Trace Amine-Associated Receptor 5 Provides Olfactory Input Into Limbic Brain Areas and Modulates Emotional Behaviors and Serotonin Transmission

Abstract

Trace amine-associated receptors (TAARs) are a class of G-protein-coupled receptors found in mammals. While TAAR1 is expressed in several brain regions, all the other TAARs have been described mainly in the olfactory epithelium and the glomerular layer of the olfactory bulb and are believed to serve as a new class of olfactory receptors sensing innate odors. However, there is evidence that TAAR5 could play a role also in the central nervous system. In this study, we characterized a mouse line lacking TAAR5 (TAAR5 knockout, TAAR5-KO) expressing beta-galactosidase mapping TAAR5 expression. We found that TAAR5 is expressed not only in the glomerular layer in the olfactory bulb but also in deeper layers projecting to the limbic brain olfactory circuitry with prominent expression in numerous limbic brain regions, such as the anterior olfactory nucleus, the olfactory tubercle, the orbitofrontal cortex (OFC), the amygdala, the hippocampus, the piriform cortex, the entorhinal cortex, the nucleus accumbens, and the thalamic and hypothalamic nuclei. TAAR5-KO mice did not show gross developmental abnormalities but demonstrated less anxiety- and depressive-like behavior in several behavioral tests. TAAR5-KO mice also showed significant decreases in the tissue levels of serotonin and its metabolite in several brain areas and were more sensitive to the hypothermic action of serotonin 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propilamino)tetralin (8-OH-DPAT). These observations indicate that TAAR5 is not just innate odor-sensing olfactory receptor but also serves to provide olfactory input into limbic brain areas to regulate emotional behaviors likely via modulation of the serotonin system. Thus, anxiolytic and/or antidepressant action of future TAAR5 antagonists could be predicted. In general, "olfactory" TAAR-mediated brain circuitry may represent a previously unappreciated neurotransmitter system involved in the transmission of innate odors into emotional behavioral responses.

Keywords: TAAR; TAAR5; antidepressant; anxiety; anxiolytic; depression; olfaction; trace amine.

Figure 1

Generation of TAAR5 knockout (TAAR5-KO) mice. (A) TAAR5-KO mice were generated using homologous recombination that produces TAAR5 gene inactivation through a replacement vector. The target gene is aligned with the targeting vector so that exon 1 (from base 1 to 320, see “Material and Methods” section) is substituted with the NEOr gene (HSV-tk, negative selection marker). (B) Genomic DNA from the recombinant embryonic stem (ES) line was assayed for homologous recombination using PCR. Amplified DNA fragments were visualized by ethidium bromide staining following agarose gel electrophoresis, as shown in the gel image on the left. The test PCRs (a set of three lanes) employed the gene-specific (GS) primer, which lies outside of and beside the targeting vector arm, paired in succession with one of three primers in the insertion fragment. The “DNA sample control” employed a primer pair intended to amplify a fragment from a nontargeted genomic locus. The “positive control” employed the GS primer paired with a primer at the other end of the arm. The table lists the primers (numbered) used in each PCR and the expected product sizes, in base pairs (bp). The diagrams to the right depict the anticipated gel image as well as the relative positions of the PCR primers. (C) Genomic DNA isolated from the indicated ES lines was digested with the indicated restriction enzymes (determined to cut outside of the construct arms). The DNA was analyzed by Southern hybridization, probing with a radiolabeled DNA fragment that hybridizes outside of and beside the construct arm. The parent ES lines (negative controls) showed bands representing the endogenous (wild-type) gene #336 allele. In contrast, the ES#3232 line showed an additional band representing the targeted allele from the expected homologous recombination event. “Additional” denotes a secondary line that was derived from the same targeting vector but was not used to generate mice. (D) Standard genotyping PCR with genomic DNA of TAAR5-WT (wild type), TAAR5-HET (heterozygous), and TAAR5-KO (knockout) mice.

Figure 2

Histological analysis of TAAR5 expression in TAAR5-KO mouse brain by LacZ staining. (A,B,E–M) Coronal sections, (C,D) Sagittal sections. (A–C) Staining in glomerular (A,B) and mitral (B,C) layers of the olfactory bulb (OB). (D) Overview of TAAR5+ regions on sagittal section of whole mouse brain (AON, anterior olfactory nucleus; CTX, cortex; HPC, hippocampus; NAc, nucleus accumbens; OT, olfactory tubercle); (E) single TAAR5+ cells in the amygdala (AMG); (F) TAAR5+ cells in the orbitofrontal cortex (OFC); (G) staining in CA1 region of the hippocampus (HPC); (H) TAAR5+ staining in the anterior olfactory nucleus (AON); (I) TAAR5+ staining in the piriform cortex (PIR); (J) single TAAR5+ cells in the thalamus (THL); (K) TAAR5+ cells in the nucleus accumbens (NAc); (L) TAAR5+ staining in the entorhinal cortex (ENT); (M) TAAR5+ cells in the ventromedial hypothalamus (HPTH). Scale bar: (A–C) 250 μM, (D) 1 mm, (E,G,K,M) 100 μM, (F,H,I,J,L) 500 μM. Arrows indicate LacZ staining.

Figure 3

Reduced anxiety and affectivity of TAAR5-KO mice. (A) Minimally altered locomotor activity of TAAR5-KO mice in the locomotor activity boxes (measured for 60 min). Increased exploration (B) and number of central entries (C) of TAAR5-KO mice in the circular open field test. (D) Increased number of nose pokes into light compartment in the light–dark transition test. Increased time spent in open arms by TAAR5-KO mice in the elevated zero-maze test (E) and the elevated plus maze test (F). Increased number of open arm entries (G) and head dippings (H) of TAAR5-KO mice in the elevated plus maze test. (I) Decreased affectivity of TAAR5-KO mice in the learned helplessness test (number of avoidances). *P < 0.05 (Student’s t-test or Mann–Whitney U test).

Figure 4

Altered 5-HT transmission in TAAR5-KO mice. (A–C) Tissue level of 5-HT, 5-HIAA, and turnover ratio 5-HIAA/5-HT in the striatum of WT and TAAR5-KO mice. (D–F) Tissue level of 5-HT, 5-HIAA, and turnover ratio 5-HIAA/5-HT in the hippocampus of WT and TAAR5-KO mice. (G–I) Tissue level of 5-HT, 5-HIAA, and turnover ratio 5-HIAA/5-HT in the hypothalamus of WT and TAAR5-KO mice. (J–L) Enhanced hypothermic effect of 5-HT1A agonist 8-hydroxy-2-(di-n-propilamino)tetralin (8-OH-DPAT; i.p.) in TAAR5-KO mice. *P < 0.05 [(A–I) Mann–Whitney U test; (J–L) two-way ANOVA followed by post hoc Bonferroni’s test for individual time point comparisons between genotypes].