Imaging elevated brain arachidonic acid signaling in unanesthetized serotonin transporter (5-HTT)-deficient mice

Abstract

Certain polymorphisms reduce serotonin (5-HT) reuptake transporter (5-HTT) function and increase susceptibility to psychiatric disorders. Heterozygous (5-HTT(+/-))-deficient mice, models for humans with these polymorphisms, have elevated brain 5-HT concentrations and behavioral abnormalities. As postsynaptic 5-HT(2A/2C) receptors are coupled to cytosolic phospholipase A(2) (cPLA(2)), which releases arachidonic acid (AA) from membrane phospholipid, 5-HTT-deficient mice may have altered brain AA signaling and metabolism. To test this hypothesis, signaling was imaged as an AA incorporation coefficient k(*) in unanesthetized homozygous knockout (5-HTT(-/-)), 5-HTT(+/-) and wild-type (5-HTT(+/+)), mice following saline (baseline) or 1.5 mg/kg s.c. DOI, a partial 5-HT(2A/2C) receptor agonist. Enzyme activities, metabolite concentrations, and head-twitch responses to DOI were also measured. Baseline k(*) was widely elevated by 20-70% in brains of 5-HTT(+/-) and 5-HTT(-/-) compared to 5-HTT(+/+) mice. DOI increased k(*) in 5-HTT(+/+) mice, but decreased k(*) in 5-HTT-deficient mice. Brain cPLA(2) activity was elevated in 5-HTT-deficient mice; cyclooxygenase activity and prostaglandin E(2) and F(2alpha) and thromboxane B(2) concentrations were reduced. Head-twitch responses to DOI, although robust in 5-HTT(+/+) and 5-HTT(+/-) mice, were markedly fewer in 5-HTT(-/-) mice. Pretreatment with para-chlorophenylalanine, a 5-HT synthesis inhibitor, restored head twitches in 5-HTT(-/-) mice to levels in 5-HTT(+/+) mice. We propose that increased baseline values of k(*) in 5-HTT-deficient mice reflect tonic cPLA(2) stimulation through 5-HT(2A/2C) receptors occupied by excess 5-HT, and that reduced k(*) and head-twitch responses to DOI reflected displacement of receptor-bound 5-HT by DOI with a lower affinity. Increased baseline AA signaling in humans having polymorphisms with reduced 5-HTT function might be identified using positron emission tomography.

Figure 1

Coronal autoradiographs of brain showing effects of DOI and 5-HTT genotype on regional AA incorporation coefficients k* in mice. Values of k* (ml/s/g brain × 10⁻⁴) are given on a color scale from 5 (blue) to 30 (red). Abbreviations: Acg, anterior cingulate cortex; Aud, auditory cortex; CPu, caudate-putamen; Hipp, hippocampus; IPC, interpeduncular nucleus; Mot, motor cortex; SN, substantia nigra; Thal, thalamus; Vis, visual cortex.

Figure 2

Difference patterns of k* responses to DOI in sagittal representation of 5-HTT mouse brain. Regions in which k* was increased significantly (p < 0.05) are solid grey, regions in which k* was decreased significantly are hatched. The 5-HTT^+/− + DOI image is compared with the 5-HTT^+/+ + saline image. The 5-HTT^+/− + saline and the 5-HTT^−/− + saline images are compared with the 5-HTT^+/+ + saline image. The 5-HTT^+/− + DOI and the 5-HTT^−/− + DOI images are compared with the 5-HTT^+/− + saline and the 5-HTT^−/− + saline, respectively. List of regions: A, amygdala; Acb, nucleus accumbens; AUD, auditory cortex; am, anteromedial thalamic nucleus; av, anteroventral thalamic nucleus; CbG, cerebellar gray matter; CbW, cerebellar white matter; Co, cochlear nucleus; CPu, caudate putamen; DLG, dorsal lateral geniculate nucleus; DR, dorsal raphe; Fr, frontal cortex; GP, globus pallidus; Hb, habenular complex; HIP, hippocampus; HYP, hypothalamus; IC, inferior colliculus; IPC, interpeduncular nucleus; LC, locus coeruleus; MI, mammillary nucleus; mG, medial geniculate nucleus; MolCbG, molecular layer of cerebellar gray matter; MOT, motor cortex; MR, median raphe; MVe, medial vestibular nucleus; OT, olfactory tubercle; PF, prefrontal cortex; PPTg, pedunculopontine tegmental nucleus; SN, substantia nigra; S, septum; SO, superior olive; Sp5, spinal trigeminal nucleus; SS, somatosensory cortex; SC, superior colliculus; SCgl, gray layer of superior colliculus; STH, subthalamic nucleus; THa, thalamus; VIS, visual cortex.

Figure 3

Effects of brain 5-HT depletion by PCPA pretreatment on DOI-induced head twitches in 5-HTT mice. At baseline (vehicle pretreatment), DOI induced fewer head twitches in 5-HTT^−/− mice compared to 5-HTT^+/+ mice, whereas DOI-induced head twitches were similar between 5-HTT^+/+ and 5-HTT^+/− mice. Pretreatment with PCPA, which depletes 5-HT levels, increased DOI-induced head twitches in 5-HTT^−/− mice, with a trend toward a significant increase in 5-HTT^+/− mice (p = 0.08), compared to their vehicle-pretreated counterparts. Data represent the means ± SEM, n = 7-13 per group. ** p < 0.01 compared to 5-HTT^+/+ mice in the same pretreatment condition; ⁺⁺⁺⁺p < 0.0001 compared to vehicle-pretreated mice of the same 5-HTT genotype.

Figure 4

5-HTP-induced head twitches in 5-HTT mice. The 5-HT precursor 5-HTP, which increases 5-HT levels, induced fewer head twitches in 5-HTT^−/− mice compared to 5-HTT^+/+ mice, with no difference between 5-HTT^+/− and 5-HTT^+/+ mice. Data represent the means ± SEM; n = 9-16 per group. * p < 0.05 compared to 5-HTT^+/+ mice.