced a reduction in the number of binding sites with no change in the affinity for the ligand. Chronic desipramine treatment did not alter the ability of NPY (0.01-25/aM) to stimulate inositol phosphate accumulation in rat frontal cortical slices as compared to saline-treated animals. The lack of change of NPY-induced inositol phosphate accumulation following chronic desipramine treatment showed that there was no change to Yt NPY-type receptors which are linked to the hydrolysis of inositol phospholipids. However, the ability of NPY (0.05-0.5/aM) to inhibit forskolin (1/aM) stimulated adenylate cyclase via Y2 NPY-type receptors in rat frontal cortical slices was significantly reduced following chronic desipramine treatment. This finding suggests that the reduction of [3H]proprionyl NPY binding in selective brain regions may be the result of an antidepressant-induced loss of Y2-type NPY receptors which are negatively linked to adenylate cyclase.
INTRODUCTION Neuropeptide Y (NPY), the 36 amino acid addition to the pancreatic polypeptide family, is found in large quantities in the rat central nervous system (CNS) 3'9. The presence of high affinity, saturable binding sites in the CNS for [125I]Bolton-Hunter NPY and [3H]proprionyl NPY suggests that NPY may play a role in synaptic transmission 18,2°,26'27"34. Although NPY exerts some of its effects by a direct receptor action, interactions with norepinephrine seem to play an important role in mediating other effects of the peptide such as the regulation of sympathetic nervous activity 17. Recently it has been suggested that NPY may be involved in the pathophysiology of depression 38. NPY exerts a powerful regulatory effect on the circadian rhythm of rat locomotor activity 1'2, eating behavior 14 and control of hypothalamic-pituitary function 29, all of which are dysregulated in depression 23. However, measurements of N P Y in cerebrospinal fluid of depressed patients have failed to provide conclusive proof for a dysregulation of the NPY system. Increases 1°, decreases 37 and no change 5 in CSF NPY concentrations from depressed
patients as compared to healthy subjects have been reported. However, this anomaly may reflect an alteration in the metabolism of N P Y between healthy subjects and depressed patients since the antibody used by Widerl6v et al. 37 detects several immunoreactive fragments which may relate to breakdown products of NPY. There are also reports of alterations in NPY concentrations following chronic, but not acute, antidepressant treatment of rats 11"25'31'35. Chronic treatment with the predominantly norepinephrine reuptake blocker, imipramine, increased NPY-immunoreactivity (NPY-ir) in frontal cortex and hypothalamus H. In contrast, chronic treatment with zimelidine, which blocks serotonin reuptake, increased NPY-ir in the frontal cortex only H. We have demonstrated this regional increase in NPY-ir with chronic antidepressant drug treatments 35. Chronic treatment with desipramine or imipramine increased NPY-ir in rat frontal cortex, hypothalamus, nucleus accumbens and hippocampus. Clorgyline, a monoamine oxidase type A inhibitor, increased NPY-ir in the hippocampus and nucleus accumbens as well as in the amygdala 35. In addition to antidepressant drug treatment, repeated, but not single electroconvulsive shock, increased NPY-ir in
Correspondence: P.S. Widdowson, Department of Psychiatry, MetroHealth Medical Center, 3395 Scranton Road, Cleveland, OH 44109, U.S.A.
0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
197 rat cortical regions a n d h i p p o c a m p u s 25'31. T h e effect of e l e c t r o c o n v u l s i v e shock t r e a t m e n t o n the increase in p e p t i d e c o n c e n t r a t i o n was highly specific for the N P Y system since t h e r e was n o a l t e r a t i o n in regional c o n c e n t r a t i o n s of g a l a n i n , vasoactive intestinal p o l y p e p t i d e ( V I P ) , s u b s t a n c e P, n e u r o k i n i n A or n e u r o t e n s i n 25. A s u b d i v i s i o n of N P Y receptors has b e e n recently p o s t u l a t e d b a s e d o n p h a r m a c o l o g i c a l studies 28'3°. Following this t e r m i n o l o g y , NPY-Y1 receptors r e q u i r e the entire N P Y m o l e c u l e for activation, while N P Y - Y 2 receptors can be activated by the 13-36 a m i n o acid C - t e r m i n a l s e q u e n c e of N P Y 3°. T h e Y l - t y p e N P Y r e c e p t o r has b e e n l i n k e d to the hydrolysis of inositol phospholipids while the Y2 N P Y r e c e p t o r is negatively c o u p l e d to a d e n y l a t e cyclase 3°. B o t h N P Y s u b t y p e s have b e e n d e m o n s t r a t e d in rat b r a i n using s e c o n d m e s s e n g e r systems as m a r k e r s . N P Y inhibits f o r s k o l i n - s t i m u l a t e d c A M P a c c u m u l a t i o n in hippocampa122 a n d striatal m e m b r a n e s 33 t h r o u g h the activation of Y2 receptors. In slices of frontal cortex, h i p p o c a m p u s a n d s t r i a t u m , N P Y stimulates the a c c u m u l a t i o n of inositol p h o s p h a t e s p r e s u m a b l y t h r o u g h activation of Y t - t y p e receptors 12. We investigated w h e t h e r c h r o n i c tricyclic antidepressant t r e a t m e n t alters N P Y b i n d i n g a n d N P Y - m e d i a t e d s e c o n d m e s s e n g e r systems in selective brain regions which m a y indicate c h a n g e s of N P Y activity. T h e action of d e s i p r a m i n e o n the N P Y system was c o m p a r e d to i m i p r a m i n e in this study b e c a u s e d e s i p r a m i n e selectively inhibits n o r a d r e n e r g i c r e u p t a k e sites 4 while i m i p r a m i n e affects n o r a d r e n e r g i c a n d s e r o t o n e r g i c r e u p t a k e sites 13. T h e c h a n g e s in N P Y - i m m u n o r e a c t i v i t y (ir) caused by c h r o n i c i m i p r a m i n e a d m i n i s t r a t i o n , as o b s e r v e d by Heilig et al. 11, m a y be d u e to a c o m b i n e d activity of i m i p r a m i n e on norepinephrine and serotonin reuptake.
MATERIALS AND METHODS Animals and treatments Male Sprague-Dawley rats (180-250 g) were chronically treated with either 0.9% saline or the tricyclic antidepressant drugs, desipramine and imipramine (5 mg/kg/twice daily/i.p.) dissolved in saline, for 14 days. Rats were maintained in groups of 4 at 20 °C with a 12 h light/dark cycle (lights on 06.00 h) and allowed free access to standard laboratory chow and water. Membrane preparation Twenty four hours after the last injection, rats were decapitated and the brains were rapidly removed and cut into 3 mm slices on an ice cold glass petri dish. Using a stereotaxic atlas for reference 2~, the hippocampus, striatum, nucleus accumbons, amygdala, hypothalamus, frontal and parieto-occipital cortex were dissected free and frozed on dry ice. The tissue was stored at -70 °C until assayed. Membranes were prepared from the tissue by homogenization in a loose-fitting motor driven teflon-glass homogenizer in ice-cold HEPES (5 mM) buffer sucrose solution (0.32 M, pH 7.4) (8 strokes up and down). The homogenates were then diluted 4-fold in the HEPES-sucrose buffer and then centrifuged at 1000 g for 10 min. The supernatant was recentrifuged at 10,000 g for 45 rain and the
resulting mitochrondrial/membrane pellet resuspended in HEPESbuffered (20 mM, pH 7.4) Krebs-Ringer solution (137 mM NaCI, 2.68 mM KCI, 2.05 mM CaCI2, 1.8 mM MgC12, 0.1% w/v ascorbic acid, 1 mg/ml glucose). Equilibrium binding was performed in 1.5 mi plastic microfuge tubes at 37 °C. [3H]Proprionyl NPY (68 Ci/mmol) (0.05-1.4 nM in 0.25 ml) (Amersham Corp., Arlington Heights, IL) in HEPESbuffered Krebs-Ringer solution containing bovine serum albumin (1.0% w/v) was added to each tube. NPY 200 nM (Bachem Inc., Torrance, CA) in 50 /~1 was added to half the tubes to define non-specific binding while the rest of the tubes received only 50/~1 buffer. The binding was initiated by the addition of 0.2 ml of protein suspension (0.1-0.2 mg) and the incubation stopped 45 min later by rapid centrifugation to separate bound NPY from the free. The supernatant was aspirated off, the pellets dried and the bottom of the plastic centrifuge tubes cut off and placed in liquid scintillation tubes. The radioactivity of the pellets was estimated by liquid scintillation spectrometry in a Beckman LS 3801 counter (counting efficiency = 46%). Data from the binding study was analyzed using LIGAND 19. Inositol phosphate hydrolysis method Twenty four hours after the last injection of either saline or desipramine (5 mg/kg/twice daily/i.p, for 14 days), rats were quickly decapitated, their brains removed and placed in ice-cold KrebsRinger buffer solution A (in mM: NaCI 120, KCI 5.5, CaCI2 2.5, NaH2PO 4 1.2, MgCI2 1.2, glucose 11, EDTA 19/~M, Na2HCO3, pH 7.4) which had been previously gassed with a 95% 02/5% CO 2 mixture. The frontal cortex and hypothalamus were dissected free on ice and the tissue cut into slices (350 x 350gm) using a MeIllwain tissue chopper. The slices were placed into 18 ml Beckman fiat-bottom polypropylene scintillation tubes containing 10 ml oxygenated solution A. The slices were then incubated in a rapidly shaking incubator (100 osciUations/min) at 37 °C for 30 min and oxygenated every 10 min with the 95% 02/5% CO 2 gas mixture. The tissue was allowed to settle to the bottom of the tube and then the buffer was aspirated off. Following the preincubation, the tissue was resuspended in 2.5 ml solution A containing 20/~Ci [3H]myoinositol (23 Ci/mmol) (Amersham, Corp., Arlington Heights, IL) and incubated for 60 min, gassing every 20 min. After prelabelling the tissue, the buffer was removed by aspiration. The tissue was then washed 3 times with warm 10 mi solution B (37 °C) containing 10 mM LiCI (solution A containing 10 mM LiCI and 110 mM NaC1). The tissue was then dispersed into 4 ml polypropylene Beckman Biovials containing 370/A solution B. The inositol phospholipid hydrolysis was initiated by the addition of NPY (0.01-25/~M) in solution B and the tissue incubated for a further 45 rain at 37 °C. The reaction was terminated by the addition of 0.5 ml solution B and the tissue washed 3 times with 0.5 ml cold solution B. 940/A of chloroform: methanol: HCI mixture (100:200:2 by vol) was added to the tissue and approximately 2000 cpm of [14C]inositol-l-phosphate (55 mCi/mmol) (Amersham Corp., Arlington Heights, IL) in 60/~1 methanol added to monitor the recovery of the inositol phosphates. The radiolabeled inositol phosphates were measured based on the method of Berridge et al. 6. The tubes were vortexed and allowed to stand for 10 min. Chloroform, 310/~1, and 310 #1 distilled water were added to the tubes and the tubes were vortexed again. The tubes were then centrifuged at 1500 g for 10 min at 4 0C. The upper aqueous layer was applied to chromatography columns (Dowex AG1 x8 100-200 mesh, formate form) which had been previously pre-equilibrated with I0 mM Tris (pH 7.4). The columns were then washed with 20 ml of 5 mM inositoi and the inositol-l-phosphate eluted with 1 ml 1 M ammonium formate: 0.1 M formic acid. The inositol-l-phosphate eluted from the columns was collected in scintillation tubes and the radioactivity [3H and tac] determined by liquid scintillation spectrometry. The average recovery of inositol1-phosphate was 65 + 2%. For the determination of the total [3H]inositol incorporated into lipid, the remaining upper phase and interphase were aspirated off and the chloroform layer evaporated to dryness. The [3H]inositol was then resuspended in 4 ml
198 scintillation fluid and the total radioactivity estimated by liquid scintillation spectrometry. The results are expressed as the fraction of the total [3H]inositol incorporated which was converted to [3H]inositol phosphate. cA MP method
Rats were sacrificed by decapitation, 24 h after the last injection of saline or desipramine (5 mg/kg/twice daily/i.p, for 14 days), their brains removed and quickly placed in ice-cold Krebs-Ringer buffer which had been previously oxygenated with 95% 02/5% CO 2. The frontal cortex and hippocampus were dissected on an ice-cold glass plate and cut into thin slices (300 x 300 gm) using a Mcllwain tissue chopper. The slices were dispersed in 40 ml oxygenated KrebsRinger buffer (KRB) at 30 °C. The slices were preincubated for 60 min washing with warm fresh oxygenated buffer every 20 min. After preincubation, slices were dispersed again in 35 ml of KRB and 2.75 ml aliquots in duplicate were placed into individual reaction vials and oxygenated continuously for 30 min. NPY (10 nM-1 #M) in 50 /d was then added to each tube and 10 min later the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthinein 150/~1 (0.5 raM) was added to each vial followed by a brief oxygenation. Finally, 10 min later forskolin (1-10/~M) in 50/~1 was added and all the tubes were oxygenated again. The reaction proceeded for 10 min and was stopped by the addition of 2.0 ml 3.1% perchloric acid. Samples were sonicated and centrifuged at 15,000 g for 15 min. Pellets were dissolved in 1.0 N NaOH and saved for subsequent protein determination. The supernatant was neutralized with excess (about 120 mg) CaCO 3 and assayed for its content of cAMP. cAMP levels were estimated by radioimmunoassay (RIANEN cAMP assay kit, New England Nuclear, DuPont, Billerica, MA) and the protein was estimated by the method of Lowry et al. 16. Statistical analysis
Statistical analysis of the data was performed using analysis of variance (ANOVA) followed by Duncan's multiple range test. Probability levels of less than 0.05 were taken to indicate a significant difference. RESULTS Chronic desipramine and imipramine treatment reduced the binding of [3H]proprionyl N P Y to m e m b r a n e s p r e p a r e d from frontal cortex, hippocampus, nucleus accumbens and hypothalamus as c o m p a r e d to the salinetreated rats (Figs. 1 and 2). The reduction of [3H]proprionyl N P Y binding following chronic antidepressant drug t r e a t m e n t was approximately equal for both desip r a m i n e and imipramine. H o w e v e r , there was no change in N P Y binding to parieto-occipital, striatal or amygdala m e m b r a n e s following desipramine and imipramine treatment (Fig. 1). Scatchard analysis of the binding in h i p p o c a m p u s and frontal cortex showed that the tricyclic antidepressant-induced reduction of N P Y binding represented a loss in NPY receptor density with no change in affinity (Figs. 2 and 3). Scatchard analysis also indicated that [3H]proprionyl N P Y b o u n d to a single site in the frontal cortical and h i p p o c a m p a l m e m b r a n e s with a Kd of about 0.3 nM. T h e r e was no evidence for multiple binding sites based on Scatchard analysis. NPY (0.01-25 /~M) significantly increased the accumulation of inositol phosphates in frontal cortical slices, o b t a i n e d from untreated rats, in a d o s e - d e p e n d e n t
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Fig. 1. Effects of chronic treatment with desipramine and imipramine on the binding of 1.25 nM [3H]proprionyl NPY to membranes prepared from the nucleus accumbens, amygdala, striatum, parietal cortex and hypothalamus. D, saline control;l~, desipramine treated; II, imipramine treated. Results are expressed as mean + S.E.M. (n = 6). *P < 0.01 as compared to saline controls.
m a n n e r (Fig. 4). Chronic t r e a t m e n t with d e s i p r a m i n e for 14 days did not significantly alter the N P Y - i n d u c e d accumulation of inositol p h o s p h a t e s in frontal cortical slices as c o m p a r e d to cortical slices o b t a i n e d from either saline treated rats or from u n t r e a t e d rats (Fig. 4). Forskolin (0.1-10 /~M) caused a significant dosed e p e n d e n t accumulation of c A M P in frontal cortical slices. Forskolin-stimulated cyclic A M P accumulation as a percentage basal concentrations: 0.1/~M forskolin, 283 + 12%; 1.0/~M forskolin, 512 + 23%; 1 0 . 0 # M forskolin, 795 + 28%. In frontal cortical slices o b t a i n e d from u n t r e a t e d rats N P Y ( 0 . 0 1 - 1 / t M ) maximally inhibited the forskolin-stimulated c A M P f o r m a t i o n in a d o s e - d e p e n dent m a n n e r by a p p r o x i m a t e l y 23% (Fig. 5). Chronic desipramine t r e a t m e n t for 14 days significantly r e d u c e d the 1/~M forskolin-stimulated c A M P formation in both h i p p o c a m p a l and frontal cortical slices by 22% (Fig. 5).
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Fig. 3. Effect of chronic desipramine and imipramine treatment on the number of binding sites and affinity for [3H]proprionyl NPY in hippocampal and frontal cortical membranes. Results are expressed as mean _+ S.E.M. (n -- 6). **P < 0.01 as compared to saline controls. [2, Saline controls; I~, desipramine treated; II, imipramine treated.
In addition, NPY (0.05-0.5/~M) failed to further reduce the forskolin-stimulated cAMP in frontal cortical and hippocampal slices obtained from desipramine-treated rats as compared to saline controls (Fig. 5).
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Fig. 5. Effect of NPY on the 1 pM forskolin-induced stimulation of adenylate cyclase in frontal cortical and hippocampal slices. Mean + S.E.M. (n = 6). *P < 0.05 significantly different to 1 /~M forskolin alone (from saline-treated or untreated rats). *Significantly different from saline-treated and untreated rats. II, untreated rats; O, saline-treated rats; 0 , desipramine-treated rats.
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Fig. 4. Effect of N P ¥ on the accumulation of inositol phosphates in rat frontal cortical slices. Mean _+ S.E.M. (n = 6-12). *P < 0.05 as compared to basal level. O, untreated rats; Fq, chronic saline-treated rats; II, chronic desipramine-treated rats.
The ability of [3H]proprionyl NPY to bind to a single site in cortical and hippocampal membranes, with an affinity constant of 0.3 nM, closely agrees with previous reports using either tritiated or iodinated NPY as a ligand t8"26'27'34. Chronic administration to rats with the tricyclic antidepressant drugs, imipramine and desipramine, produced a significant reduction in the binding of [3H]proprionyl NPY to membranes prepared from frontal cortex, hippocampus, nucleus accumbens and hypothalamus. This antidepressant-induced reduction in [3H]proprionyl NPY binding was not widespread in all the brain regions examined since membranes prepared from the parieto-occipital cortex, striatum and amygdala failed to show significant changes in [3H]proprionyl NPY binding. Scatchard analysis of the binding to frontal cortical and hippocampal membranes demonstrated that
200 the loss of [3H]proprionyl NPY binding was the result of a tricyclic antidepressant-induced loss in the receptor number with no alteration in the affinity for NPY. Desipramine and imipramine do not directly interfere with [3H]proprionyl NPY binding in rat frontal cortical membranes (P.S. Widdowson, unpublished results) suggesting that the reduction in NPY receptor density was not due to direct interaction of the tricyclic antidepressants with the NPY receptor. We have recently demonstrated that chronic administration of desipramine and imipramine to rats significantly increases NPY immunoreactivity in frontal cortex, hippocampus, nucleus accumbens and hypothalamus 35. Thus, the same brain regions show an increase in NPY immunoreactivity and a reduction in NPY receptors following chronic desipramine and imipramine treatment. We suggest that the repeated treatment of rats with desipramine and imipramine chronically interferes with NPY synthesis and release. It is possible that the antidepressant-induced reduction in [3H]proprionyi NPY binding represents the down-regulation of NPY receptors caused by chronic elevations in synaptic NPY concentrations. Repeated administration of imipramine and desipramine may increase the release of NPY from peptide stores producing a reduction in the NPY receptor density. The increase in regional NPY concentrations 35 would then reflect an increase in NPY synthesis to compensate for the increase in NPY release. Although the precise mechanism for the imipramine and desipramine-induced reduction of NPY receptors is not known, we propose that the tricyclic antidepressants act on the NPY system indirectly through their ability to increase synaptic norepinephrine concentrations 7'~5. Chronic tricyclic-induced elevations of norepinephrine concentrations may increase the release of NPY and down-regulate NPY receptors. Imipramine was approximately equipotent to desipramine in its ability to reduce NPY binding sites in the hippocampus, frontal cortex, hypothalamus and nucleus accumbens. Although imipramine is a potent inhibitor of serotonin reuptake 13 and although about 30% of chronically administered imipramine is converted to desipramine s, the similarity of these two tricyclic antidepressants in reducing NPY binding suggests that imipramine, like desipramine, acts, at least in part, through the noradrenergic system 4,~5. Repeated treatment of rats with electroconvulsive shock has been also reported to increase NPY-immunoreactivity in some cortical regions and hippocampus 25"31. However, Wahlestedt et al. 31 were unable to show a significant change in [125I]NPY binding in hippocampus, frontal or parietal cortex, although there was a 10% reduction in NPY binding in the frontal cortex 34. It is unlikely that the choice of ligands used, i.e. [3H]-
proprionyl NPY in this study and [125I] Bolton Hunter (BH) NPY used by Wahlestedt et al. 3~ is responsible for the discrepancy between the chronic tricyclic antidepressant drug treatment and electroconvulsive shock since both ligands have been shown to bind to a single class of brain receptors with approximately the same affinity ~8' 26,27.34. The reason for this discrepancy is unknown but may be related to a different mechanism of action of antidepressant drug treatment and electroconvulsive shock on the NPY system. Based on pharmacological tests and binding studies a number of investigators have suggested that multiple NPY receptors exist in central and peripheral neurons 24' 28,3o. We have provided evidence that in frontal cortex there are multiple NPY receptors based on the ability of NPY to stimulate inositol phospholipids and to inhibit adenylate cyclase. At present the lack of NPY analogues makes it impossible to accurately distinguish multiple NPY receptors. There are some reports of multiple binding sites in rat brain tissue using [~25I]BH NPY and displacing it with the structurally related peptide yy32. However, which of these putative receptor populations represent the Y1 or the Y2 type is not known but has been suggested that the Y2 NPY receptor is the predominant sub-type in the central nervous system 24. We utilized the ability of NPY to stimulate different second messenger systems, as a measure of effects of drug treatment on each NPY receptor sub-type. We demonstrated that NPY stimulates the hydrolysis of inositol phospholipids and inhibits forskolin-stimulated cAMP accumulation in slices of rat frontal cortex. This suggests that the frontal cortex possess both Y1 and Y2 receptor subtypes which have been suggested to be linked to the inositol phosphate system and the adenylate cyclase system, respectively28'3°. Chronic desipramine treatment did not alter the ability of NPY to stimulate inositol phosphate production suggesting that the YI NPY receptors, which are linked to phospholipase C 3°, are not altered by these antidepressants. Chronic desipramine treatment significantly reduced the ability of forskolin to stimulate adenylate cyclase activity in both hippocampal and frontal cortical slices. The meehanism for this desipramine-induced reduction of cAMP accumulation is unknown at present. However, chronic desipramine treatment to rats produces a widely documented down-regulation of fl-adrenoceptors and a reduction of the fl-adrenoceptor mediated stimulation of cAMP in rat brain tissue 7, presumably as a result of chronic elevations of synaptic norepinephrine 16. It is therefore plausible that the desipramine-induced reduction in forskolin-stimulated cAMP concentration represent a reduction in number of functional adenylate cyclase molecules, some of which may be linked to
201 fl-adrenoceptors which are present in large numbers in the rat cortex, Thus, following the chronic administration of desipramine, there were fewer adenylate cyclase molecules to be activated since they have been downregulated by the increased fl-adrenoceptor mediated signal as a result of the desipramine-mediated elevations of synaptic norepinephrine 15. Following the chronic desipramine administration, NPY (0.05-0.5 /~M) failed to reduce the forskolinstimulated c A M P accumulation in frontal cortical slices, as compared to forskolin alone, Although there was a significant, 25% reduction in the ability of forskolin to stimulate c A M P production in both the hippocampal and frontal cortical slices, there was still a large increase in the c A M P concentrations (330-400% above basal in the hippocampal and frontal cortical slices, respectively) following 1/~M forskolin addition for the NPY to have an effect. Only those cells which express fl-adrenoceptors would therefore be expected to have a reduced number of adenylate cyclase molecules in response to chronic desipramine administration. We have been unable to demonstrate an NPY-induced reduction in fl-adrenoceptor-stimulated c A M P accumulation in rat frontal cortex slices 36, suggesting that NPY Y2-type receptors are not located on the same cells expressing fl-adrenoceptors. Thus, the number of adenylate cyclase molecules in cells
which possess Y2-NPY receptors would not be expected to be altered following desipramine treatment, since they are not activated by desipramine treatment, rather the opposite, since Y2-type receptors reduce the stimulated c A M P concentration. The observation that N P Y failed to inhibit forskolin-induced c A M P production in the frontal cortical and hippocampal slices therefore suggests a loss in Y2 NPY receptor activity which is negatively linked to adenylate cyclase activity. Since it has been proposed that a majority of N P Y receptors in the CNS are the Y2-type 24, and that desipramine produces a large decrease in the density of N P Y receptors in selective brain regions, it would be expected that the ability of NPY to reduce c A M P concentrations would also be reduced. In summary, we have demonstrated that following repeated administration of the tricyclic antidepressants, desipramine and imipramine to rats, there is a reduction in NPY binding in selective brain regions and a reduction in the ability of NPY to inhibit forskolin-stimulated cAMP production in the frontal cortex and hippocampus. We suggest that chronic desipramine and imipramine causes a down-regulation of Y2-type NPY receptors in selective brain regions whilst Y r t y p e receptors are unchanged.
REFERENCES
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1 Albers, H.E. and Ferris, C.E, Neuropeptide Y: a role in light-dark cycle entrainment of hamster circadian rhythms, Neurosci. Lett., 50 (1984) 163-168. 2 Albers, H.E., Ferris, C.E, Leeman, S.E. and Goldman, B.D., Avian pancreatic polypeptide phase shifts hamster circadian rhythms when microinjected into the suprachiasmatic region, Science, 223 (1984) 833-835. 3 Allen, Y.S., Adrian, T.E., Allen, J.M., Tatemoto, K., Crow, T.J., Bloom, S.R. and Polak, J.R., Neuropeptide Y distribution in the rat brain, Science, 221 (1983) 877-879. 4 B/ickstrOm, I.T., Ross, S.B. and Marcusson, J.O., [3H]Desipramine binding to rat brain tissue: binding to both noradrenaline uptake sites and sites not related to noradrenaline neurons, J. Neurochem., 52 (1989) 1099-1106. 5 Berrettini, W.H., Doran, A.R., Keiso, J., Roy, A. and Pickar, D., Cerebrospinal fluid neuropeptide Y in depression and schizophrenia, Neuropsychopharmacology, 1 (1987) 81-83. 6 Berridge, M.J., Downes, C.E and Hanley, M.R., Lithium amplifies agonist dependent phosphatidylinositol responses in brain and salivary glands, Biochem. J., 206 (1982) 587-595. 7 Charney, D.S., Menkes, D.B. and Heninger, G.R., Receptor sensitivity and the mechanism of action of antidepressant treatment, Arch. Gen. Psychiat., 38 (1981) 1160-1180. 8 Daniel, W., Adamus, A., Melzacka, M., Szymura, J. and Vetulani, J., Cerebral pharmacokinetics of imipramine in rats after single and multiple dosages, Naunyn-Schmiedeberg's Arch. Pharmacol., 317 (1981) 209-213. 9 DeQuidt, M.E. and Emson, EC., Distribution of neuropeptide Y-like immunoreactivity in the rat central nervous system. I. Radioimmunoassay and chromatographic characterization, Neuroscience, 18 (1986) 527-543. 10 Ekman, R., Widerl6v, E., Walleus, H. and Lindstrom, L.H.,
Acknowledgements. This work was supported by a grant from the Metro Health Foundation (P.S.W.).
202 19 Munson, P,J. and Rodbard, D., LIGAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem., 107 (1980) 220-239. 20 Nakajima, T., Yashima, Y. and Nakamura, K., Quantitative autoradiographic localization of neuropeptide Y receptors in the rat lower brainstem, Brain Research, 380 (1986) 144-150. 21 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, Sydney, 1982. 22 Petrenko, S., Olianas, M.C., Onali, P. and Gessa, G.L., Neuropeptide Y inhibits forskolin-stimulated adenylate cyclase activity in rat hippocampus, Eur. J. Pharmacol., 136 (1987) 425-428. 23 Sachar, E.J. and Baron, M., The Biology of Affective Disorder, Ann. Rev. Neurosci., 2 (1979) 505-518. 24 Schwartz, T.W,, Fuhlendorff, J., Langeland, N., Thogersen, H., Jorgensen, J.C. and Sheikh, S.P., Y1 and Y2 receptors for NPY the evolution of PP-fold peptides and their receptors. In V. Mutt, K. Fuxe, T. H6kfelt and J.M. Lundberg (Eds.) Neuropeptide Y, Raven, New York, 1989, pp. 143-152. 25 Stenfors, C., Theodersson, E. and Mathe, A.A., Effect of repeated electroconvulsive treatment on regional concentrations of tachykinins, neurotensin, vasoactive intestinal polypeptide, neuropeptide Y and galanin in rat brain, J. Neurosci. Res., 24 (1989) 445-450. 26 Unden, A., Tatemoto, K., Mutt, V. and Bartfai, T., Neuropeptide Y receptor in the rat brain, Eur. J. Biochem., 145 (1984) 525-530. 27 Unden, A. and Bartfai, T., Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex, FEBS Left., 177 (1984) 125-128. 28 Wahlestedt, C., Yanaihara, N, and Hhkanson, R., Evidence for different pre- and postjunctional receptors for neuropeptide Y and related peptides, Regul. Pept., 13 (1986) 307-318. 29 Wahlestedt, C., Skagerberg, G., Ekman, R., Heilig, M., Sunder, F. and H~kanson, R., Neuropeptide Y (NPY) in the area of the hypothalamic paraventricular nucleus activates the pituitary-adrenocortical axis in the rat, Brain Research, 417 -
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(1987) 33-38. 30 Wahlestedt, C., Edvinson, L., Ekblad, E. and H~ikanson, R., Effects of neuropeptide Y at sympathetic neuroeffector junctions: existence of Y1 and Y2 receptors. In A. Nobin and C. Owman (Eds.), Neuronal Messengers in Vascular Function, Elsevier, Amsterdam, 1988, pp. 231-243. 3l Wahlestedt, C., Blendy, J.A., Kellar, K.J., Heilig, M., Wider16v, E. and Ekman, R., Electroconvulsive shocks increase the concentration of neocortical and hippocampal neuropeptide Y (NPY)-like immunoreactivity in the rat, Brain Research, 507 (1990) 65-68. 32 Walker, M.W. and Miller, R.J., 12SI-Neuropeptide Y and ~25I-Peptide YY bind to multiple receptor sites in rat brain, Mol. Pharmacol., 34 (1989) 779-792. 33 Westlind-Danielsson, A., Unden, A., Abens, J., Andell, S. and Bartfai, T., Neuropeptide Y receptors and the inhibition of adenylate cyclase in the human frontal and temporal cortex, Neurosci. Lett., 74 (1987) 237-242. 34 Widdowson, P.S. and Halaris, A.E., A comparison between the binding of [3H]proprionyl neuropeptide Y to rat and human frontal cortical membranes, J. Neurochem., 55 (1990) 956-962. 35 Widdowson, P.S. and Halaris, A.E., Increased levels of neuropeptide Y-immunoreactivity in rat brain limbic structures following antidepressant treatment, J. Neurochem., 52 (Suppl.) (1988) $77. 36 Widdowson, P.S. and Halaris, A.E., Effects of neuropeptide Y on alphal-and beta-adrenoceptor stimulated second messenger systems in rat frontal cortex, Peptides, 11 (1990) 661-665. 37 Widerl6v, E., Lindstom, L.M., Wahlestedt, C. and Ekman, R., Neuropeptide Y and peptide YY as possible cerebrospinal markers for major depression and schizophrenia, respectively, J. Psyehiat. Res., 22 (1988) 69-79. 38 Widerl6v, E., Heilig, M., Ekman, R. and Wahlestedt, C., Neuropeptide Y --possible involvement in depression and anxiety. In V. Mutt, K. Fuxe and T. H6kfelt (Eds.), Noble Conference on NPY, Raven, New York, 1989, pp. 331-342.
Brain Research. 539 (1991) 196-202 Elsevier
BRES 16254
Chronic desipramine treatment reduces regional neuropeptide Y binding to Ye-type receptors in rat brain P e t e r S. W i d d o w s o n 1"2 a n d A . E . Halaris 1"3 Departments of 1Psychiatry, 2Neuroscience and ~Pharmacology, Case Western Reserve University and MetroHealth Medical Center, Cleveland, OH 44109 (U.S.A.)
(Accepted 14 August 1990) Key words: Neuropeptide Y; Antidepressant drug; Yl-type receptor; Y2-type receptor; Second messenger
Chronic treatment of rats with desipramine and imipramine (5 mg/kg/twice daily/i.p.) for 14 days caused a significant reduction in the binding of [3H]proprionyl NPY to membranes prepared from frontal cortex, nucleus accumbens, hypothalamus and hippocampus. There was no change in binding of [3H]proprionyl NPY in the parieto-occipital cortex, striatum or amygdala. Scatchard analysis of binding data from frontal cortical and hippocampal membranes showed that [3H]proprionyl NPY bound to a single site with a Kd of approximately 0.3 nM. The loss of [3H]proprionyl NPY binding in hippocampal and frontal cortical membranes revealed that chronic tricyclic antidepressant treatment produced a reduction in the number of binding sites with no change in the affinity for the ligand. Chronic desipramine treatment did not alter the ability of NPY (0.01-25/aM) to stimulate inositol phosphate accumulation in rat frontal cortical slices as compared to saline-treated animals. The lack of change of NPY-induced inositol phosphate accumulation following chronic desipramine treatment showed that there was no change to Yt NPY-type receptors which are linked to the hydrolysis of inositol phospholipids. However, the ability of NPY (0.05-0.5/aM) to inhibit forskolin (1/aM) stimulated adenylate cyclase via Y2 NPY-type receptors in rat frontal cortical slices was significantly reduced following chronic desipramine treatment. This finding suggests that the reduction of [3H]proprionyl NPY binding in selective brain regions may be the result of an antidepressant-induced loss of Y2-type NPY receptors which are negatively linked to adenylate cyclase.
INTRODUCTION Neuropeptide Y (NPY), the 36 amino acid addition to the pancreatic polypeptide family, is found in large quantities in the rat central nervous system (CNS) 3'9. The presence of high affinity, saturable binding sites in the CNS for [125I]Bolton-Hunter NPY and [3H]proprionyl NPY suggests that NPY may play a role in synaptic transmission 18,2°,26'27"34. Although NPY exerts some of its effects by a direct receptor action, interactions with norepinephrine seem to play an important role in mediating other effects of the peptide such as the regulation of sympathetic nervous activity 17. Recently it has been suggested that NPY may be involved in the pathophysiology of depression 38. NPY exerts a powerful regulatory effect on the circadian rhythm of rat locomotor activity 1'2, eating behavior 14 and control of hypothalamic-pituitary function 29, all of which are dysregulated in depression 23. However, measurements of N P Y in cerebrospinal fluid of depressed patients have failed to provide conclusive proof for a dysregulation of the NPY system. Increases 1°, decreases 37 and no change 5 in CSF NPY concentrations from depressed
patients as compared to healthy subjects have been reported. However, this anomaly may reflect an alteration in the metabolism of N P Y between healthy subjects and depressed patients since the antibody used by Widerl6v et al. 37 detects several immunoreactive fragments which may relate to breakdown products of NPY. There are also reports of alterations in NPY concentrations following chronic, but not acute, antidepressant treatment of rats 11"25'31'35. Chronic treatment with the predominantly norepinephrine reuptake blocker, imipramine, increased NPY-immunoreactivity (NPY-ir) in frontal cortex and hypothalamus H. In contrast, chronic treatment with zimelidine, which blocks serotonin reuptake, increased NPY-ir in the frontal cortex only H. We have demonstrated this regional increase in NPY-ir with chronic antidepressant drug treatments 35. Chronic treatment with desipramine or imipramine increased NPY-ir in rat frontal cortex, hypothalamus, nucleus accumbens and hippocampus. Clorgyline, a monoamine oxidase type A inhibitor, increased NPY-ir in the hippocampus and nucleus accumbens as well as in the amygdala 35. In addition to antidepressant drug treatment, repeated, but not single electroconvulsive shock, increased NPY-ir in
Correspondence: P.S. Widdowson, Department of Psychiatry, MetroHealth Medical Center, 3395 Scranton Road, Cleveland, OH 44109, U.S.A.
0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
197 rat cortical regions a n d h i p p o c a m p u s 25'31. T h e effect of e l e c t r o c o n v u l s i v e shock t r e a t m e n t o n the increase in p e p t i d e c o n c e n t r a t i o n was highly specific for the N P Y system since t h e r e was n o a l t e r a t i o n in regional c o n c e n t r a t i o n s of g a l a n i n , vasoactive intestinal p o l y p e p t i d e ( V I P ) , s u b s t a n c e P, n e u r o k i n i n A or n e u r o t e n s i n 25. A s u b d i v i s i o n of N P Y receptors has b e e n recently p o s t u l a t e d b a s e d o n p h a r m a c o l o g i c a l studies 28'3°. Following this t e r m i n o l o g y , NPY-Y1 receptors r e q u i r e the entire N P Y m o l e c u l e for activation, while N P Y - Y 2 receptors can be activated by the 13-36 a m i n o acid C - t e r m i n a l s e q u e n c e of N P Y 3°. T h e Y l - t y p e N P Y r e c e p t o r has b e e n l i n k e d to the hydrolysis of inositol phospholipids while the Y2 N P Y r e c e p t o r is negatively c o u p l e d to a d e n y l a t e cyclase 3°. B o t h N P Y s u b t y p e s have b e e n d e m o n s t r a t e d in rat b r a i n using s e c o n d m e s s e n g e r systems as m a r k e r s . N P Y inhibits f o r s k o l i n - s t i m u l a t e d c A M P a c c u m u l a t i o n in hippocampa122 a n d striatal m e m b r a n e s 33 t h r o u g h the activation of Y2 receptors. In slices of frontal cortex, h i p p o c a m p u s a n d s t r i a t u m , N P Y stimulates the a c c u m u l a t i o n of inositol p h o s p h a t e s p r e s u m a b l y t h r o u g h activation of Y t - t y p e receptors 12. We investigated w h e t h e r c h r o n i c tricyclic antidepressant t r e a t m e n t alters N P Y b i n d i n g a n d N P Y - m e d i a t e d s e c o n d m e s s e n g e r systems in selective brain regions which m a y indicate c h a n g e s of N P Y activity. T h e action of d e s i p r a m i n e o n the N P Y system was c o m p a r e d to i m i p r a m i n e in this study b e c a u s e d e s i p r a m i n e selectively inhibits n o r a d r e n e r g i c r e u p t a k e sites 4 while i m i p r a m i n e affects n o r a d r e n e r g i c a n d s e r o t o n e r g i c r e u p t a k e sites 13. T h e c h a n g e s in N P Y - i m m u n o r e a c t i v i t y (ir) caused by c h r o n i c i m i p r a m i n e a d m i n i s t r a t i o n , as o b s e r v e d by Heilig et al. 11, m a y be d u e to a c o m b i n e d activity of i m i p r a m i n e on norepinephrine and serotonin reuptake.
MATERIALS AND METHODS Animals and treatments Male Sprague-Dawley rats (180-250 g) were chronically treated with either 0.9% saline or the tricyclic antidepressant drugs, desipramine and imipramine (5 mg/kg/twice daily/i.p.) dissolved in saline, for 14 days. Rats were maintained in groups of 4 at 20 °C with a 12 h light/dark cycle (lights on 06.00 h) and allowed free access to standard laboratory chow and water. Membrane preparation Twenty four hours after the last injection, rats were decapitated and the brains were rapidly removed and cut into 3 mm slices on an ice cold glass petri dish. Using a stereotaxic atlas for reference 2~, the hippocampus, striatum, nucleus accumbons, amygdala, hypothalamus, frontal and parieto-occipital cortex were dissected free and frozed on dry ice. The tissue was stored at -70 °C until assayed. Membranes were prepared from the tissue by homogenization in a loose-fitting motor driven teflon-glass homogenizer in ice-cold HEPES (5 mM) buffer sucrose solution (0.32 M, pH 7.4) (8 strokes up and down). The homogenates were then diluted 4-fold in the HEPES-sucrose buffer and then centrifuged at 1000 g for 10 min. The supernatant was recentrifuged at 10,000 g for 45 rain and the
resulting mitochrondrial/membrane pellet resuspended in HEPESbuffered (20 mM, pH 7.4) Krebs-Ringer solution (137 mM NaCI, 2.68 mM KCI, 2.05 mM CaCI2, 1.8 mM MgC12, 0.1% w/v ascorbic acid, 1 mg/ml glucose). Equilibrium binding was performed in 1.5 mi plastic microfuge tubes at 37 °C. [3H]Proprionyl NPY (68 Ci/mmol) (0.05-1.4 nM in 0.25 ml) (Amersham Corp., Arlington Heights, IL) in HEPESbuffered Krebs-Ringer solution containing bovine serum albumin (1.0% w/v) was added to each tube. NPY 200 nM (Bachem Inc., Torrance, CA) in 50 /~1 was added to half the tubes to define non-specific binding while the rest of the tubes received only 50/~1 buffer. The binding was initiated by the addition of 0.2 ml of protein suspension (0.1-0.2 mg) and the incubation stopped 45 min later by rapid centrifugation to separate bound NPY from the free. The supernatant was aspirated off, the pellets dried and the bottom of the plastic centrifuge tubes cut off and placed in liquid scintillation tubes. The radioactivity of the pellets was estimated by liquid scintillation spectrometry in a Beckman LS 3801 counter (counting efficiency = 46%). Data from the binding study was analyzed using LIGAND 19. Inositol phosphate hydrolysis method Twenty four hours after the last injection of either saline or desipramine (5 mg/kg/twice daily/i.p, for 14 days), rats were quickly decapitated, their brains removed and placed in ice-cold KrebsRinger buffer solution A (in mM: NaCI 120, KCI 5.5, CaCI2 2.5, NaH2PO 4 1.2, MgCI2 1.2, glucose 11, EDTA 19/~M, Na2HCO3, pH 7.4) which had been previously gassed with a 95% 02/5% CO 2 mixture. The frontal cortex and hypothalamus were dissected free on ice and the tissue cut into slices (350 x 350gm) using a MeIllwain tissue chopper. The slices were placed into 18 ml Beckman fiat-bottom polypropylene scintillation tubes containing 10 ml oxygenated solution A. The slices were then incubated in a rapidly shaking incubator (100 osciUations/min) at 37 °C for 30 min and oxygenated every 10 min with the 95% 02/5% CO 2 gas mixture. The tissue was allowed to settle to the bottom of the tube and then the buffer was aspirated off. Following the preincubation, the tissue was resuspended in 2.5 ml solution A containing 20/~Ci [3H]myoinositol (23 Ci/mmol) (Amersham, Corp., Arlington Heights, IL) and incubated for 60 min, gassing every 20 min. After prelabelling the tissue, the buffer was removed by aspiration. The tissue was then washed 3 times with warm 10 mi solution B (37 °C) containing 10 mM LiCI (solution A containing 10 mM LiCI and 110 mM NaC1). The tissue was then dispersed into 4 ml polypropylene Beckman Biovials containing 370/A solution B. The inositol phospholipid hydrolysis was initiated by the addition of NPY (0.01-25/~M) in solution B and the tissue incubated for a further 45 rain at 37 °C. The reaction was terminated by the addition of 0.5 ml solution B and the tissue washed 3 times with 0.5 ml cold solution B. 940/A of chloroform: methanol: HCI mixture (100:200:2 by vol) was added to the tissue and approximately 2000 cpm of [14C]inositol-l-phosphate (55 mCi/mmol) (Amersham Corp., Arlington Heights, IL) in 60/~1 methanol added to monitor the recovery of the inositol phosphates. The radiolabeled inositol phosphates were measured based on the method of Berridge et al. 6. The tubes were vortexed and allowed to stand for 10 min. Chloroform, 310/~1, and 310 #1 distilled water were added to the tubes and the tubes were vortexed again. The tubes were then centrifuged at 1500 g for 10 min at 4 0C. The upper aqueous layer was applied to chromatography columns (Dowex AG1 x8 100-200 mesh, formate form) which had been previously pre-equilibrated with I0 mM Tris (pH 7.4). The columns were then washed with 20 ml of 5 mM inositoi and the inositol-l-phosphate eluted with 1 ml 1 M ammonium formate: 0.1 M formic acid. The inositol-l-phosphate eluted from the columns was collected in scintillation tubes and the radioactivity [3H and tac] determined by liquid scintillation spectrometry. The average recovery of inositol1-phosphate was 65 + 2%. For the determination of the total [3H]inositol incorporated into lipid, the remaining upper phase and interphase were aspirated off and the chloroform layer evaporated to dryness. The [3H]inositol was then resuspended in 4 ml
198 scintillation fluid and the total radioactivity estimated by liquid scintillation spectrometry. The results are expressed as the fraction of the total [3H]inositol incorporated which was converted to [3H]inositol phosphate. cA MP method
Rats were sacrificed by decapitation, 24 h after the last injection of saline or desipramine (5 mg/kg/twice daily/i.p, for 14 days), their brains removed and quickly placed in ice-cold Krebs-Ringer buffer which had been previously oxygenated with 95% 02/5% CO 2. The frontal cortex and hippocampus were dissected on an ice-cold glass plate and cut into thin slices (300 x 300 gm) using a Mcllwain tissue chopper. The slices were dispersed in 40 ml oxygenated KrebsRinger buffer (KRB) at 30 °C. The slices were preincubated for 60 min washing with warm fresh oxygenated buffer every 20 min. After preincubation, slices were dispersed again in 35 ml of KRB and 2.75 ml aliquots in duplicate were placed into individual reaction vials and oxygenated continuously for 30 min. NPY (10 nM-1 #M) in 50 /d was then added to each tube and 10 min later the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthinein 150/~1 (0.5 raM) was added to each vial followed by a brief oxygenation. Finally, 10 min later forskolin (1-10/~M) in 50/~1 was added and all the tubes were oxygenated again. The reaction proceeded for 10 min and was stopped by the addition of 2.0 ml 3.1% perchloric acid. Samples were sonicated and centrifuged at 15,000 g for 15 min. Pellets were dissolved in 1.0 N NaOH and saved for subsequent protein determination. The supernatant was neutralized with excess (about 120 mg) CaCO 3 and assayed for its content of cAMP. cAMP levels were estimated by radioimmunoassay (RIANEN cAMP assay kit, New England Nuclear, DuPont, Billerica, MA) and the protein was estimated by the method of Lowry et al. 16. Statistical analysis
Statistical analysis of the data was performed using analysis of variance (ANOVA) followed by Duncan's multiple range test. Probability levels of less than 0.05 were taken to indicate a significant difference. RESULTS Chronic desipramine and imipramine treatment reduced the binding of [3H]proprionyl N P Y to m e m b r a n e s p r e p a r e d from frontal cortex, hippocampus, nucleus accumbens and hypothalamus as c o m p a r e d to the salinetreated rats (Figs. 1 and 2). The reduction of [3H]proprionyl N P Y binding following chronic antidepressant drug t r e a t m e n t was approximately equal for both desip r a m i n e and imipramine. H o w e v e r , there was no change in N P Y binding to parieto-occipital, striatal or amygdala m e m b r a n e s following desipramine and imipramine treatment (Fig. 1). Scatchard analysis of the binding in h i p p o c a m p u s and frontal cortex showed that the tricyclic antidepressant-induced reduction of N P Y binding represented a loss in NPY receptor density with no change in affinity (Figs. 2 and 3). Scatchard analysis also indicated that [3H]proprionyl N P Y b o u n d to a single site in the frontal cortical and h i p p o c a m p a l m e m b r a n e s with a Kd of about 0.3 nM. T h e r e was no evidence for multiple binding sites based on Scatchard analysis. NPY (0.01-25 /~M) significantly increased the accumulation of inositol phosphates in frontal cortical slices, o b t a i n e d from untreated rats, in a d o s e - d e p e n d e n t
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Fig. 1. Effects of chronic treatment with desipramine and imipramine on the binding of 1.25 nM [3H]proprionyl NPY to membranes prepared from the nucleus accumbens, amygdala, striatum, parietal cortex and hypothalamus. D, saline control;l~, desipramine treated; II, imipramine treated. Results are expressed as mean + S.E.M. (n = 6). *P < 0.01 as compared to saline controls.
m a n n e r (Fig. 4). Chronic t r e a t m e n t with d e s i p r a m i n e for 14 days did not significantly alter the N P Y - i n d u c e d accumulation of inositol p h o s p h a t e s in frontal cortical slices as c o m p a r e d to cortical slices o b t a i n e d from either saline treated rats or from u n t r e a t e d rats (Fig. 4). Forskolin (0.1-10 /~M) caused a significant dosed e p e n d e n t accumulation of c A M P in frontal cortical slices. Forskolin-stimulated cyclic A M P accumulation as a percentage basal concentrations: 0.1/~M forskolin, 283 + 12%; 1.0/~M forskolin, 512 + 23%; 1 0 . 0 # M forskolin, 795 + 28%. In frontal cortical slices o b t a i n e d from u n t r e a t e d rats N P Y ( 0 . 0 1 - 1 / t M ) maximally inhibited the forskolin-stimulated c A M P f o r m a t i o n in a d o s e - d e p e n dent m a n n e r by a p p r o x i m a t e l y 23% (Fig. 5). Chronic desipramine t r e a t m e n t for 14 days significantly r e d u c e d the 1/~M forskolin-stimulated c A M P formation in both h i p p o c a m p a l and frontal cortical slices by 22% (Fig. 5).
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In addition, NPY (0.05-0.5/~M) failed to further reduce the forskolin-stimulated cAMP in frontal cortical and hippocampal slices obtained from desipramine-treated rats as compared to saline controls (Fig. 5).
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Neuropeptide Y Concentration (pMI t~
Fig. 5. Effect of NPY on the 1 pM forskolin-induced stimulation of adenylate cyclase in frontal cortical and hippocampal slices. Mean + S.E.M. (n = 6). *P < 0.05 significantly different to 1 /~M forskolin alone (from saline-treated or untreated rats). *Significantly different from saline-treated and untreated rats. II, untreated rats; O, saline-treated rats; 0 , desipramine-treated rats.
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Fig. 4. Effect of N P ¥ on the accumulation of inositol phosphates in rat frontal cortical slices. Mean _+ S.E.M. (n = 6-12). *P < 0.05 as compared to basal level. O, untreated rats; Fq, chronic saline-treated rats; II, chronic desipramine-treated rats.
The ability of [3H]proprionyl NPY to bind to a single site in cortical and hippocampal membranes, with an affinity constant of 0.3 nM, closely agrees with previous reports using either tritiated or iodinated NPY as a ligand t8"26'27'34. Chronic administration to rats with the tricyclic antidepressant drugs, imipramine and desipramine, produced a significant reduction in the binding of [3H]proprionyl NPY to membranes prepared from frontal cortex, hippocampus, nucleus accumbens and hypothalamus. This antidepressant-induced reduction in [3H]proprionyl NPY binding was not widespread in all the brain regions examined since membranes prepared from the parieto-occipital cortex, striatum and amygdala failed to show significant changes in [3H]proprionyl NPY binding. Scatchard analysis of the binding to frontal cortical and hippocampal membranes demonstrated that
200 the loss of [3H]proprionyl NPY binding was the result of a tricyclic antidepressant-induced loss in the receptor number with no alteration in the affinity for NPY. Desipramine and imipramine do not directly interfere with [3H]proprionyl NPY binding in rat frontal cortical membranes (P.S. Widdowson, unpublished results) suggesting that the reduction in NPY receptor density was not due to direct interaction of the tricyclic antidepressants with the NPY receptor. We have recently demonstrated that chronic administration of desipramine and imipramine to rats significantly increases NPY immunoreactivity in frontal cortex, hippocampus, nucleus accumbens and hypothalamus 35. Thus, the same brain regions show an increase in NPY immunoreactivity and a reduction in NPY receptors following chronic desipramine and imipramine treatment. We suggest that the repeated treatment of rats with desipramine and imipramine chronically interferes with NPY synthesis and release. It is possible that the antidepressant-induced reduction in [3H]proprionyi NPY binding represents the down-regulation of NPY receptors caused by chronic elevations in synaptic NPY concentrations. Repeated administration of imipramine and desipramine may increase the release of NPY from peptide stores producing a reduction in the NPY receptor density. The increase in regional NPY concentrations 35 would then reflect an increase in NPY synthesis to compensate for the increase in NPY release. Although the precise mechanism for the imipramine and desipramine-induced reduction of NPY receptors is not known, we propose that the tricyclic antidepressants act on the NPY system indirectly through their ability to increase synaptic norepinephrine concentrations 7'~5. Chronic tricyclic-induced elevations of norepinephrine concentrations may increase the release of NPY and down-regulate NPY receptors. Imipramine was approximately equipotent to desipramine in its ability to reduce NPY binding sites in the hippocampus, frontal cortex, hypothalamus and nucleus accumbens. Although imipramine is a potent inhibitor of serotonin reuptake 13 and although about 30% of chronically administered imipramine is converted to desipramine s, the similarity of these two tricyclic antidepressants in reducing NPY binding suggests that imipramine, like desipramine, acts, at least in part, through the noradrenergic system 4,~5. Repeated treatment of rats with electroconvulsive shock has been also reported to increase NPY-immunoreactivity in some cortical regions and hippocampus 25"31. However, Wahlestedt et al. 31 were unable to show a significant change in [125I]NPY binding in hippocampus, frontal or parietal cortex, although there was a 10% reduction in NPY binding in the frontal cortex 34. It is unlikely that the choice of ligands used, i.e. [3H]-
proprionyl NPY in this study and [125I] Bolton Hunter (BH) NPY used by Wahlestedt et al. 3~ is responsible for the discrepancy between the chronic tricyclic antidepressant drug treatment and electroconvulsive shock since both ligands have been shown to bind to a single class of brain receptors with approximately the same affinity ~8' 26,27.34. The reason for this discrepancy is unknown but may be related to a different mechanism of action of antidepressant drug treatment and electroconvulsive shock on the NPY system. Based on pharmacological tests and binding studies a number of investigators have suggested that multiple NPY receptors exist in central and peripheral neurons 24' 28,3o. We have provided evidence that in frontal cortex there are multiple NPY receptors based on the ability of NPY to stimulate inositol phospholipids and to inhibit adenylate cyclase. At present the lack of NPY analogues makes it impossible to accurately distinguish multiple NPY receptors. There are some reports of multiple binding sites in rat brain tissue using [~25I]BH NPY and displacing it with the structurally related peptide yy32. However, which of these putative receptor populations represent the Y1 or the Y2 type is not known but has been suggested that the Y2 NPY receptor is the predominant sub-type in the central nervous system 24. We utilized the ability of NPY to stimulate different second messenger systems, as a measure of effects of drug treatment on each NPY receptor sub-type. We demonstrated that NPY stimulates the hydrolysis of inositol phospholipids and inhibits forskolin-stimulated cAMP accumulation in slices of rat frontal cortex. This suggests that the frontal cortex possess both Y1 and Y2 receptor subtypes which have been suggested to be linked to the inositol phosphate system and the adenylate cyclase system, respectively28'3°. Chronic desipramine treatment did not alter the ability of NPY to stimulate inositol phosphate production suggesting that the YI NPY receptors, which are linked to phospholipase C 3°, are not altered by these antidepressants. Chronic desipramine treatment significantly reduced the ability of forskolin to stimulate adenylate cyclase activity in both hippocampal and frontal cortical slices. The meehanism for this desipramine-induced reduction of cAMP accumulation is unknown at present. However, chronic desipramine treatment to rats produces a widely documented down-regulation of fl-adrenoceptors and a reduction of the fl-adrenoceptor mediated stimulation of cAMP in rat brain tissue 7, presumably as a result of chronic elevations of synaptic norepinephrine 16. It is therefore plausible that the desipramine-induced reduction in forskolin-stimulated cAMP concentration represent a reduction in number of functional adenylate cyclase molecules, some of which may be linked to
201 fl-adrenoceptors which are present in large numbers in the rat cortex, Thus, following the chronic administration of desipramine, there were fewer adenylate cyclase molecules to be activated since they have been downregulated by the increased fl-adrenoceptor mediated signal as a result of the desipramine-mediated elevations of synaptic norepinephrine 15. Following the chronic desipramine administration, NPY (0.05-0.5 /~M) failed to reduce the forskolinstimulated c A M P accumulation in frontal cortical slices, as compared to forskolin alone, Although there was a significant, 25% reduction in the ability of forskolin to stimulate c A M P production in both the hippocampal and frontal cortical slices, there was still a large increase in the c A M P concentrations (330-400% above basal in the hippocampal and frontal cortical slices, respectively) following 1/~M forskolin addition for the NPY to have an effect. Only those cells which express fl-adrenoceptors would therefore be expected to have a reduced number of adenylate cyclase molecules in response to chronic desipramine administration. We have been unable to demonstrate an NPY-induced reduction in fl-adrenoceptor-stimulated c A M P accumulation in rat frontal cortex slices 36, suggesting that NPY Y2-type receptors are not located on the same cells expressing fl-adrenoceptors. Thus, the number of adenylate cyclase molecules in cells
which possess Y2-NPY receptors would not be expected to be altered following desipramine treatment, since they are not activated by desipramine treatment, rather the opposite, since Y2-type receptors reduce the stimulated c A M P concentration. The observation that N P Y failed to inhibit forskolin-induced c A M P production in the frontal cortical and hippocampal slices therefore suggests a loss in Y2 NPY receptor activity which is negatively linked to adenylate cyclase activity. Since it has been proposed that a majority of N P Y receptors in the CNS are the Y2-type 24, and that desipramine produces a large decrease in the density of N P Y receptors in selective brain regions, it would be expected that the ability of NPY to reduce c A M P concentrations would also be reduced. In summary, we have demonstrated that following repeated administration of the tricyclic antidepressants, desipramine and imipramine to rats, there is a reduction in NPY binding in selective brain regions and a reduction in the ability of NPY to inhibit forskolin-stimulated cAMP production in the frontal cortex and hippocampus. We suggest that chronic desipramine and imipramine causes a down-regulation of Y2-type NPY receptors in selective brain regions whilst Y r t y p e receptors are unchanged.
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