5-HT1A autoreceptor levels determine vulnerability to stress and response to antidepressants

Abstract

Most depressed patients don't respond to their first drug treatment, and the reasons for this treatment resistance remain enigmatic. Human studies implicate a polymorphism in the promoter of the serotonin-1A (5-HT(1A)) receptor gene in increased susceptibility to depression and decreased treatment response. Here we develop a new strategy to manipulate 5-HT(1A) autoreceptors in raphe nuclei without affecting 5-HT(1A) heteroreceptors, generating mice with higher (1A-High) or lower (1A-Low) autoreceptor levels. We show that this robustly affects raphe firing rates, but has no effect on either basal forebrain serotonin levels or conflict-anxiety measures. However, compared to 1A-Low mice, 1A-High mice show a blunted physiological response to acute stress, increased behavioral despair, and no behavioral response to antidepressant, modeling patients with the 5-HT(1A) risk allele. Furthermore, reducing 5-HT(1A) autoreceptor levels prior to antidepressant treatment is sufficient to convert nonresponders into responders. These results establish a causal relationship between 5-HT(1A) autoreceptor levels, resilience under stress, and response to antidepressants.

Figure 1. A transgenic system for suppression of 5-HT_1Areceptors

(a) Mice homozygous for the regulatable tetO-1A allele, with one copy of the β-act-tTS transgene (tetO-1A^+/+ β-act-tTS⁺), express 5-HT_1A receptors in normal patterns in the brain when maintained on doxycycline, assessed by ¹²⁵I-labeled MPPI autoradiography. When maintained in the absence doxycycline, tetO-1A^+/+ β-act-tTS⁺ display no detectable 5-HT_1A receptor expression in the brain. (b) Tissue-specific expression of tTS in serotonergic raphe neurons was accomplished by placing tTS under the control of a 40kB Pet-1 mini-BAC (Pet1-tTS). (c) tetO-1A^+/+ Pet1-tTS⁺ mice were maintained on dox either throughout life (1A-High), or only until postnatal day 50 (approximately 7 weeks of age) (1A-Low). (d), (e) 1A-High and 1A-Low mice express indistinguishable levels of 5-HT_1A heteroreceptors in forebrain areas such as the hippocampus (HPC) and entorhinal cortex (EC), while 1A-Low mice display decreased 5-HT_1A expression in the dorsal (DR) and median (MR) raphe nuclei, assessed by quantitative ¹²⁵I-labeled MPPI autoradiography (N=4 mice; *** P<0.005 (DR), *P<0.05 (MR)). See also Fig S1

Figure 2. Decreased 5-HT_1Aautoreceptor response to agonist in 1A-Low mice

(a) Representative current traces from whole cell recordings in the dorsal raphe of 1A-High and 1A-Low mice in response to the 5-HT_1A agonist 5-CT. (b) Mean outward current amplitude in response to 100nm 5-CT was decreased in 1A-Low mice (N=43 1A-High and 57 1A-Low neurons, ***P<0.001). (c) Recorded neurons were filled with biocytin and processed for TPH. Photomicrographs of the dorsal raphe are shown. (d) Hypothermic response to the 5-HT_1A agonist 8-OH DPAT. In 1A-Low mice, only the 0.5 mg/kg dose caused a significant temperature change relative to the saline control. In 1A-High mice, both the 0.1 mg/kg and the 0.5 mg/kg doses elicited significantly larger temperature changes relative to control (N=4-5/dose/group; *P<0.05 and ***P<0.001 for a main effect of dose). See also Table S1

Figure 3. Increased spontaneous neuronal activity in the dorsal raphe of 1A-Low mice

Histograms depicting distribution of spontaneous firing rates for individual neurons in an in vivo anesthetized preparation of 1A-High and 1A-Low animals, with averaged action potential traces inset. The distributions are significantly different (N= 20 and 21 neurons respectively; two-tailed Mann Whitney test; P=0.0057).

Figure 4. No change in anxiety-like behavior, but altered response in stress/depression-related tests in 1A-Low mice

(a) No group differences were detected in the total exploration (i) or time spent in the center (ii) of the open field (N=21/group). (b) No group differences were detected in percent time spent in the light (i) or total path (ii) in the light/dark choice test (N=19 and 21/group). (c) 1A-High mice displayed an attenuated Stress-Induced Hyperthermic response to novel cage stress, compared to 1A-Low mice (N=11/group; ***P<0.0001). Although no differences were detected between the groups in mobility in the Tail Suspension Test (d) (N=25-26 mice/group), 1A-Low mice displayed increased mobility compared to 1A-High mice across a two-day Forced Swim Test (e) (repeated measures ANOVA across all time points, group by time interaction F_3,43=4.535, P=0.0047). Only 1A-High mice displayed decreased mobility over time on the second day of testing, and 1A-Low mice were more mobile in the final testing block (N=21, 22/group; ANOVA, between group minutes 5-6, F_1,41 = 3.953, #P= 0.0535, *P<0.05). See also Fig. S2.

Figure 5. 1A-High mice display a less active behavioral response in stressful paradigms following a repeated mild stressor

Following four weeks of a daily mild stressor, 1A-High and 1A-Low mice displayed indistinguishable behavior in the Open Field Paradigm (a) (N=13-14 mice/group), and 1A-Low mice retained a more robust temperature increase in response to novel cage stress (b) (N=6/group, *P<.05), similar to naive mice. However, after repeated stress, 1A-High mice displayed less mobility than 1A-Low mice in the Tail Suspension Test (c) (N=13-14 mice/group; *P=0.0445), and less mobility over time in a single exposure to the Forced Swim Test (d) (N=13-14 mice/group, **P=.004).

Figure 6. Robust response to fluoxetine treatment in the novelty-suppressed feeding test in 1A-Low, but not 1A-High mice

(a) and (b) 1A-High mice treated for 8 days with fluoxetine (18mg/kg/day p.o.) display no difference in latency to consume a food pellet in the middle of an aversive arena as compared to 1A-High mice treated with vehicle, while 1A-Low mice treated with fluoxetine for 8 days display a shorter latency than their vehicle controls. (c), (d) Continuation of fluoxetine treatment for 26 days failed to decrease the latency to feed in 1A-High mice, while 1A-Low mice still displayed a shorter latency to feed than their vehicle controls (N=11-13/treatment/group; *P<0.05, **P<0.01 by Mantel-Cox log-rank test). See also Fig. S3.

Figure 7. Increased serotonin levels in response to fluoxetine in 1A-Low mice

Extracellular serotonin levels measured by in vivo microdialysis in the (a) ventral hippocampus, and (c) prefrontal cortex of naïve 1A-High and 1A-Low mice, following acute challenge with fluoxetine (18mg/kg, i.p.) or saline. Total extracellular 5-HT, measured by area-under-the-curve analysis, increases in the (b) ventral hippocampus, and (d) prefrontal cortex of both 1A-High and 1A-Low mice in response to acute fluoxetine treatment. 1A-Low mice display a larger increase in extracellular 5-HT than 1A-High mice in both brain areas (N=6-9 mice/group; *P<0.05, **P<0.01, ***P<0.001).

Figure 8. Loss of 5-HT_1A agonist response in 1A-High mice treated with chronic fluoxetine

(a, i and ii) 1A-High mice treated with vehicle for 35 days display a robust hypothermic response to 0.5mg/kg 8-OH DPAT challenge, while those treated with fluoxetine for 35 days do not, demonstrating full desensitization of 5-HT_1A autoreceptors in 1A-High mice after fluoxetine treatment (N=3/group/treatment/dose; **P<0.01, main effect of dose; P=0.002, dose by time interaction; *P<0.05, **P<0.01, ***P<0.001 for between dose comparisons at each timepoint). (b, i and ii) 1A-Low mice treated with vehicle display a similarly attenuated response to those treated with fluoxetine, consistent with decreased 5-HT_1A autoreceptor levels and function (N=3/group/treatment/dose; P<0.05, dose by time interaction) in these mice.

Figure 9. Model of 5-HT_1A autoreceptor effects on the serotonergic raphe

Diagram depicts representative raphe neurons in 1A-High and 1A-Low animals, emphasizing the differences between the two groups. 1A-High mice have lower basal firing rate (indicated above the cell) and high levels of somatodendritic 5-HT_1A autoreceptor, which exert robust inhibitory effects on raphe firing. This results in increased behavioral despair in response to stress, compared to 1A-Low mice. Conversely, 1A-Low mice have higher basal firing rate and low levels of somatodendritic 5-HT_1A autoreceptors, which exert less inhibitory control over raphe firing rates. This results in less behavioral despair in response to stress, compared to 1A-High mice. While 1A-High mice do not respond behaviorally to treatment with the antidepressant fluoxetine, 1A-Low mice display a robust behavioral response. 1A-High and 1A-Low mice provide a mechanistic model for humans carrying, respectively, the G/G and C/C alleles of the Htr1aI C(-1019)G polymorphism.