Deficient serotonin neurotransmission and depression-like serotonin biomarker alterations in tryptophan hydroxylase 2 (Tph2) loss-of-function mice

Abstract

Probably the foremost hypothesis of depression is the 5-hydroxytryptamine (5-HT, serotonin) deficiency hypothesis. Accordingly, anomalies in putative 5-HT biomarkers have repeatedly been reported in depression patients. However, whether such anomalies in fact reflect deficient central 5-HT neurotransmission remains unresolved. We employed a naturalistic model of 5-HT deficiency, the tryptophan hydroxylase 2 (Tph2) R439H knockin mouse, to address this question. We report that Tph2 knockin mice have reduced basal and stimulated levels of extracellular 5-HT (5-HT(Ext)). Interestingly, cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) and fenfluramine-induced plasma prolactin levels are markedly diminished in the Tph2 knockin mice. These data seemingly confirm that low CSF 5-HIAA and fenfluramine-induced plasma prolactin reflects chronic, endogenous central nervous system (CNS) 5-HT deficiency. Moreover, 5-HT(1A) receptor agonist-induced hypothermia is blunted and frontal cortex 5-HT(2A) receptors are increased in the Tph2 knockin mice. These data likewise parallel core findings in depression, but are usually attributed to anomalies in the respective receptors rather than resulting from CNS 5-HT deficiency. Further, 5-HT(2A) receptor function is enhanced in the Tph2 knockin mice. In contrast, 5-HT(1A) receptor levels and G-protein coupling is normal in Tph2 knockin mice, indicating that the blunted hypothermic response relates directly to the low 5-HT(Ext). Thus, we show that not only low CSF 5-HIAA and a blunted fenfluramine-induced prolactin response, but also blunted 5-HT(1A) agonist-induced hypothermia and increased 5-HT(2A) receptor levels are bona fide biomarkers of chronic, endogenous 5-HT deficiency. Potentially, some of these biomarkers could identify patients likely to have 5-HT deficiency. This could have clinical research utility or even guide pharmacotherapy.

Figure 1

Functional 5-hydroxytryptamine (5-HT) deficiency in tryptophan hydroxylase 2 (Tph2) knockin mice. (a–d) Microdialysis, frontal cortex. (e–h) Microdialysis, hippocampus. (a, e) Probe location in the frontal cortex and hippocampus. Vertical black bar indicates probe membrane location. (Image modified from http://www.brain-map.org). (b, f) Basal extracellular 5-HT (5-HT_Ext) at noon and midnight (N = 9–10). (c, g) Net 5-HT_Ext release stimulated by reverse dialysis of 100 mM K⁺ via the probe (N = 8–10). (d, h) Net 5-HT_Ext release stimulated by the selective serotonin reuptake inhibitor (SSRI) escitalopram (10 mg/kg, i.p.) (N = 11–18). (i) Marbles buried in 30 min (N = 8–9). (k) Adult (12 weeks) tissue levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) (N = 7–8). (j) Neonatal (P2) tissue levels of 5-HT and 5-HIAA (N = 6–9). Data represent means±s.e.m. *P < 0.05 WT vs Tph2 knockin. (b–d, f–h) Repeated-measures (RM)-analysis of variance (ANOVA) followed by Bonferroni post hoc test. (i–k) Student’s t-test.

Figure 2

Alterations in putative 5-hydroxytryptamine (5-HT) biomarkers reported in depression and suicidality are recapitulated in tryptophan hydroxylase 2 (Tph2) knockin mice. (a) Tph2 knockin mice have decreased cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) (N = 5–6), (b) a blunted prolactin response to dexfenfluramine (DexFen, 10 mg kg⁻¹, i.p., N = 6–9), (c) an attenuated hypothermic response to the 5-HT_1A receptor (5-HT_1AR) agonist 8-OH-DPAT (1 mg kg⁻¹, i.p., N = 10) and (d) increased frontal cortex 5-HT_2A receptors (5-HT_2ARs) (N = 7). On the other hand, (e) Tph2 knockin mice have WT levels of CSF 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and of (f) plasma prolactin. (g) Tph2 knockin mice displayed a more pronounced hypothermic response to the α₂R agonist clonidine compared with WT. (h) Tph2 mice have WT levels of 5-HT_1ARs in the hippocampus. Data represent means±s.e.m. (a, d, e, h) Student’s t-test. (b, f) Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (c, g) Repeated-measures (RM)-ANOVA followed by Bonferroni post hoc test. *P < 0.05 WT vs Tph2 knockin. ^#P < 0.05 WT saline vs WT dexfenfluramine.

Figure 3

5-Hydroxytryptamine_2A receptors (5-HT_2AR), but not 5-HT_1A receptor (5-HT_1AR), function is altered in tryptophan hydroxylase 2 (Tph2) knockin mice. (a) Decreased head-twitches induced by administration of clorgyline (10 mg kg⁻¹, s.c.) 60 min before escitalopram (10 mg kg⁻¹, i.p.), a Tph2 dependent condition, in Tph2 knockin mice (N = 10). (b) Increased head-twitches induced by co-administration of 5-hydroxytryptophan (5-HTP) (100 mg kg⁻¹, s.c.) and escitalopram (10 mg kg, i.p.), a Tph2 independent condition, in Tph2 knockin mice (N = 10). (c) Increased head shakes induced by the 5-HT2_A/CR agonist DOI (3 mg kg⁻¹, i.p.), a Tph2 independent condition, in Tph2 knockin mice (N = 30–33). (d) Representative autoradiograms and (e) quantitation of [¹²⁵I]p-MPPI binding to 5-HT_1ARs in the CA1 of the hippocampus (left arrow), hypothalamus (HYP, middle arrow) and dorsal raphe (DR, right arrow) (N = 8–10). (f) Representative autoradiograms and (g) quantitation of basal and 8-OH-DPAT-stimulated [³⁵S]GTPγS binding in CA1, hypothalamus and dorsal raphe (N = 7–10). (a–c) Repeated-measures (RM)-analysis of variance (ANOVA) followed by Bonferroni post hoc test. (d–g) Student’s t-test. *P < 0.05, WT vs Tph2 knockin. ^#P < 0.05, basal vs 8-OH-DPAT treated, within genotype.

Figure 4

Basal extracellular levels of dopamine, noradrenaline and amino acid neurotransmitters are unchanged in tryptophan hydroxylase 2 (Tph2) knockin mice. (a–f) Frontal cortex (N = 7–8). (g–l) Hippocampus (N = 9–10). (a, g) Extracellular 5-hydroxytryptamine (5-HT_Ext) was decreased in Tph2 knockin mice. No significant genotype differences were found for any of the other assessed neurotransmitters (b–f, h–l). Repeated-measures (RM)- analysis of variance (ANOVA) followed by Bonferroni post hoc test. *P < 0.05 WT vs Tph2 knockin.