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. 2024 Feb 20;15(6):1185–1196. doi: 10.1021/acschemneuro.3c00758

Evidence for a Role of 5-HT-glutamate Co-releasing Neurons in Acute Stress Mechanisms

L Sophie Gullino †, Cara Fuller †, Poppy Dunn †, Helen M Collins †, Salah El Mestikawy ‡,§, Trevor Sharp †,*
PMCID: PMC10958520  PMID: 38377469

Abstract

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A major subpopulation of midbrain 5-hydroxytryptamine (5-HT) neurons expresses the vesicular glutamate transporter 3 (VGLUT3) and co-releases 5-HT and glutamate, but the function of this co-release is unclear. Given the strong links between 5-HT and uncontrollable stress, we used a combination of c-Fos immunohistochemistry and conditional gene knockout mice to test the hypothesis that glutamate co-releasing 5-HT neurons are activated by stress and involved in stress coping. Acute, uncontrollable swim stress increased c-Fos immunoreactivity in neurons co-expressing VGLUT3 and the 5-HT marker tryptophan hydroxylase 2 (TPH2) in the dorsal raphe nucleus (DRN). This effect was localized in the ventral DRN subregion and prevented by the antidepressant fluoxetine. In contrast, a more controllable stressor, acute social defeat, had no effect on c-Fos immunoreactivity in VGLUT3-TPH2 co-expressing neurons in the DRN. To test whether activation of glutamate co-releasing 5-HT neurons was causally linked to stress coping, mice with a specific deletion of VGLUT3 in 5-HT neurons were exposed to acute swim stress. Compared to wildtype controls, the mutant mice showed increased climbing behavior, a measure of active coping. Wildtype mice also showed increased climbing when administered fluoxetine, revealing an interesting parallel between the behavioral effects of genetic loss of VGLUT3 in 5-HT neurons and 5-HT reuptake inhibition. We conclude that 5-HT-glutamate co-releasing neurons are recruited by exposure to uncontrollable stress. Furthermore, natural variation in the balance of 5-HT and glutamate co-released at the 5-HT synapse may impact stress susceptibility.

Keywords: 5-HT, VGLUT3, glutamate, dorsal raphe nucleus, stress, c-Fos.

Introduction

Serotonin (5-hydroxytryptamine; 5-HT) is a key neuromodulator of emotional processing, stress sensitivity, and coping behavior.1,2 5-HT neurons in the midbrain dorsal raphe nucleus (DRN), the principal source of 5-HT innervation to the forebrain, are activated by acute inescapable stressors, such as forced swim, restraint, and footshock, as evident through increased expression of the activity-dependent immediate-early gene c-fos in 5-HT neurons.3−8 Although other forms of stress also activate 5-HT neurons,9,10 evidence suggests that stressors allowing for the least control (i.e., inescapable stressors) are associated with greater 5-HT neuron activation.9,11,12

Recently, it has become clear that 5-HT neurons are capable of releasing not only 5-HT but also glutamate. Electrophysiological evidence for 5-HT-glutamate co-release in cultured 5-HT neurons13 was followed by the discovery of the expression of type 3 vesicular glutamate transporter (VGLUT3) in 50–80% of 5-HT neurons in specific DRN subregions.14−16 More recently, electrophysiological studies have demonstrated that optogenetic activation of 5-HT neurons elicits both 5-HT and glutamate-mediated synaptic responses in different forebrain regions.17−19

Currently, the functional role of 5-HT-glutamate co-release is unclear although links to anxiety-like behavior and reward processing have been proposed based on studies of both the phenotype of VGLUT3 knockout mice19−21 and the behavioral effects of optogenetic activation of 5-HT neurons.19,20 Interestingly, in a recent chemogenetic study, activation of 5-HT neurons projecting to the prefrontal cortex from the ventral region of the DRN, an area rich in 5-HT-glutamate co-releasing neurons, increased active coping (i.e., reduced immobility) in mice exposed to swim stress.22 The latter finding suggests that glutamate co-releasing 5-HT neurons are activated by uncontrollable stressors such as swim stress, and may be involved in stress-coping behavior. This result22 also emphasizes the functional heterogeneity within DRN subregions that has been detected in previous studies.8,23,24

Here, we used c-Fos immunohistochemistry to test the prediction that 5-HT-glutamate co-releasing neurons in the DRN (particularly the ventral region) would be activated by an uncontrollable stressor, specifically swim stress. Effects were compared with a more controllable stressor, acute social defeat. Finally, behavioral experiments using a novel transgenic mouse with VGLUT3 knockout targeted to 5-HT neurons (VGLUT3 cKO5-HT mice25) examined the causal link between changes in activity of 5-HT-glutamate co-releasing neurons and stress-coping behavior.

Results and Discussion

Swim Stress Evoked c-Fos Expression in the DRN

Immunohistochemistry demonstrated an abundance of c-Fos immunoreactive neurons at the level of the DRN and median raphe nucleus (MRN) in the mouse midbrain (Figure 1). Exposure of mice to acute swim stress increased the number of c-Fos immunoreactive neurons in the DRN and MRN (effect of treatment: F(2,17) = 5.503, p = 0.014; effect of region: F(1,15) = 17.160, p < 0.001; region × treatment interaction: F(2,15) = 0.272, p = 0.766; Figures 1B and 2A). Posthoc analysis revealed that this effect was statistically significant in the DRN of swim-stressed mice compared to non-stressed controls (p = 0.017; Figure 2A). Conversely, the number of c-Fos immunoreactive cells in the MRN was not significantly different across conditions (F(2,15) = 2.065, p = 0.161; Figure 2A). These data are in accord with previous studies reporting that swim stress increased c-Fos immunoreactivity in the DRN of rats.8,23

Figure 1.

Figure 1

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C-Fos immunoreactivity in mouse midbrain following acute swim stress. (A) C-Fos immunoreactivity in a midbrain section at the level of the DRN and MRN (left) according to the stereotaxic atlas (top right) of Paxinos and Franklin.26 Higher magnification images of the DRN subregions (bottom right). (B) High-magnification images of c-Fos immunoreactivity in the ventral DRN of control mice and mice administered a single injection of either saline or fluoxetine (FLX) and exposed to swim stress. Abbreviations: dorsal raphe nucleus (DRN), median raphe nucleus (MRN), aqueduct (Aq), and medial longitudinal fasciculus (mlf).

Figure 2.

Figure 2

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Effect of acute swim stress, with or without fluoxetine, on c-Fos expression in midbrain subregions. (A) C-Fos immunoreactive neurons in the DRN and MRN. (B) C-Fos immunoreactive neurons in DRN subregions. Columns are mean ± SEM values with individual values indicated by closed circles. ** p < 0.01, *p < 0.05. Groups were control (n = 6), saline + swim stress (n = 7), and 10 mg/kg fluoxetine + swim stress (n = 7). Abbreviations as in Figure 1.

Further examination of the DRN at the subregional level (Figure 2B) revealed a statistically significant effect of both region (F(2,34) = 5.884, p = 0.006) and treatment (F(2,17) = 5.721, p = 0.013). Although the region × treatment interaction was not statistically significant (F(4,34) = 1.512, p = 0.221), likely due to the small sample size, posthoc testing was deemed justified based on previous evidence and our a priori hypothesis of preferential involvement of ventral DRN neurons in stress coping (see the Introduction section). Posthoc analysis showed a statistically significant increase in c-Fos immunoreactive neurons in the ventral DRN of swim-stressed mice compared to non-stressed controls (p = 0.002; Figures 2B and 1B) but non-significant effects in the dorsal DRN (p = 0.181) and lateral wings (p = 0.520). Pretreatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine (10 mg/kg i.p.) prevented stress-induced c-Fos expression in the ventral DRN (posthoc p = 0.028; Figure 2B). Additionally, during swim stress, fluoxetine-treated mice spent more time climbing, a measure of active coping (Mann–Whitney U = 6, p = 0.016; Supporting Information Figure 1; see later for further discussion).

Swim Stress Increased c-Fos Expression in DRN Neurons Co-expressing TPH2 and VGLUT3

Next, we investigated whether swim stress increased c-Fos immunoreactivity specifically in 5-HT-glutamate co-releasing neurons, using the same sections examined for c-Fos alone. Previous studies have revealed that VGLUT3-expressing neurons in the midbrain raphe nuclei comprise two subpopulations, one colocalizing a 5-HT marker and another only expressing VGLUT3.16,27 Here, the 5-HT-specific marker tryptophan hydroxylase 2 (TPH2) was used to distinguish these two populations (Figure 3A). In agreement with these earlier studies, somatic VGLUT3 expression was particularly evident in TPH2 immunoreactive neurons located in the ventral DRN; thus, 67.9 ± 3.04% of TPH2 immunoreactive neurons co-expressed VGLUT3 (Supporting Information Figure 2). In comparison, only sparse VGLUT3 expression was observed in TPH2 immunoreactive neurons in the dorsal DRN and lateral wings. Neurons with colocalized VGLUT3 and TPH2 were evident in the MRN although these neurons were less abundant than in the DRN; thus, in the MRN, 34.9 ± 2.9% of TPH2 immunoreactive neurons also expressed VGLUT3 (Supporting Information Figure 2).

Figure 3.

Figure 3

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Effect of swim stress, with or without fluoxetine, on c-Fos expression in neurons co-expressing TPH2 and VGLUT3 in the ventral DRN. (A) Representative image of c-Fos/TPH2/VGLUT3 triple-labeled neurons in the ventral DRN (AP= −4.6 mm). (B) Effect of swim stress on the number of c-Fos/TPH2 double-labeled neurons (left), c-Fos/TPH2/VGLUT3 triple-labeled neurons (middle), and c-Fos/TPH2 double-labeled neurons but VGLUT3 immunonegative (right). Columns represent the mean ± SEM values, with individual values indicated by closed circles. *p < 0.05. Groups were control (n = 6), saline + swim stress (n = 7), and 10 mg/kg fluoxetine + swim stress (n = 7). Abbreviations as in Figure 1.

Importantly, swim stress increased the number of c-Fos/TPH2/VGLUT3 triple-labeled neurons in the ventral DRN compared to non-stressed controls (F(2,17)= 4.896, p = 0.021; posthoc p = 0.036; Figure 3B). This effect of swim stress amounted to an increase in c-Fos in 32.3 ± 7% of TPH2/VGLUT3 immunoreactive neurons in the ventral DRN. Furthermore, compared to saline controls, pretreatment with fluoxetine prevented the stress-induced increase in c-Fos immunoreactivity in TPH2/VGLUT3 co-expressing neurons (posthoc p = 0.042; Figure 3B).

Swim stress also significantly increased the number of c-Fos/TPH2 double-labeled neurons in the ventral DRN (F(2,17) = 5.535, p = 0.014; posthoc p = 0.034) compared to non-stressed controls (26.1 ± 2.8% of TPH2 immunoreactive neurons), and this effect was also reduced by fluoxetine (F(2,17) = 5.535, p = 0.014; posthoc p = 0.023; Figure 3B). TPH2 immunoreactive neurons that were immunonegative for VGLUT3 did not show increased c-Fos expression in response to swim stress (F(2,17)= 2.115, p = 0.151; Figure 3B). The number of TPH2 immunoreactive neurons did not differ between groups (Supporting Information Figure 3A).

In comparison to the ventral DRN, swim stress had no significant effect on the number of c-Fos/TPH2/VGLUT3 triple-labeled neurons in the MRN compared to nonstressed controls (F(2,15) = 2.845, p = 0.09; Supporting Information Figure 4). Swim stress also did not significantly affect the number of c-Fos/TPH2/VGLUT3 triple-labeled neurons in the dorsal DRN (F(2,15) = 3.559, p = 0.054, trend effect driven by saline vs fluoxetine; Supporting Information Figure 4), adding further evidence that the response of these neurons to stress in the ventral DRN was subregion-specific.

Interestingly, in the MRN, swim stress did not alter the number of either c-Fos/TPH2 neurons (F(2,15) = 1.291, p = 0.304; Supporting Information Figure 4) or c-Fos/TPH2 neurons that were immunonegative for VGLUT3 (F(2,15) = 0.686, p = 0.519; Supporting Information Figure 4), but an increase was detected in the dorsal DRN (F(2,15) = 21.76, p < 0.0001, posthoc p = 0.0001 and F(2,15) = 34.62, p < 0.0001, posthoc p < 0.0001, respectively; Supporting Information Figure 4). These results are in accordance with previous studies showing that swim stress increased c-Fos in 5-HT neurons in the dorsal DRN,8 but our data now suggest that these neurons lack the capacity to co-release glutamate.

To our knowledge, this is the first report of evidence that, in the ventral DRN, 5-HT neurons with the capacity to co-release glutamate are activated by exposure to a stressor, specifically acute swim stress. The inhibitory effect of fluoxetine on this stress-evoked response is in line with electrophysiological evidence that acute SSRI administration inhibits the firing of DRN 5-HT neurons through 5-HT1A autoreceptor-mediated hyperpolarization.28−30

Social Defeat Did Not Evoke c-Fos Expression in DRN Neurons Co-expressing TPH2 and VGLUT3

Previous c-Fos studies report that 5-HT neurons in the DRN are more sensitive to uncontrollable versus controllable stressors.9,11,12,31 Acute swim stress is a well-established inescapable stressor, whereas social defeat is an example of a more controllable stressor. Thus, socially defeated animals adopt a variety of active coping strategies (e.g., flight, corner location, upright submissive postures) to minimize interactions with the opponent.32

We utilized the social defeat model to investigate the sensitivity of VGLUT3-expressing 5-HT neurons to a more controllable stressor. Here, naive intruder mice were exposed to a single episode of social defeat in the home cage of a larger territorially dominant resident. Socially defeated mice were separated from the resident after a single defeat episode that was typically limited to less than 1 min to avoid the stressor from becoming inescapable. The average latency for the resident to attack was 5.1 ± 1.7 s, and the average number of attacks per encounter was 14.9 ± 2.8, i.e., an attack every 3 s involving a combination of biting, kicking, and wrestling, prior to a clear pin down (social defeat). During the encounter, intruder mice spent most of the time moving (90 ± 3.1%) and actively avoiding the resident (distance traveled 3.4 ± 0.8 m).

Region-specific analysis showed that acute social defeat had no effect on the number of c-Fos immunoreactive neurons in the ventral DRN compared to non-stressed controls, and other DRN subregions were similarly unaffected (effect of region: F(1.815,24.50) = 0.822, p = 0.441, effect of treatment: F(1,14) = 0.064, p = 0.804, treatment × region interaction F(2, 27)= 1.123, p = 0.340; Figure 4A). Moreover, the number of c-Fos/TPH2 double-labeled neurons in the ventral DRN was not different across groups (t(13) = 1.158, p = 0.403; Figure 4B). Importantly, and in contrast to swim stress, acute social defeat did not alter the number of c-Fos/TPH2/VGLUT3 triple-labeled neurons in the ventral DRN compared to non-stressed controls (t(13) = 0.732, p = 0.167; Figure 4B).

Figure 4.

Figure 4

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Effect of acute social defeat on c-Fos expression in the DRN, including neurons co-labeled with TPH2 and VGLUT3. (A) C-Fos immunoreactive neurons in DRN subregions. (B) C-Fos/TPH2 double-labeled neurons (left), c-Fos/TPH2/VGLUT3 triple-labeled neurons (middle), and c-Fos/TPH2 double-labeled neurons immunonegative for VGLUT3 (right) in the ventral DRN. Columns represent mean ± SEM values, with individual values indicated by closed circles. Groups were nonstressed controls (n = 8) and social defeat (n = 7). Abbreviations as in Figure 1.

Social defeat also had no effect on c-Fos expression in TPH2 neurons which were VGLUT3 immunonegative (t(13) = 1.167, p = 0.264; Figure 4B), and the number of TPH2 immunoreactive neurons in the ventral DRN was also unchanged (Supporting Information Figure 3B).

The lack of effect of social defeat on c-Fos expression in the DRN is in line with previous studies exposing rodents to a single short (∼3 min) period of social defeat.33,34 Although some studies report that acute social defeat increased c-Fos expression in DRN neurons,35,36 these findings were obtained from animals exposed to the resident over a long period (∼10 min) such that the stressor likely becomes inescapable.33

Thus, the current data suggest that 5-HT neurons with the capacity to co-release glutamate are preferentially activated by an uncontrollable versus controllable stressor. These data agree with previous c-Fos studies reporting that 5-HT neurons are more sensitive to uncontrollable versus controllable footshock,9,31 but extend the findings to 5-HT-glutamate co-releasing neurons. Based on previous experiments involving localized muscimol injections, it was concluded that controllable stressors have less impact on DRN 5-HT neurons due to the inhibitory influence of the medial prefrontal cortex.11 Thus, the greater effect of swim stress versus social defeat on VGLUT3-expressing 5-HT neurons could be explained by the same mechanism.

It could be argued that the lack of effect of social defeat on DRN neurons is due to the strength of the stressor being insufficient. However, social defeat increased c-Fos expression in the periaqueductal gray (PAG). Thus, in socially defeated mice, c-Fos expression increased in the dorsal PAG compared to non-stressed controls (effect of region: F(1,14) = 181.4, p < 0.0001, effect of treatment: F(1,14) = 10.20, p = 0.007, region × treatment interaction: F(1,14) = 8.358, p = 0.012, posthoc p = 0.001; Figure 5A), and there was a trend effect in the ventrolateral region (p = 0.081). In comparison, swim stress also increased c-Fos expression in the dorsal and ventrolateral PAG (effect of region: F(1,10) = 60.77, p < 0.0001, effect of treatment: F(1,10) = 58.78, p < 0.0001, region × treatment interaction: F(1,10) = 5.597, p = 0.04, posthoc p = 0.001 and p < 0.0001; Figure 5B). PAG subregions are well-known to be both activated by stress37 and involved in stress coping.38,39 It is plausible that the preferential activation of the PAG versus the DRN by the controllable stressor could be explained by the DRN having a greater inhibitory influence from the medial prefrontal cortex.

Figure 5.

Figure 5

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Effect of acute social defeat and swim stress on c-Fos immunoreactive neurons in the PAG. (A) C-Fos immunoreactive cells in the PAG following social defeat (n = 7) versus non-stressed controls (n = 8). (B) C-Fos immunoreactive cells in the PAG following swim stress (n = 7) and swim stress with fluoxetine (n = 7) versus non-stressed controls (n = 6). Columns represent mean ± SEM values, with individual values indicated by closed circles. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Mice with VGLUT3-Deficient 5-HT Neurons Showed Increased Climbing during Swim Stress

Finally, we tested the causal role of 5-HT-glutamate co-releasing neurons in stress-coping behavior using genetically modified mice with VGLUT3 deletion targeted to 5-HT neurons (VGLUT3 cKO5-HT25). Specifically, we investigated the response of VGLUT3 cKO5-HT mice to swim stress using climbing as a measure of active coping behavior.40−42 Previous studies have shown this behavior to be increased by SSRI treatment.41,43

First, we confirmed a loss of VGLUT3 in the DRN of VGLUT3 cKO5-HT mice. Initial qPCR analysis demonstrated a 33.9 ± 5.7% reduction of VGLUT3 mRNA in the DRN of VGLUT3 cKO5-HT mice compared to wildtype controls (t(14) = 3.734, p = 0.002; Figure 6B). This effect was selective in that the VGLUT3 cKO5-HT mice did not show altered expression of the vesicular monoamine transporter 2 (VMAT2) (t(14) = 0.366, p = 0.720; Figure 6B), TPH2 (t(14) = 0.532, p = 0.603; Supporting Information Figure 5) and 5-HT1A receptors (t(14) = 0.649, p = 0.527; Supporting Information Figure 5) in the DRN. Then, immunohistochemistry confirmed a selective loss of VGLUT3 expression in DRN 5-HT neurons. Specifically, the number of TPH2/VGLUT3 co-labeled neurons in the ventral DRN of VGLUT3 cKO5-HT mice was reduced by 62.6 ± 4.8% compared to wildtype controls (t(13) = 7.879, p < 0.0001; Figure 6C). The TPH2 immunoreactive neuron count in the ventral DRN was not different between VGLUT3 cKO5-HT mice and wildtype controls (t(13) = 1.365, p = 0.195; Figure 6C), suggesting that the genetic deletion did not impact on the total number of 5-HT neurons.

Figure 6.

Figure 6

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VGLUT3 cKO5-HT mice; molecular characterization and behavioral response to swim stress. (A) Representative image of TPH2/VGLUT3 double-labeled neurons (white arrows) in the ventral DRN of control mice (top) and VGLUT3 cKO5-HT (bottom). (B) VGLUT3 and VMAT2 mRNA in the midbrain raphe region of VGLUT3 cKO5-HT mice and littermate controls. (C) Number of TPH2/VGLUT3 double-labeled neurons (left) and TPH2 neurons (right) in the ventral DRN of VGLUT3 cKO5-HT mice and littermate controls. (D) Performance of VGLUT3 cKO5-HT mice (n = 19–20) and littermate controls (n = 15) during swim stress exposure. Columns are mean ± SEM values, with individual values indicated by closed circles. ****p < 0.0001, **p < 0.01, *p < 0.05.

The incomplete depletion of VGLUT3 may reflect cross-reactivity of our antibody with non-functional VGLUT3 protein fragments that may be transcribed following the conditional knockout. Also, even though the distribution of immunolabeling with this antibody closely matched that of VGLUT3 mRNA reported in previous in situ hybridization studies,27 we cannot exclude the possibility of a low level of non-specific labeling.

Prior to the behavioral testing of VGLUT3 cKO5-HT mice, we first confirmed that pretreatment of wildtype mice with fluoxetine increased time spent climbing when exposed to swim stress (Mann–Whitney U = 6, p = 0.016; Supporting Information Figure 1). This result is in line with previous evidence that the climbing response to swim stress in mice is 5-HT-sensitive, unlike in rats where it is reported that the climbing response is also noradrenaline-dependent.41,43 Perhaps surprisingly, fluoxetine had no effect on time spent immobile (Mann–Whitney U = 15, p = 0.259; Supporting Information Figure 1), but this has also been observed previously.42,44 Although antidepressants normally reduce immobility in this paradigm, the C57BL/6 strain used here is generally less sensitive in this regard.45,46 Moreover, the small swimming chamber dimensions used here are reported to make it difficult to detect changes in immobility behavior.47,48

Interestingly, in parallel with the effects of fluoxetine, when exposed to swim stress, VGLUT3 cKO5-HT mice also spent more time climbing versus littermate controls (Mann–Whitney U = 77, p = 0.042; Figure 6D) without having altered immobility time (Mann–Whitney U = 131.5, p = 0.917; Figure 6D). Breakdown of the climbing data into smaller time bins (2 min) suggested that the VGLUT3 cKO5-HT mice showed persistent climbing over the duration of the experiment, rather than a higher level of climbing compared to their controls (Supporting Information Figure 6). Fluoxetine did not add further to the increase in time spent climbing in the VGLUT3 cKO5-HT mice, potentially because of a ceiling effect. The increase in climbing behavior in the VGLUT3 cKO5-HT mice was not associated with increased locomotor activity in that these mice showed similar levels of locomotion to their littermate controls in a separate locomotor test (effect of genotype: F(1, 34) = 0.344, p = 0.561; interaction: F(1, 34) = 0.800, p = 0.378; Figure 6D).

The increase in climbing behavior exhibited by VGLUT3 cKO5-HT mice is evidence of enhanced escape-driven active coping behavior, which typically characterizes the initial response to swim stress exposure.40 Given our above immunohistochemical evidence that swim stress activates 5-HT-glutamate co-releasing neurons, it seems as if a deficiency in co-released glutamate in VGLUT3 cKO5-HT mice promotes active coping behavior. The predicted lack of co-released glutamate in the VGLUT3 cKO5-HT mice would theoretically shift the 5-HT-glutamate balance at the synapse in favor of 5-HT. Interestingly, fluoxetine, which also increased climbing behavior, would also shift the 5-HT-glutamate balance in favor of 5-HT by selectively inhibiting 5-HT reuptake.49 In other words, a switch in 5-HT-glutamate balance in favor of 5-HT may promote active stress-coping behavior.

The latter idea is consistent with a recent report that chemogenetic activation of ventral DRN-prefrontal cortex projecting 5-HT neurons increased active coping in mice exposed to swim stress.22 Although the latter manipulation might be expected to release both 5-HT and glutamate, electrophysiological evidence from optogenetic studies18 suggests that 5-HT-glutamate co-release is frequency-dependent. Thus, glutamate was found to be preferentially released at lower frequencies (1–2 Hz), whereas 5-HT was preferentially released at higher frequencies (10–20 Hz). Therefore, chemogenetic activation may have preferentially released 5-HT resulting in increased active coping. Conversely, conditional TPH2 knockout from the same ventral DRN 5-HT neurons was found to increase immobility, supporting the hypothesis of the requirement for 5-HT in stress coping. Taken together, the evidence suggests that an altered balance of 5-HT-glutamate in favor of 5-HT (i.e., away from glutamate and toward 5-HT-signaling pathways) may increase active coping and might therefore play a critical role in the behavioral response to stress.

A caveat of this hypothesis is the current lack of consensus regarding the mechanisms by which glutamate is co-released from 5-HT synapses.50 The frequency-dependent nature of co-released glutamate and 5-HT evident in optogenetic studies18 indicates that 5-HT and glutamate are released from different vesicular pools. On the other hand, co-release from the same vesicular pools has also been suggested based on synergism between VGLUT3 and VMAT2.50 In the latter scenario, VGLUT3 would promote vesicular loading of 5-HT,51 in which case a reduction of VGLUT3 expression may decrease the vesicular content of both glutamate and 5-HT. Although this suggests that a loss of VGLUT3 in the VGLUT3 cKO5-HT mice might disrupt the balance of glutamate-5-HT co-release less than expected, it is difficult to reconcile an increase in stress coping with an overall decrease in release of 5-HT in these animals (e.g., see ref (22)). A further caveat is that the VGLUT3 cKO5-HT mice may have changes in 5-HT neuronal function, other than altered glutamate co-release, that contribute to altered stress coping in these animals. However, in these mice we found no changes in other markers of 5-HT neuronal function in the DRN, specifically mRNA encoding VMAT2, TPH2, and 5-HT1A receptors.

The theory that a shift in balance of 5-HT-glutamate in favor of 5-HT increases coping would have implications in situations where this balance is altered, for example by environmental or genetic factors affecting the expression of VGLUT3 (but also VMAT2 or SERT). Interestingly, there is evidence that the level of 5-HT-glutamate co-release may not be fixed but rather is plastic. For instance, changes in VGLUT3 expression in 5-HT neurons have been reported in rats exposed to chronic stress52 as well as during acquisition of generalized fear following acute stress.53 More generally, VGLUT3 expression is reported to vary during neurodevelopment and early postnatal life,54,55 and point mutations of the gene encoding VGLUT3 (Slc17a8) may result in a life-long alteration in VGLUT3 expression.56 If the latter changes in VGLUT3 expression occur in 5-HT neurons and affect the balance of 5-HT-glutamate at the synapse, the present data suggest that they could impact coping strategies and susceptibility to stress.

Materials and Methods

Animals

Mice were group-housed (2–6 per cage) with littermates in individually ventilated cages in a temperature-controlled room (21 °C) with a 12 h light/dark cycle. Mice had ad libitum access to food and water, and cages were lined with sawdust bedding and contained cage enrichment (sizzle nests and cardboard tube). Experiments were conducted during the light phase. Both female and male mice were used, except for the social defeat experiment which necessarily involved only males. Before each experiment, mice were habituated to handling using a cardboard tunnel to minimize background stress.57

Most experiments utilized either C57BL/6J (Charles River, age 8–10 weeks) or transgenic mice with conditional VGLUT3 deletion targeted to 5-HT neurons (SERT-Cre::vGLUT3LoxP/LoxP, C57BL/6J background, aged 8–17 weeks). The transgenic mice were generated by crossing VGLUT3loxP/loxP mice (carrying a floxed allele of the exon 2 of Slc17a8) with a serotonin transporter (SERT)-Cre line.25 SERT-Cre::VGLUT3LoxP/LoxP were compared to control littermates (SERT+/+::VGLUT3LoxP/LoxP or WT). Retired male breeder CD1 mice (Charles River, age 22–30 weeks) were employed as resident aggressor mice for the social defeat experiments.

Experiments followed the principles of the ARRIVE guidelines and were conducted according to the UK Animals (Scientific Procedures) Act of 1986 with appropriate personal and project license coverage.

Swim Stress Paradigm

Mice were randomly allocated to 1 of 3 experimental groups by stratified randomization: (i) saline, (ii) saline + swim stress, and (iii) fluoxetine (10 mg/kg) + swim stress. Mice were removed from their home cages and single-housed in a clean cage before and after undergoing single exposure to swim stress. Saline or fluoxetine was injected i.p. 30 min prior to a swim stress.

During the last 5 min prior to swim stress mice were placed in a clean but familiar cage, and their locomotor activity was recorded via an overhead camera for offline tracking using ANY-maze (Stoelting Europe) tracking software.

For swim stress, mice were placed individually for 6 min in a glass cylinder (height 25 cm, diameter 12 cm) containing water (height 20 cm) maintained at 20 °C, as described previously.58,59 A video camera was mounted in front of the cylinder, and recordings were used for offline manual scoring by an experimenter blind to treatment. Climbing and immobility were timed during the final 4 min of stress exposure. Climbing was defined as placement of the front paws on the glass walls of the cylinder above the water level,40,41 while immobility was rated as the absence of escape-oriented behaviors. After the test, the animals were towel-dried and placed in a heated cage until dry.

Ninety min after swim stress mice were deeply anesthetized prior to perfusion and collection of brain tissue for c-Fos immunohistochemistry (Figure 7). This time scale was chosen to allow for optimum c-Fos expression before tissue collection.60

Figure 7.

Figure 7

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Experimental timeline. (A) Timeline of swim stress (top) and social defeat (below) experiments. Abbreviations: fluoxetine (FLX) and dorsal raphe nucleus (DRN). Created with BioRender.com.

Social Defeat Paradigm

Male mice (C57BL/6J) were randomly allocated to two experimental groups by stratified randomization: (i) control and (ii) social defeat. On the day of social defeat mice were removed from their home cage and single-housed in a clean but familiar cage. Control mice remained in the clean cage for 90 min.61 In the “social defeat” condition, an intruder mouse was placed in the home cage of a territorially dominant, aggressive resident mouse and subject to brief social defeat (as defined below). The intruder was then separated from the resident by a perforated acrylic partition, which allowed auditory, visual, and olfactory interaction with the resident but no physical contact.61 After 90 min, mice were deeply anesthetized and perfused (see below). The resident–intruder interaction was recorded with an overhead camera for offline behavioral analysis using ANY-maze software (Stoelting Europe).

Resident Mouse Training and Selection

Resident mice were selected based on a persistent level of aggression as previously described.61 Briefly, on 3 consecutive days, an intruder mouse was placed in the cage of a resident mouse for up to 3 min or until the latter was “socially defeated”. Social defeat was defined as a clear pin down and/or a supine posture of the intruder. Each resident mouse interacted with a different intruder mouse daily. All interactions were filmed, and video analysis of the latency to attack and the number of attacks allowed the selection of resident mice that consistently attacked within the first 20 s of the resident–intruder interaction.

Immunohistochemistry and Microscopy

Mice were deeply anesthetized by i.p. injection with sodium pentobarbital (90 mg/kg; Euthatal) and intracardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were then dissected, postfixed by immersion in the same fixative for 48 h, cryoprotected in PBS containing 30% sucrose, and frozen at −80 °C until sectioning.

Cryostat-cut coronal brain sections (30 μm; Bright LOFT cryostat) were taken at the level of the DRN (Bregma: −4.6,26Figure 1A) and stored in antifreeze (30% glycerol, 30% ethylene glycol, in PBS) at −20 °C prior to processing for immunohistochemistry as previously described.62 In brief, sections were incubated overnight with the following primary antibodies: rabbit anti-c-Fos (1:1000, Abcam), goat anti-TPH2 (1:1000, Abcam), and guinea pig anti-VGLUT3 (1:500 dilution, Synaptic Systems). The secondary antibodies used for protein visualization were the following: rabbit AF488 (1:1000, Invitrogen), guinea pig Cy3 (1:1000, Jackson Immune Research), and goat AF647 (1:1000, Abcam). Cell nuclei were stained by using DAPI (1:1000, 5 min).

Images were visualized using an epi-fluorescent microscope (Olympus BMAX BX40) and acquired with ImageJ Micromanager v1.4 (500 ms exposure). Sections were imaged at 20× magnification for the ventral DRN, dorsal DRN and MRN, and at 10× for the entire DRN, lateral wings, and ventrolateral and dorsal PAG.26 Cell counting and quantification of colocalization were performed by an experimenter blind to treatment employing the ImageJ Software package.

For each mouse, the mean cell count of 3 sections was used for statistical analysis. C-Fos immunoreactive cells colocalized with DAPI immunoreactivity were defined as neurons. Colocalization of DAPI and TPH2 immunoreactivity identified 5-HT neurons, while colocalization of TPH2 and VGLUT3 identified 5-HT-glutamate co-releasing neurons.

Drugs

Fluoxetine hydrochloride (Stratech A2436-APE) was dissolved in 0.9% sodium chloride at 2 mg/mL and administered i.p. at a dose of 10 mg/kg. Control mice received saline in a volume of 2 mL/kg. All solutions were prepared fresh daily. Fluoxetine dose and administration protocol were based on previous studies.45,46

qPCR Analysis

For PCR analysis, the midbrain raphe region was dissected from frozen tissue sections (1 mm). RNA was extracted (Qiagen RNeasy Mini Kit) using the TRIzol method63 and eluted into 20 μL of RNase-free water. DNA conversion and qPCR were conducted as described previously.64 In brief, conversion to cDNA was achieved using a high-capacity cDNA reverse transcription kit (Life Technologies) and a T100 thermocycler (Bio-Rad). QPCR was performed (800 ng of RNA) using a LightCycler 480 instrument (Roche Diagnostics) with the following primers (300 nM): VGLUT3 (specifically targeting the exon 2; 5′-CGATGGGACCAATGAAGAGGA-3′ and 5′-CAGTCACAGACAGGGCGATG-3′), VMAT2 (5′-CATCACGCAGACTTGAAAGAC-3′ and 5′-CGCCTCGCCTTGCTTATCC-3′),65 TPH2 (5′-CAGGGTCGAGTACACAGAAG-3′ and 5′- CTTTCAGAAACATGGAGACG-3′)66 and 5-HT1A receptors (5′-GACAGGCGGCAACGATACT-3′ and 5′- CCAAGGAGCCGATGAGATAGTT-3′).67 GAPDH was used as the reference gene (Santa Cruz Biotechnology). Reactions (384 well-plates, 10 μL reaction volume, 5 μL PrecisionPLUS qPCR Master Mix with SYBRgreen, 25 ng cDNA) used the following cycle: enzyme activation for 2 min at 95 °C, 40 cycles of 10 s at 95 °C, 1 min at 60 °C, then held at 4 °C. Samples were run in triplicate and 2–ΔΔCT was calculated for each sample, where ΔCT = CTtarget gene – CTreference gene. Data were analyzed as fold-change in gene expression relative to the control group.

Statistical Analysis

The Shapiro–Wilk test for normality was applied to all data sets. If data were normally distributed, then the t-test and one-way or two-way ANOVA were used followed by Tukey’s or Šídák’s posthoc tests as appropriate. Specifically, when c-Fos data was analyzed across multiple regions, repeated-measures two-way ANOVA was employed for balanced data, whereas a repeated-measure mixed-effect model was used for data sets with missing values. If the data were non-parametric, then a single or multiple Mann–Whitney test was employed, with Holm–Šídák correction for multiple comparisons. GraphPad Prism was used for all analysis and plotting of graphs. Data are presented as mean ± standard error of the mean (SEM) values; p < 0.05 was considered statistically significant.

Acknowledgments

L.S.G. was supported by a studentship from the Oxford-MRC Doctoral Training Programme (grant ref MR/N013468/1). H.M.C. was supported by a Wellcome Trust Ph.D. Studentship in Basic Science (grant no. 219982/Z/19/Z).

Glossary

Abbreviations

5-HT

5-hydroxytryptamine

SSRI

selective serotonin reuptake inhibitor

FLX

fluoxetine

DRN

dorsal raphe nucleus

MRN

median raphe nucleus

PAG

periaqueductal gray

TPH2

tryptophan hydroxylase 2

VGLUT3

vesicular glutamate transporter 3

VMAT2

vesicular monoamine transporter

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.3c00758.

  • Behavior of wildtype mice exposed to swim stress with and without fluoxetine. Colocalization of TPH2 and VGLUT3 in neurons of mouse raphe regions. TPH2 expression in the ventral DRN of mice exposed to swim stress and social defeat. Effect of swim stress on c-Fos expression in DRN neurons co-expressing TPH2 and VGLUT3 in the MRN and dorsal DRN. Additional molecular characterization of DRN of VGLUT3 cKO5-HT mice. Behavioral response of VGLUT3 cKO5-HT mice to swim stress in 2 min time bins (PDF)

Author Contributions

L.S.G. performed experiments, analyzed the data, and contributed to writing the manuscript. C.F. and P.D. performed ex vivo tissue analysis. H.M.C. contributed to behavioral experiments. S.E.M. contributed to manuscript preparation. T.S. contributed to the conception and design of the work, drafting, and revising the manuscript, and interpretation of data.

The authors declare no competing financial interest.

Special Issue

Published as part of ACS Chemical Neurosciencevirtual special issue “Serotonin Research 2023”.

Supplementary Material

cn3c00758_si_001.pdf (736.8KB, pdf)

References

  1. Chaouloff F.; Berton O.; Mormède P. Serotonin and Stress. Neuropsychopharmacology 1999, 21 (1), 28–32. 10.1038/sj.npp.1395332. [DOI] [PubMed] [Google Scholar]
  2. Cools R.; Roberts A. C.; Robbins T. W. Serotoninergic Regulation of Emotional and Behavioural Control Processes. Trends Cognit. Sci. 2008, 12 (1), 31–40. 10.1016/j.tics.2007.10.011. [DOI] [PubMed] [Google Scholar]
  3. Roche M.; Commons K. G.; Peoples A.; Valentino R. J. Circuitry Underlying Regulation of the Serotonergic System by Swim Stress. J. Neurosci. 2003, 23 (3), 970–977. 10.1523/JNEUROSCI.23-03-00970.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Silveira M. C. L.; Sandner G.; Graeff F. G. Induction of Fos Immunoreactivity in the Brain by Exposure to the Elevated Plus-Maze. Behav. Brain Res. 1993, 56 (1), 115–118. 10.1016/0166-4328(93)90028-O. [DOI] [PubMed] [Google Scholar]
  5. Senba E.; Matsunaga K.; Tohyama M.; Noguchi K. Stress-Induced c-Fos Expression in the Rat Brain: Activation Mechanism of Sympathetic Pathway. Brain Res. Bull. 1993, 31 (3), 329–344. 10.1016/0361-9230(93)90225-Z. [DOI] [PubMed] [Google Scholar]
  6. Hale M. W.; Hay-Schmidt A.; Mikkelsen J. D.; Poulsen B.; Bouwknecht J. A.; Evans A. K.; Stamper C. E.; Shekhar A.; Lowry C. A. Exposure to an Open-Field Arena Increases c-Fos Expression in a Subpopulation of Neurons in the Dorsal Raphe Nucleus, Including Neurons Projecting to the Basolateral Amygdaloid Complex. Neuroscience 2008, 157 (4), 733–748. 10.1016/j.neuroscience.2008.09.050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bonapersona V.; Schuler H.; Damsteegt R.; Adolfs Y.; Pasterkamp R. J.; Van Den Heuvel M. P.; Joëls M.; Sarabdjitsingh R. A. The Mouse Brain after Foot Shock in Four Dimensions: Temporal Dynamics at a Single-Cell Resolution. Proc. Natl. Acad. Sci. U.S.A. 2022, 119 (8), e2114002119 10.1073/pnas.2114002119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kelly K. J.; Donner N. C.; Hale M. W.; Lowry C. A. Swim Stress Activates Serotonergic and Nonserotonergic Neurons in Specific Subdivisions of the Rat Dorsal Raphe Nucleus in a Temperature-Dependent Manner. Neuroscience 2011, 197, 251–268. 10.1016/j.neuroscience.2011.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Amat J.; Matus-Amat P.; Watkins L. R.; Maier S. F. Escapable and Inescapable Stress Differentially and Selectively Alter Extracellular Levels of 5-HT in the Ventral Hippocampus and Dorsal Periaqueductal Gray of the Rat. Brain Res. 1998, 797, 12–22. 10.1016/S0006-8993(98)00368-0. [DOI] [PubMed] [Google Scholar]
  10. Lee E. H. Y.; Lin H. H.; Yin H. M. Differential Influences of Different Stressors upon Midbrain Raphe Neurons in Rats. Neurosci. Lett. 1987, 80, 115–134. 10.1016/0304-3940(87)90506-4. [DOI] [PubMed] [Google Scholar]
  11. Amat J.; Baratta M. V.; Paul E.; Bland S. T.; Watkins L. R.; Maier S. F. Medial Prefrontal Cortex Determines How Stressor Controllability Affects Behavior and Dorsal Raphe Nucleus. Nat. Neurosci. 2005, 8 (3), 365–371. 10.1038/nn1399. [DOI] [PubMed] [Google Scholar]
  12. Maier S. F.; Watkins L. R. Stressor Controllability and Learned Helplessness: The Roles of the Dorsal Raphe Nucleus, Serotonin, and Corticotropin-Releasing Factor. Neurosci. Biobehav. Rev. 2005, 29 (4–5), 829–841. 10.1016/j.neubiorev.2005.03.021. [DOI] [PubMed] [Google Scholar]
  13. Johnson M. D. Synaptic Glutamate Release by Postnatal Rat Serotonergic Neurons in Microculture. Neuron 1994, 12 (2), 433–442. 10.1016/0896-6273(94)90283-6. [DOI] [PubMed] [Google Scholar]
  14. Schäfer M.-H.; Varoqui H.; Defamie N.; Weihe E.; Erickson J. D. Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons. J. Biol. Chem. 2002, 277 (52), 50734–50748. 10.1074/jbc.M206738200. [DOI] [PubMed] [Google Scholar]
  15. Gras C.; Herzog E.; Bellenchi G. C.; Ronique Bernard V.; Ravassard P.; Pohl M.; Gasnier B.; Giros B.; Mestikawy S. El. A Third Vesicular Glutamate Transporter Expressed by Cholinergic and Serotoninergic Neurons. J. Neurosci. 2002, 22 (13), 5442–5451. 10.1523/JNEUROSCI.22-13-05442.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fremeau R. T.; Burman J.; Qureshi T.; Tran C. H.; Proctor J.; Johnson J.; Zhang H.; Sulzer D.; Copenhagen D. R.; Storm-Mathisen J.; Reimer R. J.; Chaudhry F. A.; Edwards R. H. The Identification of Vesicular Glutamate Transporter 3 Suggests Novel Modes of Signaling by Glutamate. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (2), 14488–14493. 10.1073/pnas.222546799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Varga V.; Losonczy A.; Zemelman B. V.; Borhegyi Z.; Nyiri G.; Domonkos A.; Hangya B.; Holderith N.; Magee J. C.; Freund T. F. Fast Synaptic Subcortical Control of Hippocampal Circuits. Science 2009, 326 (5951), 449–453. 10.1126/science.1178307. [DOI] [PubMed] [Google Scholar]
  18. Sengupta A.; Bocchio M.; Bannerman D. M.; Sharp T.; Capogna M. Control of Amygdala Circuits by 5-HT Neurons via 5-HT and Glutamate Cotransmission. J. Neurosci. 2017, 37 (7), 1785. 10.1523/JNEUROSCI.2238-16.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu Z.; Zhou J.; Li Y.; Hu F.; Lu Y.; Ma M.; Feng Q.; Zhang J. en.; Wang D.; Zeng J.; Bao J.; Kim J. Y.; Chen Z. F.; ElMestikawy S.; Luo M. Dorsal Raphe Neurons Signal Reward through 5-HT and Glutamate. Neuron 2014, 81 (6), 1360–1374. 10.1016/j.neuron.2014.02.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wang H. L.; Zhang S.; Qi J.; Wang H.; Cachope R.; Mejias-Aponte C. A.; Gomez J. A.; Mateo-Semidey G. E.; Beaudoin G. M. J.; Paladini C. A.; Cheer J. F.; Morales M. Dorsal Raphe Dual Serotonin-Glutamate Neurons Drive Reward by Establishing Excitatory Synapses on VTA Mesoaccumbens Dopamine Neurons. Cell Rep. 2019, 26 (5), 1128–1142.e7. 10.1016/j.celrep.2019.01.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Amilhon B.; Lepicard È.; Renoir T.; Mongeau R.; Popa D.; Poirel O.; Miot S.; Gras C.; Gardier A. M.; Gallego J.; Hamon M.; Lanfumey L.; Gasnier B.; Giros B.; El Mestikawy S. VGLUT3 (Vesicular Glutamate Transporter Type 3) Contribution to the Regulation of Serotonergic Transmission and Anxiety. J. Neurosci. 2010, 30 (6), 2198–2210. 10.1523/JNEUROSCI.5196-09.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ren J.; Friedmann D.; Xiong J.; Liu C. D.; Ferguson B. R.; Weerakkody T.; DeLoach K. E.; Ran C.; Pun A.; Sun Y.; Weissbourd B.; Neve R. L.; Huguenard J.; Horowitz M. A.; Luo L. Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-Systems. Cell 2018, 175 (2), 472–487.e20. 10.1016/j.cell.2018.07.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hale M. W.; Shekhar A.; Lowry C. A. Stress-Related Serotonergic Systems: Implications for Symptomatology of Anxiety and Affective Disorders. Cell Mol. Neurobiol. 2012, 32 (5), 695–708. 10.1007/s10571-012-9827-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lowry C. A.; Johnson P. L.; Hay-Schmidt A.; Mikkelsen J.; Shekhar A. Modulation of Anxiety Circuits by Serotonergic Systems. Stress 2005, 8 (4), 233–246. 10.1080/10253890500492787. [DOI] [PubMed] [Google Scholar]
  25. Mansouri-Guilani N.; Bernard V.; Vigneault E.; Vialou V.; Daumas S.; El Mestikawy S.; Gangarossa G. VGLUT3 Gates Psychomotor Effects Induced by Amphetamine. J. Neurochem. 2019, 148 (6), 779–795. 10.1111/jnc.14644. [DOI] [PubMed] [Google Scholar]
  26. Paxinos G.; Franklin K. B. J.. The Mouse Brain in Stereotaxic Coordinates; Academic Press, 2001; Vol. 296. [Google Scholar]
  27. Hioki H.; Nakamura H.; Ma Y. F.; Konno M.; Hayakawa T.; Nakamura K. C.; Fujiyama F.; Kaneko T. Vesicular Glutamate Transporter 3-Expressing Nonserotonergic Projection Neurons Constitute a Subregion in the Rat Midbrain Raphe Nuclei. J. Comp. Neurol. 2010, 518 (5), 668–686. 10.1002/cne.22237. [DOI] [PubMed] [Google Scholar]
  28. Rasmussen K.; McCreary A. C.; Shanks E. A. Attenuation of the Effects of Fluoxetine on Serotonergic Neuronal Activity by Pindolol in Rats. Neurosci. Lett. 2004, 355 (1–2), 1–4. 10.1016/j.neulet.2003.10.039. [DOI] [PubMed] [Google Scholar]
  29. El Mansari M.; Sánchez C.; Chouvet G.; Renaud B.; Haddjeri N. Effects of Acute and Long-Term Administration of Escitalopram and Citalopram on Serotonin Neurotransmission: An in Vivo Electrophysiological Study in Rat Brain. Neuropsychopharmacology 2005, 30 (7), 1269–1277. 10.1038/sj.npp.1300686. [DOI] [PubMed] [Google Scholar]
  30. Gartside S. E.; Umbers V.; Hajós M.; Sharp T. Interaction between a Selective 5-HT1A Receptor Antagonist and an SSRI in Vivo: Effects on 5-HT Cell Firing and Extracellular 5-HT. Br. J. Pharmacol. 1995, 115 (6), 1064–1070. 10.1111/j.1476-5381.1995.tb15919.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Grahn R. E.; Will M. J.; Hammack S. E.; Maswood S.; Mcqueen M. B.; Watkins L. R.; Maier S. F. Activation of Serotonin-Immunoreactive Cells in the Dorsal Raphe Nucleus in Rats Exposed to an Uncontrollable Stressor. Brain Res. 1999, 826, 35–43. 10.1016/S0006-8993(99)01208-1. [DOI] [PubMed] [Google Scholar]
  32. Diaz V.; Lin D. Neural Circuits for Coping with Social Defeat. Curr. Opin. Neurobiol. 2020, 60, 99–107. 10.1016/j.conb.2019.11.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matsuda S.; Peng H.; Yoshimura H.; Wen C.; Fukuda T.; Sakanaka M. Persistent C-Fos Expression in the Brains of Mice with Chronic Social Stress. Neurosci. Res. 1996, 26, 157–170. 10.1016/S0168-0102(96)01088-7. [DOI] [PubMed] [Google Scholar]
  34. Martinez M.; Calvo-Torrent A.; Herbert J. Mapping Brain Response to Social Stress in Rodents with C-Fos Expression: A Review. Stress 2002, 5 (1), 3–13. 10.1080/102538902900012369. [DOI] [PubMed] [Google Scholar]
  35. Numa C.; Nagai H.; Taniguchi M.; Nagai M.; Shinohara R.; Furuyashiki T. Social Defeat Stress-Specific Increase in c-Fos Expression in the Extended Amygdala in Mice: Involvement of Dopamine D1 Receptor in the Medial Prefrontal Cortex. Sci. Rep. 2019, 9 (1), 16670 10.1038/s41598-019-52997-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Gardner K. L.; Thrivikraman K. V.; Lightman S. L.; Plotsky P. M.; Lowry C. A. Early Life Experience Alters Behavior during Social Defeat: Focus on Serotonergic Systems. Neuroscience 2005, 136 (1), 181–191. 10.1016/j.neuroscience.2005.07.042. [DOI] [PubMed] [Google Scholar]
  37. Lino-de-Oliveira C.; de Oliveira R. M. W.; Pádua Carobrez A.; de Lima T. C. M.; Bel E. A. Del.; Guimarães F. S. Antidepressant Treatment Reduces Fos-like Immunoreactivity Induced by Swim Stress in Different Columns of the Periaqueductal Gray Matter. Brain Res. Bull. 2006, 70 (4–6), 414–421. 10.1016/j.brainresbull.2006.07.007. [DOI] [PubMed] [Google Scholar]
  38. Paul E. D.; Johnson P. L.; Shekhar A.; Lowry C. A. The Deakin/Graeff Hypothesis: Focus on Serotonergic Inhibition of Panic. Neurosci. Biobehav. Rev. 2014, 46 (P3), 379–396. 10.1016/j.neubiorev.2014.03.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Deakin J. F. W.; Graeff F. G. 5-HT and Mechanisms of Defence. J. Psychopharmacol. 1991, 5 (4), 305–315. 10.1177/026988119100500414. [DOI] [PubMed] [Google Scholar]
  40. Commons K. G.; Cholanians A. B.; Babb J. A.; Ehlinger D. G. The Rodent Forced Swim Test Measures Stress-Coping Strategy, Not Depression-like Behavior. ACS Chem. Neurosci. 2017, 8 (5), 955–960. 10.1021/acschemneuro.7b00042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Costa A. P. R.; Vieira C.; Bohner L. O. L.; Silva C. F.; Santos E. C. da S.; De Lima T. C. M.; Lino-de-Oliveira C. A Proposal for Refining the Forced Swim Test in Swiss Mice. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 2013, 45, 150–155. 10.1016/j.pnpbp.2013.05.002. [DOI] [PubMed] [Google Scholar]
  42. Perona M. T. G.; Waters S.; Hall F. S.; Sora I.; Lesch K. P.; Murphy D. L.; Caron M.; Uhl G. R. Animal Models of Depression in Dopamine, Serotonin, and Norepinephrine Transporter Knockout Mice: Prominent Effects of Dopamine Transporter Deletions. Behav. Pharmacol. 2008, 19 (5–6), 566–574. 10.1097/FBP.0b013e32830cd80f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Carratalá-Ros C.; López-Cruz L.; Martínez-Verdú A.; Olivares-García R.; Salamone J. D.; Correa M. Impact of Fluoxetine on Behavioral Invigoration of Appetitive and Aversively Motivated Responses: Interaction With Dopamine Depletion. Front. Behav. Neurosci. 2021, 15, 700182 10.3389/fnbeh.2021.700182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Lucki I.; Dalvi A.; Mayorga A. J. Sensitivity to the Effects of Pharmacologically Selective Antidepressants in Different Strains of Mice. Psychopharmacology 2001, 155 (3), 315–322. 10.1007/s002130100694. [DOI] [PubMed] [Google Scholar]
  45. Tang M.; He T.; Meng Q. Y.; Broussard J. I.; Yao L.; Diao Y.; Sang X. B.; Liu Q. P.; Liao Y. J.; Li Y.; Zhao S. Immobility Responses between Mouse Strains Correlate with Distinct Hippocampal Serotonin Transporter Protein Expression and Function. Int. J. Neuropsychopharmacol. 2014, 17 (11), 1737–1750. 10.1017/S146114571400073X. [DOI] [PubMed] [Google Scholar]
  46. Jin Z. L.; Chen X. F.; Ran Y. H.; Li X. R.; Xiong J.; Zheng Y. Y.; Gao N. N.; Li Y. F. Mouse Strain Differences in SSRI Sensitivity Correlate with Serotonin Transporter Binding and Function. Sci. Rep. 2017, 7 (1), 8631 10.1038/s41598-017-08953-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sunal R.; Gümüşel B.; Kayaalp S. O. Effect of Changes in Swimming Area on Results of “Behavioral Despair Test”. Pharmacol., Biochem. Behav. 1994, 49 (4), 891–896. 10.1016/0091-3057(94)90239-9. [DOI] [PubMed] [Google Scholar]
  48. Rosas-Sánchez G. U.; German-Ponciano L. J.; Rodríguez-Landa J. F. Considerations of Pool Dimensions in the Forced Swim Test in Predicting the Potential Antidepressant Activity of Drugs. Front. Behav. Neurosci. 2022, 15, 757348 10.3389/fnbeh.2021.757348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Fischer A. G.; Jocham G.; Ullsperger M. Dual Serotonergic Signals: A Key to Understanding Paradoxical Effects?. Trends Cognit. Sci. 2015, 19 (1), 21–26. 10.1016/j.tics.2014.11.004. [DOI] [PubMed] [Google Scholar]
  50. Trudeau L. E.; El Mestikawy S. Glutamate Cotransmission in Cholinergic, GABAergic and Monoamine Systems: Contrasts and Commonalities. Front. Neural Circuits 2018, 12, 113 10.3389/fncir.2018.00113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. El Mestikawy S.; Wallén-Mackenzie Å.; Fortin G. M.; Descarries L.; Trudeau L.-E. From Glutamate Co-Release to Vesicular Synergy: Vesicular Glutamate Transporters. Nat. Rev. Neurosci. 2011, 12 (4), 204–216. 10.1038/nrn2969. [DOI] [PubMed] [Google Scholar]
  52. Prakash N.; Stark C. J.; Keisler M. N.; Luo L.; Der-Avakian A.; Dulcis D. Serotonergic Plasticity in the Dorsal Raphe Nucleus Characterizes Susceptibility and Resilience to Anhedonia. J. Neurosci. 2020, 40 (3), 569–584. 10.1523/JNEUROSCI.1802-19.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Li H.-Q.; Jiang W.; Ling L.; Gupta V.; Chen C.; Pratelli M.; Godavarthi S. K.; Spitzer N. C.. Generalized Fear Following Acute Stress Is Caused by Change in Co-Transmitter Identity of Serotonergic Neurons bioRxiv 2023, 10.1101/2023.05.10.540268. [DOI]
  54. Gras C.; Vinatier J.; Amilhon B.; Guerci A.; Christov C.; Ravassard P.; Giros B.; El Mestikawy S. Developmentally Regulated Expression of VGLUT3 during Early Post-Natal Life. Neuropharmacology 2005, 49 (6), 901–911. 10.1016/j.neuropharm.2005.07.023. [DOI] [PubMed] [Google Scholar]
  55. Boulland J. L.; Qureshi T.; Seal R. P.; Rafiki A.; Gundersen V.; Bergersen L. H.; Fremeau R. T.; Edwards R. H.; Storm-Mathisen J.; Chaudhry F. A. Expression of the Vesicular Glutamate Transporters during Development Indicates the Widespread Corelease of Multiple Neurotransmitters. J. Comp. Neurol. 2004, 480 (3), 264–280. 10.1002/cne.20354. [DOI] [PubMed] [Google Scholar]
  56. Ramet L.; Zimmermann J.; Bersot T.; Poirel O.; De Gois S.; Silm K.; Sakae D. Y.; Mansouri-Guilani N.; Bourque M. J.; Trudeau L. E.; Pietrancosta N.; Daumas S.; Bernard V.; Rosenmund C.; El Mestikawy S. Characterization of a Human Point Mutation of VGLUT3 (p.A211V) in the Rodent Brain Suggests a Nonuniform Distribution of the Transporter in Synaptic Vesicles. J. Neurosci. 2017, 37 (15), 4181–4199. 10.1523/JNEUROSCI.0282-16.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Gouveia K.; Hurst J. L. Improving the Practicality of Using Non-Aversive Handling Methods to Reduce Background Stress and Anxiety in Laboratory Mice. Sci. Rep. 2019, 9 (1), 20305 10.1038/s41598-019-56860-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Porsolt R. D.; Pichon Le.; Jalfre M.; Depression M.; New A. Animal Model Sensitive to Antidepressant Treatments. Nature 1977, 266 (5604), 730–732. [DOI] [PubMed] [Google Scholar]
  59. Bogdanova O. V.; Kanekar S.; D’Anci K. E.; Renshaw P. F. Factors Influencing Behavior in the Forced Swim Test. Physiol. Behav. 2013, 118, 227–239. 10.1016/j.physbeh.2013.05.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Kovács K. J. Invited Review C-Fos as a Transcription Factor: A Stressful (Re)View from a Functional Map. Neurochem. Int. 1998, 33 (4), 287–297. 10.1016/S0197-0186(98)00023-0. [DOI] [PubMed] [Google Scholar]
  61. Golden S. A.; Covington H. E.; Berton O.; Russo S. J. A Standardized Protocol for Repeated Social Defeat Stress in Mice. Nat. Protoc. 2011, 6 (8), 1183–1191. 10.1038/nprot.2011.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Sengupta A.; Holmes A. A Discrete Dorsal Raphe to Basal Amygdala 5-HT Circuit Calibrates Aversive Memory. Neuron 2019, 103 (3), 489–505.e7. 10.1016/j.neuron.2019.05.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Rio D. C.; Ares M.; Hannon G. J.; Nilsen T. W. Purification of RNA Using TRIzol (TRI Reagent). Cold Spring Harb. Protoc. 2010, 2010 (6), pdb-prot5439 10.1101/pdb.prot5439. [DOI] [PubMed] [Google Scholar]
  64. Radford-Smith D. E.; Probert F.; Burnet P. W. J.; Anthony D. C. Modifying the Maternal Microbiota Alters the Gut–Brain Metabolome and Prevents Emotional Dysfunction in the Adult Offspring of Obese Dams. Proc. Natl. Acad. Sci. U.S.A. 2022, 119 (9), e2108581119 10.1073/pnas.2108581119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Hwang D. Y.; Hong S.; Jeong J. W.; Choi S.; Kim H.; Kim J.; Kim K. S. Vesicular Monoamine Transporter 2 and Dopamine Transporter Are Molecular Targets of Pitx3 in the Ventral Midbrain Dopamine Neurons. J. Neurochem. 2009, 111 (5), 1202–1212. 10.1111/j.1471-4159.2009.06404.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Vogelgesang S.; Niebert S.; Renner U.; Möbius W.; Hülsmann S.; Manzke T.; Niebert M. Analysis of the Serotonergic System in a Mouse Model of Rett Syndrome Reveals Unusual Upregulation of Serotonin Receptor 5b. Front. Mol. Neurosci. 2017, 10, 61 10.3389/fnmol.2017.00061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Gorlova A.; Ortega G.; Waider J.; Bazhenova N.; Veniaminova E.; Proshin A.; Kalueff A. V.; Anthony D. C.; Lesch K. P.; Strekalova T. Stress-Induced Aggression in Heterozygous TPH2Mutant Mice Is Associated with Alterations in Serotonin Turnover and Expression of 5-HT6 and AMPA Subunit 2A Receptors. J. Affective Disord. 2020, 272, 440–451. 10.1016/j.jad.2020.04.014. [DOI] [PubMed] [Google Scholar]

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