VGLUT3 (vesicular glutamate transporter type 3) contribution to the regulation of serotonergic transmission and anxiety

Abstract

Three different subtypes of H(+)-dependent carriers (named VGLUT1-3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT(1A)-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.

Figure 1.

VGLUT3 is poorly present in serotonergic fibers in the raphe. a–c, Immunohistochemistry for VGLUT3 (green), 5HT (blue) and VMAT2 (red) in the DRN (a, b) and MRN (c). VGLUT3-positive terminals almost never colocalise with 5-HT and VMAT2 in the DRN (a) or the MRN (c), whereas the supra-ependymal 5-HT axons contain both VMAT2 and VGLUT3 (a1–a4). VGLUT3 is also found in some serotonergic somas in large organelles containing both 5-HT and VMAT2 (b1–b4). (d–f) Immunohistochemistry for VGLUT3 (green), 5HT (blue) and SERT (red) in the DRN (d, e) and MRN (f). VGLUT3-positive terminals never colocalized with SERT in DRN (d, e) or MRN (f, at higher magnification in f1–f4), except in 5-HT supra-ependymal axons (e1–e4). Scale bars: a, c, d, f, 40 μm; a1–a4, b1–b4, e1–e4, 20 μm, in f1–f4, 10 μm.

Figure 2.

VGLUT3 deletion does not modify 5-HT outflow in the raphe. Extracellular levels of 5-HT were measured in the DRN by in vivo microdialysis coupled to HPLC after injection of saline or paroxetine (i.p., 10 mg/kg). a, The time course of saline (solid lines) or paroxetine (dashed lines)-induced 5-HT outflow (measured every 15 min during 90 min) did not differ between Vglut3^+/+ and Vglut3^−/− animals (Repeated-measures ANOVA, F = 0.241, p = 0.9593). b, No difference was found between genotypes in 5-HT outflow after saline injection. In both genotypes, paroxetine induced a marked augmentation of 5-HT extracellular levels (n = 7, Mann–Whitney, Vglut3^+/+, p = 0.0017, Vglut3^−/−, p = 0.0181).

Figure 3.

Raphe 5HT_1A autoreceptors are desensitized in Vglut3^−/− mice. a, Characteristic pacemaker activity of 5-HT neurons was similar between wild-type (+/+) and mutant (−/−) animals (Vglut3^−/−, 1.98 ± 0.19 spikes/s, n = 14; Vglut3^+/+, 1.99 ± 0.13 spikes/s, n = 12). Integrated firing rate histograms show that the inhibitory effect of ipsapirone on the electrical activity of DRN 5-HT neurons is less important in Vglut3^−/− mice when compared with wild-type animals. b, Concentration–response curves of ipsapirone-induced inhibition of the firing of DRN 5-HT neurons. The dotted lines illustrate the graphical determination of ipsapirone EC₅₀ values (abscissa) in both groups (Vglut3^+/+, EC₅₀ = 4.19 ± 0.52 10⁻⁸ m, n = 10; Vglut3^−/−, EC₅₀ = 6.32 ± 0.51 10⁻⁸ m, n = 10; two-tailed t test, p = 0.009). The concentration–response curve of ipsapirone-induced inhibition in Vglut3^−/− mice was significantly different from that in Vglut3^+/+ mice (n = 10, two-way ANOVA, F = 8.59, p = 0.0038). c, Autoradiograms of brain sections at the level of the DRN, labeled by [³⁵S]GTP-γ-S (50 pm) in the absence (basal) or the presence (stimulated) of 10⁻⁵ m 5-CT. The concentration-response curves show a desensitization of 5HT_1A autoreceptors in Vglut3^−/− mice (black circles) when compared with Vglut3^+/+ mice (white circles). [³⁵S]GTP-γ-S labeling was indeed significantly lower in mutant animals (two-way ANOVA, F = 10.23, p = 0.004; Bonferroni, 10⁻⁷ m p < 0.005, 10⁻⁶ m p < 0.01). OD, optical density. d, The 5HT_1A autoreceptor antagonist 8-OH-DPAT induced a loss of body temperature in both genotypes. At the lowest dose (0.06 mg/kg, s.c.) the loss of temperature was not significant in wild-type or mutant mice (n = 7 and n = 8, respectively). At the 0.1 mg/kg dose, the temperature loss in Vglut3^−/− mice (n = 7, −1.2 ± 0.1°C) was significantly less important than in Vglut3^+/+ mice (n = 7, −2.1 ± 0.2°C, Mann–Whitney, p = 0.0045). This hyposensitivity of Vglut3^−/− mice to 8-OH-DPAT-induced hypothermia was confirmed at the dose of 0.3 mg/kg (Vglut3^−/−, n = 7, −1.8 ± 0.3°C, Vglut3^+/+, n = 8, −3.2 ± 0.4°C, Mann–Whitney, p = 0.0278).

Figure 4.

Colocalization of VGLUT3 and VMAT2 in subsets of 5-HT fibers and terminals within limbic areas. VGLUT3-positive terminals (green) are colocalized with VMAT2 (red) and 5-HT (blue) in the prefrontal cortex (Prl Cx) in layer I/III and V/VI (a, b), in the CA1 and CA3 fields of pyramidal cells (c, d) of the hippocampus, in the dentate gyrus (DG) of the hippocampus (e), in stratum radiatum (f), in the hippocampal fissure (g), and in the lateral septum (LS) (h). In contrast, VGLUT3-positive (green) 5-HT (blue) terminals rarely coexpressed SERT (red) in the LS (i), Prl Cx (j), and CA3 field (k). Regions boxed in a, b, f, and k are enlarged and the different channels (red, green, blue and merge) are shown. In some terminals, VGLUT3 is colocalized with VMAT2 but not with 5-HT (arrowheads in a4, b4, and f4). Scale bars in a–e, g, i, j, k, 40 μm; in h, 20 μm; in a1–a4, b1–b4, f1–f4, k1–k4, 10 μm.

Figure 5.

VGLUT3 deletion reduces hippocampal extracellular levels of 5-HT. a–g, Levels of 5-HT, DA, NA and metabolites were measured using HPLC coupled to electrochemical detection. Levels of all neurotransmitters and metabolites were unchanged between wild-type (white bars) and mutant (black bars) animals. However, the 5-HIAA/5-HT ratio was augmented in Vglut3^−/− mice hippocampi (+25%, Mann–Whitney, p = 0.0056), reflecting a slightly increased 5HT turnover in this structure. h–i, Microdialysis measurements of extracellular 5-HT levels in the ventral hippocampus of Vglut3^−/− and Vglut3^+/+ mice. h, 5-HT outflow reached a lower plateau in Vglut3^−/− mice when compared with Vglut3^+/+ mice (Repeated-measures ANOVA, F = 5.905, p < 0.0001). i, As in the raphe, basal 5-HT outflow in the hippocampus of wild-type and mutant animals was not different (fmol per 20 μl: Vglut3^+/+, 3.54 ± 1.72, Vglut3^−/−, 3.64 ± 1.12; repeated-measures ANOVA, F = 0.052, p = 0.82). Following paroxetine injection (10 mg/kg, i.p.), 5-HT outflow was significantly increased in both genotypes (Mann–Whitney, Vglut3^+/+, p < 0.0001, n = 9; Vglut3^−/−, p < 0.0001, n = 10). Moreover, paroxetine-induced 5-HT outflow was significantly lower in mutant mice (Mann–Whitney test, p = 0.0011).

Figure 6.

VGLUT3 stimulates vesicular 5-HT loading. a, [³H]5-HT reserpine-sensitive vesicular accumulation is augmented by l-glutamate (10 mm) in the rat hippocampus (Hi, +29%, Mann–Whitney, p = 0.0369) and cortex (Cx, +34%, Mann–Whitney, p = 0.0369), but not in the striatum (St). b, The stimulatory effect of glutamate on 5-HT vesicular uptake had the pharmacological profile of a vesicular glutamate transporter. Cortical reserpine-sensitive 5-HT vesicular uptake was measured in the presence (+) or absence (−) of different compounds. Reserpine-sensitive [³H]5-HT was significantly stimulated only by l-glutamate (+22%, Mann–Whitney, p = 0.0021). c, Glutamate effect on [³H]5-HT uptake into cortical vesicles from Vglut3^+/+ and Vglut3^−/− mice. l-Glutamate (10 mm) stimulates uptake into wild-type (+35%, Mann–Whitney, p = 0.009) but not VGLUT3-deficient vesicles.

Figure 7.

VGLUT3 deletion increases anxiety. a, Total immobility time out of 6 min of recording after saline, paroxetine or intraperitoneal reboxetine injection in the tail suspension test. Wild-type (+/+) and mutant (−/−) mice showed no difference in immobility time after intraperitoneal saline injection. The decrease of immobility time after injection of paroxetine or reboxetine was globally the same between both genotypes. b, Anxiety levels appeared higher in mutant mice when assessed in the elevated plus maze. Horizontal and vertical exploration were measured during 6 min. Compared with wild type littermates, Vglut3^−/− mice spent less time in the open and bright arms (n = 8, Mann–Whitney, p = 0.0184). Vertical exploration was also reduced in mutant mice as shown by the lower number of rearings (Mann–Whitney, p = 0.0260). c, The number of glass marbles buried by Vglut3^+/+ (n = 23) and Vglut3^−/− (n = 25) mice was measured during 1 h. Mutant animals buried the marbles faster than did wild-type animals (repeated-measure ANOVA, F = 3.975, p < 0.0001), reflecting a higher level of anxiety in the presence of novel objects. d, Left: When tested in an unfamiliar environment (new cage, NC), Vglut3^−/− mice (n = 20) started to feed later (t = 2.066, p = 0.0457) compared with Vglut3^+/+ mice (n = 20). No difference between genotypes was observed when animals where tested in their home cage (HC). Right: Novelty-suppressed feeding behavior after 5-HT depletion. The higher latency to feed in an unfamiliar environment observed in Vglut3^−/− mice was conserved in groups that received intraperitoneal saline injections (Vglut3^+/+, n = 11, Vglut3^−/−, n = 8, p = 0.0353). After 5-HT depletion by PCPA injection (300 mg/kg, i.p. daily during 3 d), latency to feed in Vglut3^−/− mice (n = 8) was reduced to the same level as Vglut3^+/+ mice (n = 12). e, USVs were recorded during 20 min in 8-d-old Vglut3^+/+ and Vglut3^−/− pups. Total USV durations were calculated over consecutive 30 s periods for each animal during the 20 min of the test and values are expressed as the average duration of USV trains over 30 s. In response to the stress of being separated from mother and littermates, mutant pups emitted more vocalizations than wild type pups (Fisher's PLSD test, p = 0.0128).

Figure 8.

Schematic representation of VGLUT3 localization and functions in 5-HT neurons. a, A majority of 5-HT neurons from the DRN and MRN express VGLUT3 mRNA. In these nuclei, serotonergic terminals generally lack VGLUT3 (type 1, VMAT2/SERT 5-HT terminals). Intracellular 5HT is thus loaded in synaptic vesicles by VMAT2, and released in the synaptic cleft by exocytosis. 5-HT transmission is stopped by reuptake via the plasma membrane transporter SERT. VGLUT3 in DRN and MRN is almost exclusively expressed by purely glutamatergic terminals, whose origins remain to be elucidated. These VGLUT3-positive terminals exert a light inhibition on serotonergic tone through either intracellular or extracellular pathways. A particular situation is observed along the border of the third ventricle/aqueduct where supra-ependymal serotonergic axons express VMAT2/VGLUT3/SERT (type 2, VMAT2/VGLUT3/SERT 5-HT terminals). b, In serotonergic projection areas such as the hippocampus (Hi), the lateral septum (LS) and the prelimbic cerebral cortex (PrlCx), a combination of two types of 5-HT terminals is found in equivalent proportions. “Classical” 5-HT terminals (type 1) coexist with 5-HT terminals expressing VGLUT3 and lacking the plasma membrane transporter SERT (type 3, VMAT2/VGLUT3 5-HT terminals). Vesicular glutamate uptake via VGLUT3 allows vesicular-filling synergy thus increasing serotonergic release. Moreover, these VGLUT3-positive 5HT terminals lack SERT further strengthening the extent and duration of 5-HT transmission. Whether glutamate released by these terminals bind to presynaptic or postsynaptic receptor is still unresolved. This combination of vesicular and plasma membrane transporters within subsets of 5-HT terminals allows different intensity of serotonergic transmission. These three types of 5-HT terminals thus allow a scaling of 5-HT transmission with Type1 < Type 2 < Type 3 terminals in terms of signal intensity.