5-HT1A receptor antagonism decreases motor activity and influences dopamine and serotonin metabolization pathways, primarily in cingulate cortex and striatum

Introduction

The serotonin (5-HT)1A receptor (5-HT1AR) is the main inhibitory 5-HTR subtype in the mammalian brain (for review see[1](https://www.nature.com/articles/s41598-025-26268-7#ref-CR1 “Carhart-Harris, R. L. & Nutt, D. J. Serotonin and brain function: A tale of two receptors. J. Psychopharmacol. 31, 1091–1120. https://doi.org/10.1177/0269881117725915

(2017).“)) and is implicated in disorders like major depression and anxiety. In both diseases, conflicting 5-HT1AR binding patterns have been reported, with reductions[2](#ref-CR2 “Sullivan, G. M. et al. Brain serotonin1A receptor binding in major depression is related to psychic and somatic anxiety. Biol. Psychiatry 58, 947–954. https://doi.org/10.1016/j.biopsych.2005.05.006

(2005).“),[3](#ref-CR3 “Lanzenberger, R. R. et …

Introduction

(2005).“),[3](#ref-CR3 “Lanzenberger, R. R. et al. Reduced serotonin-1A receptor binding in social anxiety disorder. Biol. Psychiatry. 61, 1081–1089. https://doi.org/10.1016/j.biopsych.2006.05.022

(2007).“),[4](https://www.nature.com/articles/s41598-025-26268-7#ref-CR4 “Drevets, W. C. et al. Serotonin-1A receptor imaging in recurrent depression: Replication and literature review. Nucl. Med. Biol. 34, 865–877. https://doi.org/10.1016/j.nucmedbio.2007.06.008

(2017).“) or increases5,[6](https://www.nature.com/articles/s41598-025-26268-7#ref-CR6 “Kaufman, J. A. et al. Quantification of the serotonin 1A receptor using PET: Identification of a potential biomarker of major depression in males. Neuropsychopharmacology 40, 1692–1699. https://doi.org/10.1038/npp.2015.15

(2015).“), suggesting complex and presumably region-dependent roles.

N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarbox-amide maleate (WAY100,635;[7](https://www.nature.com/articles/s41598-025-26268-7#ref-CR7 “Houle, S., DaSilva, J. N. & Wilson, A. A. Imaging the 5-HT(1A) receptors with PET: WAY-100635 and analogues. Nucl. Med. Biol. 27, 463–466. https://doi.org/10.1016/s0969-8051(00)00112-8

(2000).“)) is a widely used 5-HT1AR antagonist in rodent studies. However, systemic WAY100,635 either reduced ambulation (0.4 mg/kg intraperitoneally [i.p.],[8](https://www.nature.com/articles/s41598-025-26268-7#ref-CR8 “Nikolaus, S. et al. WAY100,635 decreases motor/exploratory behaviors and nigrostriatal and mesolimbocortical dopamine D2/3 receptor binding in the adult rat. Pharmacol. Biochem. Behav. 215, 173363. https://doi.org/10.1016/j.pbb.2022.173363

(2022).“)) or had no effect (0.3 mg/kg subcutaneously [s.c.],[9](https://www.nature.com/articles/s41598-025-26268-7#ref-CR9 “Bickerdike, J., Fletcher, A. & Marsden, C. A. Attenuation of CCK-induced aversion in rats on the elevated x-maze by the selective 5-HTIA receptor antagonists (+)WAY100135 and WAY100635. Neuropharmacology 34, 805–811. https://doi.org/10.1016/0028-3908(95)00037-7

(1995).“); 0.4 mg/kg i.p.,[10](#ref-CR10 “Carey, R., Damianopoulos, E. & de Palma, G. The 5-HT antagonist WAY 100635 can block the low-dose 1A locomotor stimulant effects of cocaine. Brain Res. 862, 242–246. https://doi.org/10.1016/s0006-8993(00)02084-9

(2000).“),[11](#ref-CR11 “Müller, C. P., Carey, R. J., De Souza Silva, M. A., Jocham, G. & Huston, J. P. Cocaine increases serotonergic activity in the hippocampus and nucleus accumbens in vivo: 5-HT1a-receptor antagonism blocks behavioral but potentiates serotonergic activation. Synapse 45, 67–77. https://doi.org/10.1002/syn.10083

(2002).“),[12](https://www.nature.com/articles/s41598-025-26268-7#ref-CR12 “Müller, C. P. et al. The selective serotonin(1A)-receptor antagonist WAY 100635 blocks behavioral stimulating effects of cocaine but not ventral striatal dopamine increase. Behav. Brain Res. 134, 337–346. https://doi.org/10.1016/s0166-4328(02)00042-6

(2002).“)). Rearing was either increased (0.4–2.0 mg/kg i.p.,[8](https://www.nature.com/articles/s41598-025-26268-7#ref-CR8 “Nikolaus, S. et al. WAY100,635 decreases motor/exploratory behaviors and nigrostriatal and mesolimbocortical dopamine D2/3 receptor binding in the adult rat. Pharmacol. Biochem. Behav. 215, 173363. https://doi.org/10.1016/j.pbb.2022.173363

(2022).“),[13](https://www.nature.com/articles/s41598-025-26268-7#ref-CR13 “Jackson, D. M., Wallsten, C. E., Jerning, E., Hu, P. S. & Deveney, A. M. Two selective 5-HT1A receptor antagonists, WAY-100 635 and NDL-249, stimulate locomotion in rats acclimatised to their environment and alter their behaviour: A behavioural analysis. Psychopharmacology 139, 300–310. https://doi.org/10.1007/s002130050721

(1998).“)) or remained unaltered (0.3 mg/kg s.c.,[9](https://www.nature.com/articles/s41598-025-26268-7#ref-CR9 “Bickerdike, J., Fletcher, A. & Marsden, C. A. Attenuation of CCK-induced aversion in rats on the elevated x-maze by the selective 5-HTIA receptor antagonists (+)WAY100135 and WAY100635. Neuropharmacology 34, 805–811. https://doi.org/10.1016/0028-3908(95)00037-7

(2002).“)). Also, object recognition studies were inconsistent, with either no effect (0.3 and 1 mg/kg, i.p.,[14](https://www.nature.com/articles/s41598-025-26268-7#ref-CR14 “Pitsikas, N., Tsitsirigou, S., Zisopoulou, S. & Sakellaridis, N. The 5-HT1A receptor and recognition meomory. Possible modulation of its behavioral effects by the nitrergic system. Behav. Brain Res. 159, 287–293. https://doi.org/10.1016/j.bbr.2004.11.007

(2005).“)) or increased exploration of a novel object (0.01—0.05 mg/kg s.c.,[15](https://www.nature.com/articles/s41598-025-26268-7#ref-CR15 “Pitsikas, N., Rigamonti, A. E., Cella, S. G. & Muller, E. E. The 5-HT 1A receptor antagonist WAY 100635 improves rats performance in different models of amnesia evaluated by the object recognition task. Brain Res. 983, 215–222. https://doi.org/10.1016/s0006-8993(03)03091-9

(2003).“); 1 mg/kg i.p.,[16](https://www.nature.com/articles/s41598-025-26268-7#ref-CR16 “Carey, R. J., Shanahan, A., Daminaopoulos, E. N., Müller, C. P. & Huston, J. P. Behavior selectively elicited by novel stimuli: Modulation by the 5-HT1A agonist-8-OHDPAT and antagonist WAY100,635. Behav. Pharmacol. 19, 361–364. https://doi.org/10.1097/FBP.0b013e3283096848

(2008).“)).

5-HT is metabolized by monoamine oxidase (MAO) to 5-hydroxyindole acetic acid (5-HIAA;17). DA metabolism involves MAO and catechol-0-methyltransferase (COMT), degrading DA to dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT), respectively[18](https://www.nature.com/articles/s41598-025-26268-7#ref-CR18 “Westerink, B. H. C. Sequence and significance of dopamine metabolism in the rat brain. Neurochem. Int. 7, 221–227. https://doi.org/10.1016/0197-0186(85)90108-1

(1985).“), which both are further converted to homovanillic acid (HVA;19).

Microdialysis studies in rats show that systemic WAY100,635 did not alter 5-HT concentrations in nucleus accumbens (NAC; 0.4 mg/kg intraperitoneally [i.p.],[11](https://www.nature.com/articles/s41598-025-26268-7#ref-CR11 “Müller, C. P., Carey, R. J., De Souza Silva, M. A., Jocham, G. & Huston, J. P. Cocaine increases serotonergic activity in the hippocampus and nucleus accumbens in vivo: 5-HT1a-receptor antagonism blocks behavioral but potentiates serotonergic activation. Synapse 45, 67–77. https://doi.org/10.1002/syn.10083

(2002).“)), hippocampus (HIPP; 0.4 mg/kg i.p.,[11](https://www.nature.com/articles/s41598-025-26268-7#ref-CR11 “Müller, C. P., Carey, R. J., De Souza Silva, M. A., Jocham, G. & Huston, J. P. Cocaine increases serotonergic activity in the hippocampus and nucleus accumbens in vivo: 5-HT1a-receptor antagonism blocks behavioral but potentiates serotonergic activation. Synapse 45, 67–77. https://doi.org/10.1002/syn.10083

(2002).“)) or frontal cortex (FC; 0.16 mg/kg subcutaneously [s.c.],[20](https://www.nature.com/articles/s41598-025-26268-7#ref-CR20 “Millan, M. J. et al. WAY 100,635 enhances both the ‘antidepressant’ actions of duloxetine and its influence on dialysate levels of serotonin in frontal cortex. Eur. J. Pharmacol. 341, 165–167. https://doi.org/10.1016/s0014-2999(97)01445-3

(1998).“)), but elevated 5-HIAA levels in the HIPP (0.4 mg/kg i.p.,[11](https://www.nature.com/articles/s41598-025-26268-7#ref-CR11 “Müller, C. P., Carey, R. J., De Souza Silva, M. A., Jocham, G. & Huston, J. P. Cocaine increases serotonergic activity in the hippocampus and nucleus accumbens in vivo: 5-HT1a-receptor antagonism blocks behavioral but potentiates serotonergic activation. Synapse 45, 67–77. https://doi.org/10.1002/syn.10083

(2002).“)). DA findings are inconsistent: in caudateputamen (CP), DA release increased (0.1–0.5 mg/kg s.c.,[21](https://www.nature.com/articles/s41598-025-26268-7#ref-CR21 “Ichikawa, J. & Meltzer, H. Y. The effect of serotonin(1A) receptor agonism on antipsychotic drug-induced dopamine release in rat striatum and nucleus accumbens. Brain Res. 858, 252–263. https://doi.org/10.1016/s0006-8993(99)02346-x

(2000).“)) or remained unchanged (0.1 mg/kg s.c.,[22](https://www.nature.com/articles/s41598-025-26268-7#ref-CR22 “Lucas, G., De Deurwaerdère, P., Porras, G. & Spampinato, U. Endogenous serotonin enhances the release of dopamine in the striatum only when nigro-striatal dopaminergic transmission is activated. Neuropharmacology 39, 1984–1995. https://doi.org/10.1016/s0028-3908(00)00020-4

(2000).“)), while DA levels were neither affected in NAC (0.1, 0.2 and 0.5 mg/kg s.c., [21](https://www.nature.com/articles/s41598-025-26268-7#ref-CR21 “Ichikawa, J. & Meltzer, H. Y. The effect of serotonin(1A) receptor agonism on antipsychotic drug-induced dopamine release in rat striatum and nucleus accumbens. Brain Res. 858, 252–263. https://doi.org/10.1016/s0006-8993(99)02346-x

(2000).“); 0.4 mg/kg i.p.,[12](https://www.nature.com/articles/s41598-025-26268-7#ref-CR12 “Müller, C. P. et al. The selective serotonin(1A)-receptor antagonist WAY 100635 blocks behavioral stimulating effects of cocaine but not ventral striatal dopamine increase. Behav. Brain Res. 134, 337–346. https://doi.org/10.1016/s0166-4328(02)00042-6

(2002).“); 0.16 mg/kg s.c.,[23](https://www.nature.com/articles/s41598-025-26268-7#ref-CR23 “DevroyeHaddjeri, C. N. et al. Opposite control of mesocortical and mesoaccumbal dopamine pathways by serotonin2B receptor blockade: Involvement of medial prefrontal cortex serotonin1A receptors. Neuropharmacology 119, 91–99. https://doi.org/10.1016/j.neuropharm.2017.04.001

(2017).“) nor in FC (0.16 mg/kg s.c., [20](https://www.nature.com/articles/s41598-025-26268-7#ref-CR20 “Millan, M. J. et al. WAY 100,635 enhances both the ‘antidepressant’ actions of duloxetine and its influence on dialysate levels of serotonin in frontal cortex. Eur. J. Pharmacol. 341, 165–167. https://doi.org/10.1016/s0014-2999(97)01445-3

(1998).“)). Also, DOPAC levels remained unaltered (0.4 mg/kg i.p.,[12](https://www.nature.com/articles/s41598-025-26268-7#ref-CR12 “Müller, C. P. et al. The selective serotonin(1A)-receptor antagonist WAY 100635 blocks behavioral stimulating effects of cocaine but not ventral striatal dopamine increase. Behav. Brain Res. 134, 337–346. https://doi.org/10.1016/s0166-4328(02)00042-6

(2002).“)). However, in an imaging study, WAY100,635 (0.4 mg/kg i.p) reduced D2/3R binding in CP, thalamus (THAL), FC, parietal cortex and ventral HIPP (vHIPP), indicating elevated DA[8](https://www.nature.com/articles/s41598-025-26268-7#ref-CR8 “Nikolaus, S. et al. WAY100,635 decreases motor/exploratory behaviors and nigrostriatal and mesolimbocortical dopamine D2/3 receptor binding in the adult rat. Pharmacol. Biochem. Behav. 215, 173363. https://doi.org/10.1016/j.pbb.2022.173363

(2022).“).

We previously demonstrated that network analysis can detect changes in regional neurotransmitter processing following pharmacological interventions[24](https://www.nature.com/articles/s41598-025-26268-7#ref-CR24 “Nikolaus, S. et al. 2,5-Dimethoxy-4-iodoamphetamine and altanserin induce region-specific shifts in dopamine and serotonin metabolization pathways in the rat brain. Pharmacol. Biochem. Behav. 242, 173823. https://doi.org/10.1016/j.pbb.2024.173823

(2024).“). The 5-HT2AR antagonist altanserin (ALT) suppressed DA metabolism in the THAL, while the agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) extended this suppression to mesolimbic and nigrostriatal regions associated with the THAL. Both compounds suppressed 5-HT metabolism in THAL; additionally, DOI exerted suppression in NAC and CING, whereas ALT promoted metabolization in dorsal HIPP (dHIPP). On the basis of these findings, we hypothesized that also the 5-HT1AR antagonist WAY100,635 would influence DA and 5-HT release and metabolism in regions of the nigrostriatal and mesolimbic system, the direction and extent of these alterations being relevant for understanding the role of 5-HT1AR in neuropsychiatric disorders.

Given the inconsistent neurochemical effects of WAY100,635, we extended the investigations of its impact on DA, 5-HT, their metabolites (DOPAC, 3-MT, HVA, 5-HIAA) to further regions of the rat monoaminergic system (CING, CP, NAC, THAL, dHIPP, vHIPP, brainstem [BS], cerebellum [CER]). Building on our previous work[24](https://www.nature.com/articles/s41598-025-26268-7#ref-CR24 “Nikolaus, S. et al. 2,5-Dimethoxy-4-iodoamphetamine and altanserin induce region-specific shifts in dopamine and serotonin metabolization pathways in the rat brain. Pharmacol. Biochem. Behav. 242, 173823. https://doi.org/10.1016/j.pbb.2024.173823

(2024).“), we analyzed enzyme-specific metabolic changes and behavioral effects by means of a modified novelty preference test assessing recognition memory for object and place[25](https://www.nature.com/articles/s41598-025-26268-7#ref-CR25 “Chao, O. Y., Huston, J. P., Nikolaus, S. & De Souza Silva, M. A. Concurrent assessment of memory for object and place: Evidence for different preferential importance of perirhinal cortex and hippocampus and for promnestic effect of a neurokinin-3 R agonist. Neurobiol. Learning Mem. 130, 149–158. https://doi.org/10.1016/j.nlm.2016.02.007

(2016).“).

Materials and methods

Animals

Studies were conducted on a total of 25 male Wistar rats (ZETT, Heinrich-Heine University, Düsseldorf, Germany), weighing 415 ± 28 g (mean ± standard deviation [S.D.]; age: 3 – 4 months). All rats underwent both behavioral and neurochemical measurements (WAY100,635: n = 12, SAL: n = 13). From the analysis of neurochemical findings, however, those data were excluded, which exceeded the respective group means by more than twice the S.D. Animals were randomly assigned to the treatment groups.

Rats were maintained in standard makrolon cages (59 × 38 × 20 cm, 3 animals per cage) in a climate cabinet (Scantainer, Scanbur BK, Karslunde, Denmark; temperature: 20° C, air humidity: 70%) with an artificial light–dark cycle (lights on at 6:00 a.m., lights off at 6:00 p.m.) and food and water freely available. The study was approved by the regional authority (reference number of ethical approval: AZ 81–02.04.2017.A450; Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen, Recklinghausen, Germany) and carried out in accordance with the European Communities Council Directive (86/609/EEC), the German Law on the Protection of Animals and ARRIVE guidelines.

Drug treatment

Rats received either WAY100,635 (Sigma-Aldrich, Taufkirchen, Germany; dissociation constant [Kd]: 0.28 nM,[26](https://www.nature.com/articles/s41598-025-26268-7#ref-CR26 “Chemel, B. R., Roth, B. L., Armbruster, B., Watts, V. J. & Nichols, D. E. WAY100,635 is a potent dopamine D4 receptor agonist. Psychopharmacology 188, 244–251. https://doi.org/10.1007/s00213-006-0490-4

(2006).“), molecular weight: 538.64 g/mol; dose: 0.4 mg/kg i.p., injection volume: 1 ml/kg, concentration: 0.4 mg/ml) or isotonic saline (SAL; B. Braun Melsungen AG, Melsungen, Germany; molecular weight: 58.5 g/mol; dose: 1 ml/kg i.p.). The dose of 0.4 mg/kg was chosen based on our previous study[8](https://www.nature.com/articles/s41598-025-26268-7#ref-CR8 “Nikolaus, S. et al. WAY100,635 decreases motor/exploratory behaviors and nigrostriatal and mesolimbocortical dopamine D2/3 receptor binding in the adult rat. Pharmacol. Biochem. Behav. 215, 173363. https://doi.org/10.1016/j.pbb.2022.173363

(2022).“), which had shown effects of WAY100,635 on DA levels in a variety of regions including CP, THAL, FC, parietal cortex and vHIPP. Moreover, the dose of 0.4 mg/kg was previously shown to block cocaine-induced increases of locomotion[10](#ref-CR10 “Carey, R., Damianopoulos, E. & de Palma, G. The 5-HT antagonist WAY 100635 can block the low-dose 1A locomotor stimulant effects of cocaine. Brain Res. 862, 242–246. https://doi.org/10.1016/s0006-8993(00)02084-9

(2002).“).

Rat behavior

Recognition memory for object and place (for review see[27](https://www.nature.com/articles/s41598-025-26268-7#ref-CR27 “Chao, O. Y., Nikolaus, S., Yang, Y. M. & Huston, J. P. Neuronal circuitry for recognition memory of object and place in rats and mice. Neurosci. Biobehav. Rev. 141, 104855. https://doi.org/10.1016/j.neurobiorev.2022.104855

(2022).“)) was assessed together with motor/exploratory behaviors as previously described (see, e.g.,[28](https://www.nature.com/articles/s41598-025-26268-7#ref-CR28 “Nikolaus, S. et al. The 5-HT1A receptor agonist 8-OH-DPAT modulates motor/exploratory activity, recognition memory and dopamine transporter binding in the dorsal and ventral striatum. Neurobiol. Learn. Mem. 205, 107848. https://doi.org/10.1016/j.nlm.2023.107848

(2023).“)). On two consecutive days, the rats were placed for 10 min into an open field (PhenoTyper®, Noldus Information Technology, Wageningen, Netherlands; dimensions: 45 × 45 × 56 cm, illumination: 19 lx) for the purpose of habituation. One day later, they were first subjected to a 5-min sample trial and a 5-min test trial with two identical polyhedrons (material: lead, length: 9.5 cm, width: 7 cm, height: 4 cm; color: green) located opposite to each other at the middle of the left and right side of the apparatus at a distance of 5 cm to the respective wall. After completion of the sample trial, the rats were injected WAY100,635 or SAL. Thirty min post-challenge, a 5-min test trial was performed with one of the polyhedrons moved to the middle of the back side of the apparatus and the other polyhedron replaced by a cylinder (material: lead, diameter: 5.6 cm, height: 8 cm; color: grey). Placement of the cylinder at the left and the right side of the apparatus was counterbalanced. Duration (sec) and frequency (n) of object exploration in sample- and test-trial were determined by registering physical contact with snout or fore-paws. Increased exploration of the novel object (cylinder) in the test trial indicates preferential memory for what, while increased exploration of the displaced object (polyhedron) indicates preferential memory for where[25](https://www.nature.com/articles/s41598-025-26268-7#ref-CR25 “Chao, O. Y., Huston, J. P., Nikolaus, S. & De Souza Silva, M. A. Concurrent assessment of memory for object and place: Evidence for different preferential importance of perirhinal cortex and hippocampus and for promnestic effect of a neurokinin-3 R agonist. Neurobiol. Learning Mem. 130, 149–158. https://doi.org/10.1016/j.nlm.2016.02.007

(2016).“). Evaluation was performed with EthoVision® XT (Noldus Information Technology, Wageningen, Netherlands) with the experimenter (S.N.) blinded to the precedent treatment.

In the test trial, also durations (sec) and frequencies (n) of the following behavioral parameters were registered: (1) ambulation (as measure of motor activity), (b) sitting, (c) grooming, (d) rearing freely and against the walls of the open field (as a general measure of active exploration not related to the objects), (e) explorative movements of head, neck and shoulders in the open field (also not related to the objects). Furthermore, based on the movement of the animal’s center point, EthoVision XT automatically determined the distance in cm covered by the rat.

Behavioral tests were performed on all rats after either WAY100,635 or SAL. The behavioral results obtained after WAY100,635 have been previously published in relation to regional DA transporter binding[29](https://www.nature.com/articles/s41598-025-26268-7#ref-CR29 “Nikolaus, S. et al. 5-HT1A and 5-HT2A receptor effects on recognition memory, motor/ exploratory behaviors, emotionality and regional dopamine transporter binding in the rat. Behav. Brain Res. 469, 115051. https://doi.org/10.1016/j.bbr.2024.115051

(2024).“). Behavioral studies were conducted in the light phase between 9:00 a.m. and 5:00 p.m.

Neurochemistry

After completion of the test trial (35 min post-challenge), animals were sacrificed with an overdose of pentobarbital natrium (Narcoren®, Boehringer Ingelheim Pharma GmbH & Co.KG, Ingelheim am Rhein, Germany, concentration: 0.16 g/ml, dose: 2.5 ml/kg i.p.). Subsequently, rats were decapitated and the brains taken out. From both hemispheres, CING, CP, NAC, THAL, dHIPP, vHIPP, BS and CER were dissected. Tissue samples were homogenized with an ultrasonic cell disruptor (Microsom™, Misonix, Farmingdale, USA) in 500 ml of 0.05 M perchloric acid (Sigma-Aldrich, Taufkirchen, Germany) and centrifuged at 12,000 revolutions/min (rpm) for 20 min at 4 °C (Eppendorf Centrifuge 5417 R, Hamburg, Germany). Subsequently, the samples were filtered by centrifugation at 2000 rpm for 2 min at 4 °C and stored at -80 °C until analysis.

The levels of DA, 5-HT, the DA metabolites DOPAC, 3-MT and HVA and the 5-HT metabolite 5-HIAA were determined with high performance liquid chromatography (HPLC). Monoamines were analyzed, using a Nucleosil 120-5C18 column (Macherey & Nagel, Düren, Germany). The mobile phase consisted of the following compounds dissolved in 1 l of bidistilled water: 14.14 g chloroacetic acid (0.15 M; Sigma-Aldrich, Taufkirchen, Germany), 4.66 g sodium hydroxide (0.12 M; Mallinckrodt Baker Inc, Phillipsburg, USA), 0.30 g potassium chloride (4 mM; Merck, Darmstadt, Germany), 0.20 g sodium octylsulfate (0.86 mM; Sigma-Aldrich, Taufkirchen, Germany), 0.25 g ethylenediaminetetraacetic acid (0.67 mM; VWR International GmbH, Darmstadt, Germany), 35 ml acetonitrile (0.9 mM; VWR International GmbH, Darmstadt, Germany) and 18 ml tetrahydrofuran (2 mM; VWR International GmbH, Darmstadt, Germany). The pH was adjusted to 3.0 by adding either chloracetic acid or sodium hydroxide.

The electrochemical detector (DECADE Elite, Antec Scientific, Leiden, The Netherlands) was set at a potential of 530 mV versus an Ag/AgCl (ISAAC) reference electrode (temperature: 35 °C, range: 10 nA). Neurotransmitter and metabolite levels were analyzed with Chrom Perfect Software (Chrom Perfect Version 5.5.6, Justice Laboratory Software, Denville, USA).

Statistical analysis

Motor/exploratory behaviors and recognition memory for object and place. Each parameter in the test trial of each treatment group was tested for normality of distribution with the Kolmogorov–Smirnov test. With the exception of grooming duration and frequency, and both duration and frequency of polyhedron exploration (p < 0.005), all parameters were normally distributed (0.087 ≤ p ≤ 0.830). If normally distributed, parameters were compared between groups with independent t tests (α = 0.05), whereas not-normally distributed parameters were compared with Mann–Whitney rank sum tests (α = 0.05).

Neurochemistry. Means and S.D.’s of transmitter and metabolite concentrations (pg/mg tissue, wet weight) in each brain region were computed for each treatment group. Moreover, for each treatment group the means and S.D.’s of the following turnover ratios in each brain region were computed: DOPAC/DA, 3-MT/DA, HVA/DOPAC, HVA/3-MT, 5-HIAA/5-HT.

For each compound and turnover ratio, two-way ANOVAS were calculated for the factors “treatment” and “brain region” as well as for their interaction (“treatment x brain region”). Pairwise multiple comparisons (Holm-Sidak method, overall α = 0.05) were performed separately for the factors “treatment” and “brain region” as well as for “treatment” with respect to the individual regions and for “brain region” with respect to the individual treatments.

Statistic calculations were performed with SigmaStat (version 3.5, Systat Software Inc., Erkrath, Germany).

Correlation analysis. The association between neurotransmitter concentrations and behavioral parameters (overall activity, duration and frequency of polyeder and cylinder exploration, ambulation, sitting, rearing, head-shoulder motility, grooming) was assessed by computing Pearson product moment correlation coefficients (r). Calculations were performed with SigmaStat (version 3.5, Systat Software Inc., Erkrath, Germany).

Network analysis. With this mode of analysis, the network structure of variables - pre-defined so-called “nodes” - can be analysed by estimating path coefficients, which describe the direction (positive or negative) and the “strength” of the individual connections. Thereby, firstly, covariance matrices were estimated with gaussian graphical models employing graphical L1 (lasso) regularized regression in order to decrease matrix sparsity[30](https://www.nature.com/articles/s41598-025-26268-7#ref-CR30 “Friedman, J., Hastie, T. & Tibshirani, R. Sparse inverse covariance estimation with the graphical lasso. Biostatistics 9, 432–441. https://doi.org/10.1093/biostatistics/kxm045

(2008).“), thus increasing the number of pairwise interactions. Covariance is defined as the mean of multiplication of corresponding S.D.’s of a pair of variables, and, consequently, indicates the direction of the linear relationship between these variables. By dividing the covariances of each pair of variables by the product of their S.D.’s, standardized path correlation coefficients (between -1 and + 1) are obtained, which, thus, indicate the strength in addition to the direction of their association. Here, we separately assessed the associations between the concentrations of neurotransmitter and metabolites in the individual brain regions after treatment with either WAY100,635 or SAL. Network analyses were computed with JASP (version 0.18.1, JASP Team*,* © 2023 University of Amsterdam).

Results

Motor/exploratory behaviors and object recognition

Overall activity (t, 3.252, p = 0.029) and frequency of head-shoulder motility (t, 3.204, p = 0.004) were reduced after WAY100,635 relative to SAL (Fig. 1A and I). All other parameters were not different between groups (0.0331 ≤ t ≤ 1.747, 0.094 ≤ p ≤ 0.974; see Figs. 1B-H and Figs. J-O).

Fig. 1

Overall activity (A), ambulation duration (B), ambulation frequency (C), sittíng duration (D), sitting frequency (E), rearing duration (F), rearing frequency (G), duration of head-shoulder motility (H), frequency of head-shoulder motility (I), grooming duration (J), grooming frequency (K), duration of polyhedron exploration (L), frequency of polyhedron exploration (M), duration of cylinder exploration (N) and frequency of cylinder exploration (O) after challenge with saline (SAL; 0.9% solution; 1 ml/kg i.p.) and WAY100,635 (0.4 mg/kg i.p.). Given are mean values and standard deviations (S.D.). The significant p values given in the figure were obtained with independent t tests (α ≤ 0.05).

Neurochemistry

Neurochemical results are given in Table 1 and 2 as well as in Figs. 2 and 3. Results of network analyses are given in Figs. 4,5,6,7,8,9 and 10.

Table 1 Concentrations (pg/mg brain tissue, wet weight; means ± standard deviations [S.D.s]) of dopamine (DA), serotonin (5-HT) and metabolites (3,4-dihydroxyphenylacetic acid [DOPAC], 3-methoxytyramine [3-MT], homovanillic acid [HVA], 5-hydroxyindole-acetic acid [5-HIAA]) in the individual brain regions (cingulate [CING], caudateputamen [CP], nucleus accumbens [NAC], thalamus [THAL], dorsal hippocampus [dHIPP], ventral hippocampus [vHIPP], brainstem [BS], cerebellum [CER]) after treatment with saline (SAL), and WAY100,635 as obtained with high performance liquid chromatography.

Table 2 Turnover ratios (means ± standard deviations [S.D.s]) of dopamine (DA) and serotonin (5-HT). Given are the ratios DOPAC/DA, 3-MT/DA, HVA/DOPAC, HVA/3-MT and 5-HIAA/5-HT in the individual brain regions (cingulate [CING], caudateputamen [CP], nucleus accumbens [NAC], thalamus [THAL], dorsal hippocampus [dHIPP], ventral hippocampus [vHIPP], brainstem [BS], cerebellum [CER]) after treatment with saline (SAL) and WAY100,635.

Fig. 2

Concentrations of (A) dopamine (DA), (B) homovanillic acid (HVA). (C) serotonin (5-HT) and (D) 5-hydroxyindole-acetic acid (5-HIAA) in cingulate (CING), caudateputamen (CP), nucleus accumbens (NAC), thalamus (THAL), dorsal hippocampus (dHIPP), ventral hippocampus (vHIPP) and cerebellum after challenge with saline (SAL; 0.9% solution; 1 ml/kg and WAY100,635 (0.4 mg/kg i.p.). Given are mean values and standard deviations (S.D.). The circles represent the individual rats. The significant p values given in the figure were obtained with two-way ANOVAS and post hoc Holm-Sidak tests (α ≤ 0.05).

Fig. 3

(A) DOPAC/DA, (B) 3-MT/DA and (C) HVA/3-MT turnover ratios in cingulate (CING), caudateputamen (CP), nucleus accumbens (NAC), thalamus (THAL), dorsal hippocampus (dHIPP), ventral hippocampus (vHIPP) and cerebellum after challenge with saline (SAL; 0.9% solution; 1 ml/kg and WAY100,635 (0.4 mg/kg i.p.). Given are mean values and standard deviations (S.D.). The circles represent the individual rats. The significant p values given in the figure were obtained with two-way ANOVAS and post hoc Holm-Sidak tests (α ≤ 0.05).

Fig. 4

Top: Path coefficient matrix obtained obtained for the cingulate with EBICglasso modelling of neurotransmitter (DA, 5-HT) and metabolite (DOPAC, HVA, 3-MT, 5-HIAA) concentrations after treatment with SAL (13 out of 15 possible connections, sparsity: 0.133) and WAY100,635 (2 out of 15 possible connections, sparsity: 0.867). Bottom: Connections between DA, 5-HT and metabolites (DOPAC, 3-MT, HVA, 5-HIAA) in cingulate after SAL, and WAY100,635. Positive and negative associations are represented by blue and red lines, respectively. The thickness of the lines indicates the strength of the individual connections.

Fig. 5

Top: Path coefficient matrix obtained obtained for the caudateputamen with EBICglasso modelling of neurotransmitter (DA, 5-HT) and metabolite (DOPAC, HVA, 3-MT, 5-HIAA) concentrations after treatment with SAL (11 out of 15 possible connections, sparsity: 0.267) and WAY100,635 (12 out of 15 possible connections, sparsity: 0.200). Bottom: Connections between DA, 5-HT and metabolites (DOPAC, 3-MT, HVA, 5-HIAA) in cingulate after SAL and WAY100,635. Positive and negative associations are represented by blue and red lines, respectively. The thickness of the lines indicates the strength of the individual connections.

Fig. 6

Top: Path coefficient matrix obtained obtained for the nucleus accumbens with EBICglasso modelling of neurotransmitter (DA, 5-HT) and metabolite (DOPAC, HVA, 3-MT, 5-HIAA) concentrations after treatment with SAL (14 out of 15 possible connections, sparsity: 0.067) and WAY100,635 (3 out of 15 possible connections, sparsity: 0.800). Bottom: Connections between DA, 5-HT and metabolites (DOPAC, 3-MT, HVA, 5-HIAA) in nucleus accumbens after SAL and WAY100,635. Positive and negative associations are represented by blue and red lines, respectively. The thickness of the lines indicates the strength of the individual connections.

Fig. 7

Top: Path coefficient matrix obtained obtained for the thalamus with EBICglasso modelling of neurotransmitter (DA, 5-HT) and metabolite (DOPAC, HVA, 3-MT, 5-HIAA) concentrations after treatment with SAL (13 out of 15 possible connections, sparsity: 0.133) and WAY100,635 (15 out of 15 possible connections, sparsity: 0.000). Bottom: Connections between DA, 5-HT and metabolites (DOPAC, 3-MT, HVA, 5-HIAA) in thalamus after SALand WAY100,635. Positive and negative associations are represented by blue and red lines, respectively. The thickness of the lines indicates the strength of the individual connections.

Fig. 8

Top: Path coefficient matrix obtained for dorsal and ventral hippocampus with EBICglasso modelling of neurotransmitter (DA, 5-HT) and metabolite (DOPAC, HVA, 3-MT, 5-HIAA) concentrations after treatment with SAL (dorsal hippocampus: 10 out of 15 possible connections, sparsity: 0.333; ventral hippocampus: 11 out of 15 connections, sparsity: 0.267) and WAY100,635 (dorsal hippocampus: 12 out of 15 possible connections, sparsity: 0.200; ventral hippocampus: 0 out of 15 connections, sparsity: 1.00). Bottom: Connections between DA, 5-HT and metabolites (DOPAC, 3-MT, HVA, 5-HIAA) in dorsal and ventral hippocampus after SAL and WAY100,635. Positive and negative associations are represented by blue and red lines, respectively. The thickness of the lines indicates the strength of the individual connections.

Fig. 9

Top: Path coefficient matrix obtained for the brainstem with EBICglasso modelling of neurotransmitter (DA, 5-HT) and metabolite (DOPAC, HVA, 3-MT, 5-HIAA) concentrations after treatment with SAL (10 out of 15 possible connections, sparsity: 0.333) and WAY100,635 (10 out of 15 possible connections, sparsity: 0.333). Bottom: Connections between DA, 5-HT and metabolites (DOPAC, 3-MT, HVA, 5-HIAA) in brainstem after SAL and WAY100,635. Positive and negative associations are represented by blue and red lines, respectively. The thickness of the lines indicates the strength of the individual connections.

Fig. 10

Top: Path coefficient matrix obtained for the cerebellum with EBICglasso modelling of neurotransmitter (DA, 5-HT) and metabolite (DOPAC, HVA, 3-MT, 5-HIAA) concentrations after treatment with SAL (12 out of 15 possible connections, sparsity: 0.200) and WAY100,635 (5 out of 15 possible connections, sparsity: 0.667). Bottom: Connections between DA, 5-HT and metabolites (DOPAC, 3-MT, HVA, 5-HIAA) in cerebellum after SAL and WAY100,635. Positive and negative associations are represented by blue and red lines, respectively. The thickness of the lines indicates the strength of the individual connections.

Cingulate. The analysis of HPLC data revealed an increase of HVA levels (Table. 1, Fig. 2B) in CING after WAY100,635 relative to SAL (p = 0.005; “treatment “: F1,7,7, 0.0176, p = 0.895; “brain region”: F1,7,7, 30.281, p < 0.001; “treatment” x “brain region”: F1,7,7, 30.281, p < 0.001).

Turnover of DA to DOPAC (Table. 2, Fig. 3A) was reduced after WAY100,635 compared to SAL (p ≤ 0.0001; “treatment”: F1,7,7, 3.508, p = 0.063, “brain region”: F1,7,7 = 13.087, p < 0.001, “treatment x brain region”: F1,7,7, 3.088, p = 0.006). Likewise, turnover of DA to 3-MT (Table. 2, Fig. 3B) was lower after WAY100,635 (p ≤ 0.0001; “treatment”: F1,7,7, 3.508, p = 0.063, “brain region”: F1,7,7, 13.087, p < 0.001, “treatment x brain region”: F1,7,7, 3.088, p = 0.006). 5-HIAA concentrations (Table. 1, Fig. 2D) were decreased after WAY100,635 (p ≤ 0.0001; “treatment”: F1,7,7, 6.751, p < 0.010; “brain region”: F1,7,7, 42.018, p < 0.001; “treatment x brain region”: F1,7,7, 2.943, p = 0.006).

In network analysis, after SAL, DA and DOPAC, and 5-HT and 5-HIAA showed strong negative connections with lower mean neurotransmitter than metabolite levels, indicating rapidly ongoing MAO-mediated degradation. DA and 3-MT exhibited a weaker positive association with low mean DA and high mean 3-MT, reflecting also active metabolization by COMT at the time of sacrifice. DOPAC and HVA were negatively associated with high DOPAC and low HVA, suggesting a delay in COMT-mediated HVA formation. 3-MT and HVA were positively associated, with the only slightly higher mean HVA level indicating slow but ongoing MAO-mediated degradation (Table. 1, Fig. 4).

After WAY100,635, the DA-DOPAC connection became positive, suggesting ongoing MAO-mediated conversion, while DA-3-MT, DOPAC-HVA, and 3-MT-HVA associations disappeared. Reduced DOPAC/DA and 3-MT/DA ratios together with higher DA and DOPAC levels relative to SAL indicate that initially high DA levels underwent rapid degradation by MAO, incurring also high levels of the primary metabolite. At sacrifice, the positive DA-DOPAC connection was still visible, but with a subdued turnover ratio likely due to saturation. An initially rapid and then abated degradation is also suggested by elevated HVA along with a decreased 3-MT/DA ratio relative to SAL. The increase of HVA reveals that the parallel steps of COMT- and MAO-mediated degradation had also taken place, but, at sacrifice, were not reflected any more by path coefficients between the individual nodes. This may be due to saturation effects at least in part of the animals, as reflected by the comparatively high interindividual variation. The negative 5-HT-5-HIAA association was lost, with the high 5-HIAA variability also suggesting suppressed MAO-mediated degradation at least in part of the animals, which is in line with the reduced 5-HIAA levels after WAY100,635.

Caudateputamen. DA concentrations (Table. 1, Fig. 2A) after WAY100,635 were higher relative to SAL (p ≤ 0.001; “treatment”: F1,7,7, 0.954, p = 0.330; “brain region”: F1,7,7, 12.084, p < 0.001; “treatment x brain region”: F1,7,7, 7.667, p < 0.001). Moreover, WAY100,635 decreased the turnover of 3-MT to HVA (Table. 2, Fig. 3B) relative to SAL (p = 0.002; “treatment”: F1,7,7, 0.599, p = 0.440, “brain region”: F1,7,7, 19.90, p < 0.001, “treatment x brain region”: F1,7,7, 1.748, p = 0.103).

After SAL, DA and DOPAC showed a weak positive association with higher mean DOPAC than DA, indicating ongoing MAO-mediated conversion. DA and 3-MT exhibited a stronger, weakly negative association with higher DA than 3-MT, suggesting delayed COMT-mediated turnover. DOPAC and HVA were positively associated with higher DOPAC than HVA, indicating decelerated but ongoing COMT-mediated conversion. No 3-MT-HVA connection was observed. The high HVA variability implies suppressed MAO-mediated degradation in some animals. 5-HT and 5-HIAA showed a weak positive connection with higher 5-HIAA than 5-HT, reflecting ongoing MAO-mediated conversion (Table. 1, Fig. 5).

After WAY100,635, DA-DOPAC and DA-3-MT associations strengthened and became positive, indicating ongoing MAO- and COMT-mediated conversion. DOPAC-HVA retained a strong positive connection, with the higher DOPAC than HVA level indicating a retardation of COMT-mediated turnover. The introduction of a strong, negative 3-MT-HVA connection with lower 3-MT than HVA indicates a predominance of MAO-mediated metabolization to HVA. The elevation of DA after WAY100,635 may reflect either initial DA increase with early saturation or a suppression or retardation of turnover. The positive 5-HT-5-HIAA connection was stronger, indicating enhanced MAO-mediated conversion.

Nucleus accumbens. After WAY100,635, DA levels (Table. 1, Fig. 2A) exceeded those obtained after SAL (p = 0.019; “treatment”: F1,7,7, 0.954, p = 0.330; “brain region”: F1,7,7, 12.084, p < 0.001; “treatment x brain region”: F1,7,7, 7.667, p < 0.001).

After SAL, DA was positively associated with DOPAC and 3-MT, and 5-HT with 5-HIAA, indicating ongoing MAO- and COMT-mediated DA degradation to DOPAC and 3-MT, respectively, and MAO-mediated 5-HT degradation to 5-HIAA. 3-MT was positively and DOPAC negatively associated with HVA with mean DOPAC higher and mean 3-MT lower than HVA, indicating ongoing MAO-mediated 3-MT-to-HVA but delayed COMT-mediated DOPAC-to-HVA conversion (Table. 1, Fig. 6).

After WAY100,635, a positive DA-HVA connection persisted, suggesting that prior metabolization steps had occurred, but were not captured by path coefficients for DA-DOPAC, DOPAC-HVA, DA-3-MT, and 3-MT-HVA at the time of sacrifice. Although mean DOPAC was high, so was the interindividual variation, which implies suppression of MAO-mediated degradation in some animals. The loss of DA-3-MT and DOPAC-HVA connections with higher DA and DOPAC than HVA, suggest reduced COMT- and MAO-mediated metabolism, contributing to the elevated DA levels compared to SAL. Elevated ventrostriatal DA after WAY100,635, may reflect either initial DA increase with subsequent saturation or a suppression or retardation of turnover. The positive 5-HT-5-HIAA association was lost, indicating inhibited MAO-mediated conversion.

Thalamus. DA concentration (Table. 1, Fig. 2A) after WAY100,635 was lower than after SAL (p ≤ 0.001; “treatment”: F1,7,7, 0.954, p = 0.330; “brain region”: F1,7,7, 12.084, p < 0.001; “treatment x brain region”: F1,7,7, 7.667, p < 0.001). Also, 5-HIAA levels (Table. 1, Fig. 2D) were diminished after WAY100,635 (p = 0.015; treatment”: F1,7,7, 6.751, p < 0.010; “brain region”: F1,7,7, 42.018, p < 0.001; “treatment x brain region”: F1,7,7, 2.943, p = 0.006).

After SAL, DA was positively associated with DOPAC but negatively and weaker with 3-MT, while 3-MT was positively associated with HVA. This indicates ongoing MAO- and COMT-mediated conversion of DA to DOPAC and 3-MT, respectively, with rapid, subsequent MAO-catalyzed turnover of 3-MT to HVA (as mean HVA exceeded mean 3-MT). 5-HT was positively associated with 5-HIAA, reflecting ongoing MAO-mediated 5-HT metabolization (Table. 1, Fig. 7).

After WAY100,635, DA-DOPAC and DOPAC-HVA (both positive) as well as DA-3-MT (negative) and 3-MT-HVA (positive) associations strengthened. This suggests, for one, ongoing MAO- and COMT-mediated DA degradation via the pathway DA → DOPAC → HVA. Higher DA than 3-MT and slightly higher HVA than 3-MT, however, indicate decelerated COMT-mediated DA-to-3-MT but accelerated MAO-mediated 3-MT-to-HVA conversion, which may explain the reduction in thalamic DA after WAY100,635. The 5-HT-5-HIAA association was slightly strengthened, although the decline in 5-HT concentration and turnover ratio may account for the decrease of 5-HIAA relative to SAL (Table. 1 and 2, Fig. 7).

Dorsal and ventral hippocampus. No differences in concentrations or turnover ratios were observed between WAY100,635 and SAL (Table. 1 and 2; 0.118 ≤ p ≤ 0.995).

After SAL, dHIPP showed no DA-DOPAC, but a weak positive DA-3-MT connection, suggesting low COMT-mediated DA degradation. Conversely, the vHIPP displayed a weak positive DA-DOPAC connection, indicating minor MAO-mediated DA degradation. In dHIPP, DOPAC-HVA was strongly positive with no 3-MT-HVA link, whereas, in vHIPP, DOPAC-HVA was weakly positive with a strong negative 3-MT-HVA connection, indicating a COMT-dominated second-step degradation in dHIPP and a MAO-dominated second-step degradation in vHIPP. In dHIPP, 5-HT was strongly positively associated with 5-HIAA, indicating MAO-mediated degradation, absent in vHIPP (Table. 1, Fig. 8).

After WAY100,635, no changes in metabolite concentrations or turnover ratios occurred in dHIPP or vHIPP versus SAL. In dHIPP, DA showed a weak negative DA-DOPAC and strong positive DA-3-MT connection; DOPAC-HVA weakened, and a negative 3-MT-HVA connection emerged. Mean DOPAC and 3-MT exceeded DA, but not HVA, indicating ongoing MAO- and COMT-mediated degradation with rapid further metabolization. The 5-HT-5-HIAA association in dHIPP slightly strengthened, reflecting ongoing MAO-mediated conversion. In vHIPP, no connections were detected. However, low DA, slightly higher DOPAC and 3-MT, and much higher HVA and 5-HIAA concentrations imply prior MAO- and COMT-mediated steps,

Introduction

Introduction

Materials and methods

Animals

Drug treatment

Rat behavior

Neurochemistry

Statistical analysis

Results

Motor/exploratory behaviors and object recognition

Neurochemistry

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