Neonatal DSP-4 treatment impairs 5-HT 1B receptor reactivity in adult rats. Behavioral and biochemical studies
Marzena Ferdyn-Drosik1, Przemys³aw Nowak1, Kamila Bojanek1, Micha³ Ba³asz1, Jacek Kasperski2, Dariusz Skaba3, Rafa³ Muchacki1, Richard M. Kostrzewa4
Department of Pharmacology, Department of Prosthetic Dentistry,!Department of Conservative Dentistry and Endodontics Division of Dental Propedeutics, Medical University of Silesia, H. Jordana 38, PL 41-808 Zabrze, Poland
"Department of Pharmacology, Quillen College of Medicine, East Tennessee State University, Johnson City,
TN 37614, USA
Correspondence: Przemys³aw Nowak, e-mail: pnowak@sum.edu.pl
Abstract:
To examine the effect of a central noradrenergic lesion on the reactivity of the 5-HT1Breceptor we compared intact male rats with rats in which noradrenergic nerve terminals were largely destroyed with the neurotoxin DSP-4 (50 mg/kg × 2, on the 1st and 3rd days of postnatal life). When rats attained 10 weeks of age, control and DSP-4 rats were divided into two subgroups receiving either saline or the serotonin (5-HT) synthesis inhibitor (p-chlorophenylalanine; p-CPA; 100 mg/kg). Employing an elevated plus maze test, we demonstrated that CP 94,253 (5-propoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-pyrrolo[3,2-b]pyridine hydrochloride)(4.0 mg/kg;
5-HT1Bagonist) induced an anxiogenic-like action in control rats; however, it failed to elicit this effect in the DSP-4 group. Surpris- ingly, inp-CPA pretreated rats anxiogenic-like activity was observed both in control and DSP-4 treated rats. CP 94,253 significantly attenuated 5-HT synthesis in the medial prefrontal cortex (mPFC) of control rats, and SB 216641 (N-{3-[3-(dimethyl- amino)ethoxy]-4-methoxyphenyl}-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl)-[1,1’-biphenyl]-4-carboxamide hydrochloride) (4.0 mg/kg; 5-HT1Bantagonist) was able to antagonize this effect. Conversely, CP 94,253 failed to significantly inhibit the 5-HT synthesis rate in DSP-4-treated rats. In the microdialysis study CP 94,253 induced long-lasting attenuation of 5-HT release in the mPFC of control rats but had no effect in DSP-4 rats. These data lead to the proposal that presynaptic 5-HT1Bautoreceptors under- went desensitization in DSP-4 treated rats.
Key words:
DSP-4, lesion, 5-HT *receptor, anxiety, rats
Introduction
Serotonin (5-HT) plays a key modulatory role in a plethora of functions of the central nervous system in both the physiological and disease states [4]. There are 14 different 5-HT receptors, among them 5-HT1A and 5-HT1Breceptors play a crucial role in regulating
5-HT neurotransmission. Both 5-HT1A and 5-HT1B receptors are autoreceptors on 5-HT-containing neu- rons and heteroreceptors on several neurons that do not contain 5-HT, e.g., norepinephrine (NE), dopamine (DA), etc. [7, 25, 42, 48, 53, 62].
It is generally accepted that there is a mutual inter- action between norepinephrine (NE) and 5-HT. First, numerous brain regions are innervated by both sero-
Pharmacological Reports 2010, 62, 608620 ISSN 1734-1140
Copyright © 2010 by Institute of Pharmacology Polish Academy of Sciences
toninergic and noradrenergic neurons originating from the dorsal raphe nuclei (DRN) and median raphe nuclei (MRN) and locus coeruleus (LC), respectively [35, 60]. Second, LC, the major NE brainstem nu- cleus, sends projections to the DRN, while the DRN projects to the LC, creating ample opportunity for cross-modulation [21, 49].a2-Receptors are known to exert an inhibitory influence on ascending sero- toninergic pathways, partly by activation of a2-auto- receptors, thereby suppressing a tonic, excitatory in- fluence ofa1-autoreceptors on serotoninergic perikarya in raphe nuclei [39]. Inhibitory a2-heteroceptors are localized on terminals of serotoninergic neurons in corticolimbic structures [22], and this inhibitory influ- ence of a2-agonists on serotoninergic pathways con- tributes to sedative and potential anxiolytic actions.
Conversely, serotoninergic neurons are known to ex- ert tonic inhibition on the noradrenergic nervous sys- tem. Microdialysis studies have shown that 5-HT1A, 5-HT2C, and 5-HT3 receptor agonists reduce NE re- lease [51]. In addition, 5-HT enhances a tonic inhibi- tory influence on noradrenergic neurons indirectly through postsynaptic 5-HT2A receptors located on GABA interneurons in the LC [57, 58]. However, the role of 5-HT1B heteroreceptors on the noradrenergic system has not been elucidated [51]. 5-HT1Bterminal autoreceptors appear to be engaged in the adaptation of DRN neurons to 5-HT-selective antidepressants, which are also effective in many anxiety disorders [53]. 5-HT1B mRNA is selectively downregulated in DRN by either fluoxetine or paroxetine [2]. Further- more, learned helplessness in rats (an animal model of depression) is associated with a reversible deficit in 5-HT release in prefrontal cortex as well as an in- crease in 5-HT1BmRNA in DRN [40, 47]. However, in most cases it has not been possible to ascribe the 5-HT1Beffects to a particular population of neurons because 5-HT1Breceptors on various neuronal termi- nals are intermingled in practically all forebrain areas.
Clark et al. [12] have developed a viral-mediated gene transfer strategy to express hemagglutinin-tagged 5-HT1Band separately manipulate autoreceptors and heteroreceptors in DRN. They found that overactivity of 5-HT1Bautoreceptors in DRN neurons may be an important mediator of the pathological responses to anxiogenic stimulus. These observations imply that increased 5-HT1B autoreceptor activity induces de- pressive and related anxiety symptoms and that down- regulation of 5-HT1B autoreceptors localized in the DRN by sustained (at least 8 week) antidepressants
administration may be crucial in normalizing sero- toninergic neurotransmission. In this way it would re- lieve the symptoms of depression or anxiety [8]. Kos- towski et al. [29] demonstrated that electrical stimula- tion of the LC (either unilateral or bilateral) reduced immobility. For example, it produced an antidepres- sant effect similar to that produced by desipramine.
Conversely, lesion of the LC failed to influence ani- mal behavior in the immobility test. These results are in line with electrophysiological data showing that the stimulation of LC NE neurons enhanced the firing rate of DRN 5-HT neurons, while the lesion of LC NE neurons had no effect [23, 56]. More recently, Harro et al. [23] found that seven days after DSP-4 treat- ment (50 mg/kg, ip), rats exhibited a decrease rather than an increase in immobility time, assessed by the forced swimming test (anti-depressive effect). These effects seemingly contrast with the current view of the role of the noradrenergic system in depression (im- paired and functional diminution in NE neurotrans- mission). One explanation of this phenomenon could be the downregulation of 5-HT1B autoreceptors in DSP-4 treated rats serving as a “self-protecting” mecha- nism to preserve functional control (e.g., preventing manifestation of depressive-like behavior). However, to the best of our knowledge, there are no data con- cerning the influence of neonatal DSP-4 treatment on the reactivity of the 5-HT1Breceptors. Lesion studies demonstrated that severe 5-HT depletion (with 5,7- DHT) in adult rats produces large increases in the number of 5-HT1B/1Dbinding sites in the substantia nigra and the shell of the nucleus accumbens, whereas no changes took place in the globus pallidus [13].
Upregulation of 5-HT1Breceptors was also observed in the hypothalamus in rats lesioned with 5,7-DHT [37]. The data concerning noradrenergic system lesion, both in neonates and adulthood, are very obscure. Only Laporte et al. [32] reported that noradrenergic de- struction with DSP-4 (in adult rats) results in a signifi- cant increase in the specific binding of [3H]8-OH- DPAT to 5-HT1Aand [125I]GTI to 5-HT1Breceptors in the dorsal horn.
It was demonstrated that NE (directly or indirectly) as well as its transporter (NET) are involved in the ac- tivation of adrenergic receptors and increase the ad- renergic neuron differentiation [55, 63]. It was also found that the depletion of NE by DSP-4 results in a reduction in the proliferation of adult hippocampal granule cell progenitors [31]. However, there is no data on how NE affects 5-HT1B receptor develop-
ment. Neonatally administered DSP-4 is known to produce marked and permanent alterations of the postnatal development of the central noradrenergic, serotoninergic and other systems in the brain [27, 30, 52]. From our previous study we found that chemical lesioning of noradrenergic neurons with DSP-4 greatly altered the function of histaminergic [44], do- paminergic [45] and GABA-erigc systems in the brain [5, 6]. In an attempt to better understand the control of the serotoninergic system by noradrenergic inputs, we recently examined the effects of neonatal DSP-4 treat- ment on the 5-HT1A mediated neurotransmission in adulthood [15, 16]. Here we extend our study to ex- amine the impact of noradrenergic lesion on the reac- tivity of 5-HT1Breceptors. As it was noted, the inter- play between both aminergic systems is involved in common physiological aspects of serotonergic and noradrenergic neurons, and these interactions play a role in the reported superior clinical efficacy of anti- depressants acting on both neurotransmitter systems.
For this reason we believe that this study provides possible implications for understanding how the nora- drenergic dysfunction affects serotoninergic system activity.
Materials and Methods
Animals
Male and female Wistar rats (University Animal De- partment; Katowice, Poland) were housed under con- trolled environmental conditions in a well-ventilated room, at 22 ± 2°C and under a 12 h light : 12 h dark cycle (lights on 7:00 a.m.). Animals received food and water ad libitum. Offspring rats were weaned at 21 days and segregated by sex.
The central noradrenergic system of male newborn rats was lesioned with N-(-2-chloroethyl)-N-ethyl-2- bromobenzylamine (DSP-4) (Sigma, St. Louis, MO, USA). Rats were injected on the 1st and 3rd days of postnatal life with either DSP-4 (50 mg/kg, sc) or 0.9% NaCl (1.0 ml/kg, sc). DSP-4 was dissolved in 0.9% NaCl immediately before injection. Rats contin- ued to be housed as above until 8–10 weeks, for fur- ther experimentation. Experiments were carried out in the morning in only male rats, handled in accordance with the principles and guidelines described in the
NIH booklet Care and Use of Laboratory Animals.
All procedures were reviewed and approved by the Local Bioethical Committee for Animal Care.
Elevated plus maze
Anxiety-like behavior in the elevated plus maze was measured according to Pellow et al. [46]. This appara- tus was composed of two opposing open arms (50 × 10 cm) without sides and two opposing open arms of the same size with 40 cm high walls. The apparatus was attached to a central square (10 cm2). The maze was thus shaped as plus sign. All surfaces were black.
The entire device was placed 50 cm above the floor.
Rats from both test groups (control and DSP-4) were divided into four subgroups receiving CP 94,253 (5-HT1Breceptor agonist) or SB 216641 (5-HT1Bre- ceptor antagonist) according to the following sched- ule:
1. saline (1.0 ml/kg,ip) twice, 30 min interval, 2. saline (1.0 ml/kg, ip) 30 min before CP 94,253 (4.0 mg/kg,ip),
3. SB 216641 (4.0 mg/kg, ip) 30 min before saline (1.0 ml/kg,ip),
4. SB 216641 (4.0 mg/kg,ip) 30 min before CP 94,253 (4.0 mg/kg,ip).
Immediately after the last injection, rats were re- turned to their home cage for 30 min before the 5-min plus maze test. Each rat was then placed in the central square of the plus maze, facing an enclosed arm. An arm entry was defined as all four paws entering an arm. The following scores were recorded: number of entries into closed arms and time spent in enclosed arms. The time and the number of entries into the open arms were measured. The total number of open and closed arm entries reflected general motor activ- ity. The maze was cleaned after each rat was tested.
The same procedure was performed on animals (con- trol and DSP-4) that formerly were pretreated with the 5-HT synthesis inhibitor,p-chlorophenylalanine (p-CPA;
100 mg/kg,ip) twice at 24 h intervals, approximately 48 h prior to behavioral testing.
The doses of 5-HT1B ligands were chosen on the basis of our pilot studies and the literature data. Oth- ers also demonstrated that CP 94,253 in the range of 1.0–5.6 mg/kg altered exploration in the open arms of the plus-maze without altering overall locomotor ac- tivity [34]. Higher doses of CP 94,253 stimulates mo- tor activity [15] and obscures data interpretation.
The observer did not know the group to which each rat belonged during testing. The experimenter used one rat from each group, followed by a second rat from each group, and then a third, etc.
Assay of biogenic amines and their metabolites
Animals were treated with CP 94,253 (4.0 mg/kg,ip) and/or SB 216641 (4.0 mg/kg, ip) according to the same paradigm as described in the elevated plus maze test. Thirty minutes after the last injection, the rats of all tested groups were decapitated. The mPFC was rapidly dissected and placed on dry ice, weighed and stored at –70°C, pending assay. Samples were ho- mogenized for 15–20 s in ice-cold trichloroacetic acid (0.1 M) containing 0.05 mM ascorbic acid. After cen- trifugation (5,000 × g, 5 min), supernatants were fil- tered through 0.2 µm cellulose membranes (Titan MSF Microspin filters, Scientific Resources Inc., Eatontown, GB) and injected onto the HPLC/ED column.
Levels of DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT, 5-hydroxyindolea- cetic acid (5-HIAA) and NA were assayed [36, 43].
The mobile phase was composed of 75 mM NaH2PO4 (Avocado, Research Chemicals Ltd.), 1.7 mM 1-octane- sulfonic acid (Avocado, Research Chemicals Ltd.), 5 µM EDTA (Avocado, Research Chemicals Ltd.), 100 µl triethylamine (Sigma), and 9.5% acetonitrile (Lab-Scan), pH 3 adjusted with phosphoric acid (Fluka). The flow rate was maintained at 0.7 ml/min, at a temperature of 22°C, and the oxidation potential was fixed at +700 mV, 10 nA/V sensitivity. Peaks were automatically integrated by universal chroma- tographic interface UCI-100 (Dionex, Germany). The instrumentation included an electrochemical detector model 141 with flow cell, piston pump model 302 with head 5SC, manometric module model 802 (Gil- son, France), thermostat for STH 595 column (Dionex, Germany), precolumn Hypersil BDS C18, 10 × 4 mm, 3 µm and chromatographic column Hy- persil BDS C18, 250 × 4.6 mm, 3 µm (ThermoQuest, GB).
5-HTP accumulation assay (an index of 5-HT synthesis rate)
The synthesis rate of 5-HT was assessed by the indi- rect method of Carlsson et al. [9], based on the accu- mulation rate of 5-hydroxytryptophan (5-HTP) after injection of the aromatic amino acid decarboxylase
inhibitor hydroxybenzylhydrazine (NSD-1015). Rats from control and DSP-4 groups were divided into 4 subgroups receiving CP 94,253 (4.0 mg/kg,ip) and/or SB 216641 (4.0 mg/kg, ip) according to the same paradigm as described in the elevated plus maze test.
Thirty minutes after the last injection, rats were in- jected with NSD-1015 (100 mg/kg,ip). After 30 min the rats were decapitated, and brain tissue samples were dissected and analyzed by HPLC for 5-HTP as described above.
Brain microdialysis
Microdialysis in freely moving rats was used to char- acterize the 5-HT response to the 5-HT1B receptor agonist (CP 94,253) in the mFPC. Male Wistar rats were anesthetized with relanium (Polfa, 10 mg/kg,ip) and ketamine (Parke-Davis, 80 mg/kg,ip), and placed in a stereotaxic frame. The dermis overlying the skull was incised, and the dermis was retracted to expose the skull plate. A small burr hole was drilled to allow implantation of a dialysis probe with 4 mm active membrane (ID 75 µm, OD 150 µm, Polymicron Tech- nologies Inc., USA) into the right mPFC (A +3.0, R +0.6, V –5.0 in relation to bregma; according to Paxi- nos and Watson). Two stainless steel screws were mounted near the probe and fastened to the skull with dental cement (Duracryl Plus, Spofa, Praha). On the following day the free ends of the probe were con- nected with teflon tubes and continuously perfused with artificial cerebrospinal fluid (aCSF) (Na+145 mM, K+2.7 mM, Ca2+1.2 mM, Cl-151.7) at a flow rate of 2.0 µl/min (Microdialysis pump, Harvard Apparatus Model 22, GB) [43]. Because the microdialysate level of 5-HT represents a balance between release and re- uptake of the neurotransmitters, a selective 5-HT re- uptake inhibitor (zimelidine) (SSRI) was added at a concentration of 1 µM to the CSF. In this way the microdialysate content of 5-HT reflects mainly the re- lease of transmitter. Samples were collected every 20 min and injected directly onto a 3 µm 150 × 3 mm column (MD 150/RP-18, ESA, USA), using a mobile phase con- sisting of 1.7 mM 1-octanesulfonic acid, 25 µM EDTA, 100 µl triethylamine/1000 ml, and 10% acetonitrile in 75 mM phosphate buffer at pH 3 and flow rate of 0.6 ml/min. A guard cell (+250 mV), and flow-through electrochemical cell (E1 +250; E2 –175) were used for analysis, with a Coulochem (ESA, USA) data analysis system to integrate peak areas of 5-HT. When dialysate 5-HT levels were constant (at about 1.5 h
from the beginning of perfusion), rats were injected with CP 94,253 (4.0 mg/kg, ip). Final results of the 5-HT analysis, in picograms (pg) in 20 µl capacity of loop, were expressed as a percent of the mean of the 3 pre-CP 94,253 samples. At the end of the study, the position of the probe in the mPFC was verified.
Statistical analysis
Group differences were assessed by an analysis of variance (ANOVA) and the post-ANOVA test of Newman-Keuls. Group differences in microdialysis studies were analyzed by Student’st-test. A p < value 0.05 was taken as the level of significant difference.
Results
Elevated plus maze
Basal anxiety, assessed as time spent on open arms af- ter saline treatment did not differ between control and
DSP-4 treated rats. In addition, CP 94,253 (4.0 mg/kg, ip) was without effect on measured parameters for control and DSP-4 treated rats. Only SB 216641 (4.0 mg/kg,ip) significantly increased the time spent on open arms in control rats in comparison to the DSP-4 group (2-way ANOVA, groups [F = 4.665, p < 0.035]; substances [F = 3.571, p < 0.020]; both factors [F = 0.580, p < 0.630]). Neither CP 94,253 nor SB 216641 altered the number of entries into open arms in control and DSP-4-lesioned rats (groups [F = 3.018, p < 0.088]; substances [F = 2.041, p < 0.119];
both factors [F = 0.869, p < 0.869]) (Fig. 1).
CP 94,253 (4.0 mg/kg,ip) increased the time spent on closed arms in control rats (vs. saline treatment), being without effect in DSP-4 lesioned animals. The 5-HT1Bantagonist (SB 216641) alone did not alter the time in closed arms but when administered 30 min be- fore CP 94,253, the effect (i.e., time spent on closed arms) was attenuated (groups [F = 0.495, p < 0.484];
substances [F = 9.211, p < 0.00005]; both factors [F = 0.618, p < 0.606]). Neither CP 94,253 nor SB 216641 altered the number of entries into closed arms in con- trol and DSP-4-lesioned rats (groups [F = 0.076,
0 3 6 9 12 15
0.9% NaCl CP 94253 SB 216641 CP 94253 + SB 216641
0 50 100 150 200 250 300
0.9% NaCl CP 94253 SB 216641 CP 94253 + SB 216641
* OPEN ARMS
time(s)numberofentries
Fig. 1. Effect of neonatal DSP-4 (50 mg/kg,sc) lesioning on the time spent in open arms and the number of entries in open arms of elevated plus maze after CP 94,253 (4.0 mg/kg,ip) or SB 216641 (4.0 mg/kg,ip) treatment of adult rats (n = 6–8) (the mean ± SEM).
Explanations:¨ – control, ¢ – DSP-4,
* p < 0.05; control (saline)vs. DSP-4
0 50 100 150 200 250 300
0.9% NaCl CP 94253 SB 216641 CP 94253 + SB 216641
* *
time(s)
0 3 6 9 12 15
0.9% NaCl CP 94253 SB 216641 CP 94253 + SB 216641
numberofentries
CLOSED ARMS
Fig. 2. Effect of neonatal DSP-4 (50 mg/kg, sc) lesioning on the time spent in closed arms and the number of entries in closed arms of elevated plus maze after CP 94,253 (4.0 mg/kg,ip) or SB 216641 (4.0 mg/kg, ip) treatment of adult rats (n = 6–8) (the mean ± SEM).
Explanations:¨ – control, ¢ – DSP-4,
* p < 0.05; control (saline)vs. control (CP 94,253); control (CP 94,253)vs.
control (CP 94,253 + SB 216641)
0 50 100 150 200 250 300
0.9% NaCl CP 94253 SB 216641 CP 94253 + SB 216641
time(s)
OPEN ARMS ( -CPA)p
time(s)
0 3 6 9 12 15
0.9% NaCl CP 94253 SB 216641 CP 94253 + SB 216641
numberofentriesnumberofentries
Fig. 3. Effect of neonatal DSP-4 (50 mg/kg,sc) lesioning on the time spent in open arms and the number of en- tries in open arms of elevated plus maze after CP 94,253 (4.0 mg/kg,ip) or SB 216641 (4.0 mg/kg,ip) admini- stration to adult rats pretreated with p-CPA (100 mg/kg, ip two times every 24 h) (n = 6–8) (the mean ± SEM). Ex- planations:¨ – control, ¢ – DSP-4
p < 0.783]; substances [F = 1.053, p 0.376]; both fac- tors [F = 0.889, p < 0.453]) (Fig. 2).
In p-CPA pretreated rats neither CP 94,253 (4.0 mg/kg,ip) nor SB 216641 (4.0 mg/kg, ip) altered the time spent in open arms in control and DSP-4 group (groups [F = 0.205, p 0.652]; substances [F = 6.786, p
< 0.0006]; both factors [F = 0.530, p < 0.663]). Addi- tionally, 5-HT1Aligands did not affect numbers of en- tries into open arms (groups [F = 0.171, p < 0.681];
substances [F = 9.551, p < 0.0004]; both factors [F = 0.487, p < 0.692]) (Fig. 3).
CP 94,253 increased the time spent in closed arms inp-CPA pretreated control and DSP-4 groups (groups [F = 2.438, p 0.124]; substances [F = 4.906, p < 0.0044];
both factors [F = 0.242, p < 0.865]) (Fig. 4). SB 216641 alone did not alter the time spent in closed arms. In addition, CP 94,253 and SB 216641 did not alter the numbers of entries into closed arms, suggest-
0 50 100 150 200 250 300
0.9% NaCl CP 94253 SB 216641 CP 94253 + SB 216641
*
*
CLOSED ARMS ( -CPA)p
0 3 6 9 12 15
0.9% NaCl CP 94253 SB 216641 CP 94253 + SB 216641
time(s)numberofentries
Fig. 4. Effect of neonatal DSP-4 (50 mg/kg,sc) lesioning on time spent in closed arms and number of entries in closed arms of elevated plus maze after CP 94,253 (4.0 mg/kg,ip) or SB 216641 (4.0 mg/kg, ip) administration to adult rats pretreated with p-CPA (100 mg/kg, ip two times every 24 h) (n = 6–8) (the mean ± SEM). Explanations:¨ – control, ¢ – DSP-4, * p < 0.05; control (saline) vs. control (CP 94,253); DSP-4 (saline) vs. DSP-4 (CP 94,253)
Tab. 1. Effect of neonatal DSP-4 (50 mg/kg,sc) lesioning on the concentration of NA, 5-HT, 5-HIAA, DA, DOPAC and HVA in the mPFC after CP 94,253 (4.0 mg/kg,ip) or SB 216641 administration to adult rats (n = 6–8) (the mean ± SEM)
Groups Substances NA
(ng/g of wet tissue)
5-HT (ng/g of wet tissue)
5-HIAA (ng/g of wet tissue)
DA (ng/g of wet tissue)
DOPAC (ng/g of wet tissue)
HVA (ng/g of wet tissue) CONTROL 0.9% NaCl 247.8 ± 27.3 351.8 ± 39.3 157.5 ± 18.7 161.0 ± 55.8 45.7 ± 10.7 68.8 ± 15.4
CP 94253 215.8 ± 11.9 370.4 ± 19.0 124.2 ± 11.6 196.6 ± 54.5 52.0 ± 6.4 94.5 ± 8.1 SB 216641 239.7 ± 19.3 373.2 ± 36.9 150.9 ± 16.0 194.8 ± 12.7 53.7 ± 11.7 85.9 ± 17.6 CP 94253 + SB 216641 223.2 ± 18.7 367.7 ± 75.7 108.6 ± 9.0 153.8 ± 52.3 33.3 ± 3.2 58.5 ± 7.6 DSP-4 0.9% NaCl 15.4 ± 5.5* 362.8 ± 48.8 159.0 ± 28.7 124.8 ± 66.9 40.8 ± 13.9 82.8 ± 12.4
CP 94253 11.0 ± 4.3* 342.7 ± 33.4 132.6 ± 22.5 85.9 ± 15.9 31.6 ± 5.2 72.1 ± 7.4 SB 216641 30.3 ± 10.9* 303.0 ± 23.0 114.0 ± 7.2 188.8 ± 94.3 55.7 ± 21.2 91.5 ± 23.3 CP 94253 + SB 216641 28.2 ± 4.1* 292.7 ± 40.8 89.9 ± 11.8 81.3 ± 26.4 30.1 ± 8.5 70.9 ± 12.3
* p < 0.05; controlvs. DSP-4
Tab. 2. Effect ofp-CPA (100 mg/kg ´ 2) pretreatment on the concentration of NA, 5-HT, 5-HIAA, DA, DOPAC and HVA in the mPFC of adult con- trol and DSP-4 (50 mg/kg,sc) rats (n = 6–8) (the mean ± SEM)
Groups NA
(ng/g of wet tissue)
5-HT (ng/g of wet tissue)
5-HIAA (ng/g of wet tissue)
DA (ng/g of wet tissue)
DOPAC (ng/g of wet tissue)
HVA (ng/g of wet tissue) Medial prefrontal cortex
(after saline ml/kg,ip)
CONTROL 247.8 ± 27.3 351.8 ± 39.3 157.5 ± 18.7 161.0 ± 55.8 45.7 ± 10.7 68.8 ± 15.4 DSP-4 15.4 ± 5.5 362.8 ± 48.8 159.0 ± 28.7 124.8 ± 66.9 40.8 ± 13.9 82.8 ± 12.4 Medial prefrontal cortex
(afterp-CPA 100 mg/kg, ip) CONTROL 194.0 ± 28.3 38.6 ± 5.9* 11.8 ± 5.2* 174.4 ± 33.2 41.1 ± 5.6 72.2 ± 7.4 DSP-4 14.9 ± 3.0 29.8 ± 3.4* 17.9 ± 9.2* 91.3 ± 12.4 22.9 ± 4.6* 59.4 ± 4.5*
* p < 0.05; control (saline)vs. control (p-CPA) and DSP-4 (saline) vs. DSP-4 (p-CPA)
0 50 100 150 200 250 300
0.9 % NaCl CP 94253 SB 216641 CP 94253 + SB 216641
ng/g wet tissue
*
Fig. 5. Effect of neonatal DSP-4 (50 mg/kg,sc) lesioning on the 5-HT synthesis rate in the mPFC after CP 94,253 (4.0 mg/kg, ip) or SB 216641 (4.0 mg/kg,ip) administration to adult rats (n = 5) (the mean ± SEM). Explanations:¨ – control, ¢ – DSP-4, * p < 0.05; control (saline) vs. control (CP 94,253)
ing that the behavioral effect observed in control and DSP-4 treated rats was not due to a decrease in ex- ploratory activity (groups [F = 1.265, p < 0.265]; sub- stances [F = 0.07, p < 0.974]; both factors [F = 0.821, p < 0.488]) (Fig. 4).
Tissue assay
In the mPFC, DSP-4 reduced the NA content by
~95%, without altering the levels of DA, 5-HT and their metabolites. Neither CP 94,253 nor SB 216641 altered NA, DA, DOPAC, HVA, 5-HT or 5-HIAA lev- els in the mPFC of control and DSP-4 groups (Tab. 1).
In control and DSP-4 groupsp-CPA (2 × 100 mg/kg, ip) reduced the mPFC levels of 5-HT and 5-HIAA each by approximately 90%. However, in the DSP-4 group alone,p-CPA treatment also reduced the levels of the DA metabolites DOPAC and HVA by 50% and 14%, respectively (Tab. 2).
5-HT synthesis rate
There was no difference in the 5-HT synthesis rate in mPFC of control versus DSP-4 rats (after saline injec- tion). While SB 216641 had no effect on the 5-HT synthesis rate, CP 94,253 (4.0 mg/kg,ip) reduced the 5-HT synthesis rate of control rats by ~33%, but had no in DSP-4 treated rats (Fig. 5).
In vivo microdialysis study
The baseline extraneuronal (i.e., microdialysate) level of 5-HT in mPFC was reduced by more than 40% in DSP-4 rats (i.e., 0.37 ± 0.23 pg/20 µl)versus control (0.56 ± 0.26 pg/20 µl) (differences not significant). In control rats CP 94,253 (4.0 mg/kg,ip) increased the microdialysate 5-HT level by ~40% at 20-min, and this was followed by a gradual decline in 5-HT levels by ~40% below baseline, over the next 120 min (Fig.
6). In DSP-4 rats CP 94,253 increased 5-HT levels in the microdialysate to an even greater extent (~100%) at 20-min, and this was similarly followed by a grad- ual decline in 5-HT levels slightly below baseline over the next 120 min. Because of the high variability, there was no difference in 5-HT between control and DSP-4 rats.
Discussion
The results of the current study demonstrate that neo- natal DSP-treatment did not affect numbers of entries or time spent in the closed arms of the elevated plus maze and open arms. This finding indicates that “ba- sal” anxiety (after saline) was not altered in adult rats in which mPFC content was reduced from birth by
~95%. However, as has been noted, the selective
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
0 0 20 40 60 80 100 120 140 160 min
% (derived from pg/20 µl)
CP 94253
Fig. 6. Effect of neonatal DSP-4 (50 mg/kg,sc) lesioning on the 5-HT microdialysate concentration in the mPFC after CP 94,253 (4.0 mg/kg, ip) administration to adult rats (n = 5) (the mean ± SEM). Explanations: o ––o control,¸ ––¸ DSP-4
5-HT1B receptor agonist CP 94,253 (4.0 mg/kg, ip) produced anxiogenic-like behavior. This effect was observed only in control animals, and the effect was blocked by SB 216641. Moreover, because the number of entries in closed arms in both tested groups was not modified by a 5-HT1Bagonist, the anxiogenic effect of CP 94,253 does not appear to be related to an effect on exploratory activity. Lin and Parsons [34]
also found that CP 94,253; (1.0–5.6 mg/kg) dose- dependently decreased the amount of exploration in open arms of the plus-maze without altering overall locomotor activity. Furthermore, they showed that this effect was reversed by pretreatment with the se- lective 5-HT1B/1Dantagonist GR 127935, which alone had no effect on plus-maze behavior. This finding and the present study contrast with the study done by Chojnacka-Wójcik et al. [11] who reported that CP 94,253 produced an anxiolytic-like effect in rats.
However, in the latter study a Vogel conflict drinking test was employed with a lower dose of CP 94,253.
Summing up, the behavioral data of the present study indirectly suggest that an enhancement in 5-HT neu- rotransmission is necessary to produce an anxiolytic- like effect because a decrease in 5-HT neurotransmis- sion evoked by 5-HT1Bagonist administration elicited anxiogenic-like behavior in control rats.
In the present study, CP 94,253 did not alter the time spent in closed arms of the maze in DSP-treated rats. To the best of our knowledge, there are no other reports of an effect of 5-HT1Bagonists in DSP-4 rats used for anxiety behavioral testing. Anxiolytic-like effects of chronic antidepressant treatment (desi- pramine, phenelzine) were modified in DSP-4 treated rats, while anxiolytic-like effects of barbiturates and benzodiazepines were not altered [20].
Despite the many observations of 5-HT systems in modulating anxiety behaviors, p-CPA did not modify the anxiogenic effects of CP 94,253 in control rats de- spite the marked reduction in 5-HT and 5-HIAA in mPFC (Tab. 2). However, p-CPA unmasked the anxiogenic-like “potential” of CP 94,253 in DSP-4 rats, increasing the time spent in closed arms. Appar- ently, a 5-HT1Bagonist response does not depend on 5-HT system integrity. We assume that 5-HT1Bhetero- receptors on dopaminergic, noradrenergic, or other nerves are more closely associated with the effect. In contrast, in DSP-4 lesioned rats the 5-HT system ap- pears to “suppress” the anxiogenic-like actions of CP 94,253 presumably via presynaptic 5-HT1B autore-
ceptors. Functional inactivation of 5-HT1Bautorecep- tors by p-CPA-induced 5-HT depletion may unmask anxiogenic CP 94,253 properties in these animals.
There is a body of evidence suggesting that 5-HT1B receptors control 5-HT levels mainly in the ventral hippocampus [18, 50]. However, some reports dem- onstrated that 5-HT1Bmediated neurotransmission is also crucial for 5-HT levels in the frontal cortex or hy- pothalamus – the structures deeply involved in anxi- ety, stress and depression [17, 26]. 5-HT-ergic path- ways innervating such brain regions as the frontal cor- tex, amygdala, hypothalamus and hippocampus have been found to possess some similarities; they are acti- vated by anxiogenic stimuli, including psychosocial stress, conditioned fear and conflict procedures [38].
For these reasons, the mPFC was chosen for micro- dialysis assay in our studies. CP 94,253 (4.0 mg/kg, ip) induced inhibition of 5-HT synthesis in the mPFC, and reduction of 5-HT levels in microdialysate was observed only in control rats. 5-HT1Bagonists also re- duce 5-HT release in other brain regions [1, 28]. It is likely that 5-HT1B autoreceptor desensitization oc- curred in DSP-4 rats and that this effect accounted for the diminished “biochemical response” to CP 94,253 challenge. However, one must be cognizant of the fact that zimelidine (selective 5-HT reuptake inhibitor, SSRI) addition into aCSF could lead to activation of 5-HT auto- and hetero-receptors that are normally protected by clearance of extracellular 5-HT. SSRIs, which acutely increase extracellular 5-HT levels, pro- duce a decrease in the DRN 5-HT firing rate in rats due to negative feedback on inhibitory 5-HT1Asoma- todendritic autoreceptors [3]. Despite these disadvan- tages of zimelidine usage, it is worth noting that con- trol and DSP-4 treated rats were perfused in exactly the same manner. Furthermore, SSRIs stabilize very low levels of 5-HT in mPFC microdialysates, thereby improving the assay conditions. As we show, the ex- traneuronal (i.e., microdialysate) baseline concentra- tion of 5-HT in mPFC was reduced by more than 40%
in DSP-4 rats (i.e., 0.37 ± 0.23 pg/20 µl) versus con- trol (0.56 ± 0.26 pg/20 µl). Such a reduction (although still not significant) may suggest a decrease in sponta- neous 5-HT release following the lesion, and an up- regulation of 5-HT1Areceptors rather than their de- sensitization. However, noradrenergic fibers exert tonic excitatory stimulation of the spontaneous firing rate of 5-HT neurons within the raphe nucleiin vivo.
Stimulation of the cell body a1-adrenoreceptors re- sults in increased serotoninergic cell firing [59],
whereas firing is suppressed by stimulation ofa2-adre- noreceptors located on noradrenergic terminals [56].
The depletion of noradrenergic nerve terminals in the raphe nuclei observed after DSP-4 treatment [10]
could simply result in the lack of facilitatory NE con- trol on serotoninergic neurons, especially in DRN re- ceiving direct noradrenergic projections from the LC area. This would consequently result in lowering of the 5-HT microdialysate concentration in the mPFC of the DSP-4 group. However, these considerations are in opposition with electrophysiological studies which revealed that the lesion of NE neurons does not affect the firing activity of 5-HT neurons [23, 56].
Conversely, findings presented by Cassano et al. [10]
shed some light on our microdialysis results. They demonstrated that DSP-4 treatment reduced the level of NE in the LC with a concomitant increase in 5-HT concentration. This is particularly interesting knowing the inhibitory effect of 5-HT on LC NE neurons. Such an increase in 5-HT in the LC may thus result in an at- tenuated release of NE in the DRN, and in turn, de- creased 5-HT neurotransmission at the nerve terminal (e.g., in mPFC). It must also be added that 5-HT1Band 5-HT1D receptors display remarkably similar phar- macology, and the ligands used in our study posses some affinity to 5-HT1Dreceptors. For these reasons we cannot rule out the involvement of these receptors in influencing the effects obtained in the present work.
In summary, this is the first report showing that a neonatal lesion of noradrenergic nerves in the brain is associated with a reduction in reactivity of a 5-HT1B receptor agonist in adulthood. Previously, we reported that 5-HT1A autoreceptor desensitization occurs in rats similarly lesioned as neonates with DSP-4 [13].
In a counterpart study, Anthony et al. [2] found that 5-HT1BmRNA is selectively downregulated in DRN by either fluoxetine or paroxetine. Microdialysis stud- ies by Lifschytz et al. [33] and Newman et al. [41]
shown that fluoxetine (5.0 mg/kg,ip daily for 7 or 15 days) desensitized 5-HT1Aand 5-HT1Bautoreceptors in the frontal cortex and hypothalamus. Because 5-HT1A and 5-HT1B autoreceptor desensitization is thought to underlie the therapeutic effects of SSRIs (for review see Watson and Dawson [60]), we set forth the hypothesis that the neonatal noradrenergic lesion in rats leads to similar neurobiological conse- quences as far as 5-HT1Aand 5-HT1Breceptor status is concerned. Perhaps, it is a “self-protective” mecha- nism which has been developed during brain matura-
tion (after DSP-4 neonatal insult) for preserving func- tional brain integrity (e.g., preventing manifestation of depressive-like behavior) in DSP-4 treated rats.
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Received:
May 8, 2009; in revised form: November 19, 2009.
