Conservation of 5-HT1A receptor-mediated autoinhibition of serotonin (5-HT) neurons in mice with altered 5-HT homeostasis

Abstract

Firing activity of serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) is controlled by inhibitory somatodendritic 5-HT1A autoreceptors. This autoinhibitory mechanism is implicated in the etiology of disorders of emotion regulation, such as anxiety disorders and depression, as well as in the mechanism of antidepressant action. Here, we investigated how persistent alterations in brain 5-HT availability affect autoinhibition in two genetically modified mouse models lacking critical mediators of serotonergic transmission: 5-HT transporter knockout (Sert-/-) and tryptophan hydroxylase-2 knockout (Tph2-/-) mice. The degree of autoinhibition was assessed by loose-seal cell-attached recording in DRN slices. First, application of the 5-HT1A-selective agonist R(+)-8-hydroxy-2-(di-n-propylamino)tetralin showed mild sensitization and marked desensitization of 5-HT1A receptors in Tph2-/- mice and Sert-/- mice, respectively. While 5-HT neurons from Tph2-/- mice did not display autoinhibition in response to L-tryptophan, autoinhibition of these neurons was unaltered in Sert-/- mice despite marked desensitization of their 5-HT1A autoreceptors. When the Tph2-dependent 5-HT synthesis step was bypassed by application of 5-hydroxy-L-tryptophan (5-HTP), neurons from both Tph2-/- and Sert-/- mice decreased their firing rates at significantly lower concentrations of 5-HTP compared to wildtype controls. Our findings demonstrate that, as opposed to the prevalent view, sensitivity of somatodendritic 5-HT1A receptors does not predict the magnitude of 5-HT neuron autoinhibition. Changes in 5-HT1A receptor sensitivity may rather be seen as an adaptive mechanism to keep autoinhibition functioning in response to extremely altered levels of extracellular 5-HT resulting from targeted inactivation of mediators of serotonergic signaling.

Keywords: 5-HT1A receptor; autoinhibition; dorsal raphe nucleus; knockout; serotonin transporter; tryptophan hydroxylase-2.

FIGURE 1

Comparison of 5-HT neuron basal firing rates across genotypes. Data are shown as mean ± SD (number of cells shown in parentheses). Analysis of data using Kruskal–Wallis test revealed statistically significant differences among groups [H ₍₄₎ = 16.67, p = 0.002]. Dunn’s multiple comparison post hoc test resulted in being significant for Sert-/- vs. Tph2-/- (**p < 0.01) and wt vs. Tph2-/- (*p < 0.05).

FIGURE 2

Sensitivity of 5-HT neurons to R(+)-8-OH-DPAT differs across genotypes. Time courses of firing rate changes in response to increasing concentrations of R(+)-8-OH-DPAT of individual 5-HT neurons in brain slices obtained from wt (A,B), Tph2-/- (C,D), and Sert-/- mice (E,F). Traces show action current of corresponding neurons recorded. (G) Dots represent log EC₅₀ of concentration–responses from individual experiments. Red lines report mean ± SD of values. One-way ANOVA followed by Tukey’s multiple comparison test showed statistically significant differences across genotypes [F _(4,53) = 48.38, p < 0.0001]. Asterisks indicate level of statistical significance between the indicated genotypes (for Sert-/-, vs. all the other four genotypes): ***p < 0.001, *p < 0.05. (H) Average concentration–response curves obtained from all the experiments. Each data point corresponds to the mean from several neurons (numbers in parentheses). For the sake of clarity, error bars are shown only for Sert-/- mice and Tph2-/- mice in a single direction. Data are normalized on average baseline firing rates recorded before R(+)-8-OH-DPAT application. Note that, curves for Sert-/- mice did not achieve full inhibition of firing (see E).

FIGURE 3

Autoinhibition by endogenous 5-HT synthesized from Trp is conserved across genotypes. (A) Trp (30 μM) produced a 5-HT_1A receptor-mediated decrease in the firing rate of a 5-HT neuron from Sert-/- mice. Application of a selective 5-HT_1A receptor antagonist, WAY-100635 (50 nM; representative of three experiments) fully antagonized Trp effect, confirming that autoinhibition was mediated by 5-HT_1A autoreceptors. (B) Application of 30 μMTrp significantly suppressed firing activity of DRN serotonergic neurons in all the genotypes tested (p < 0.05; Wilcoxon signed rank test). Comparison of the effect of Trp among genotypes revealed no statistically significant differences among genotypes [H ₍₃₎ = 3.336, p = 0.3427; Kruskal–Wallis test]. Data are shown as mean ± SD. The number of neurons recorded for each genotype is shown at the bottom of the histograms.

FIGURE 4

Quantification of autoinhibition capacity of 5-HT neurons across genotypes by concentration–response curves for 5-HTP. Time courses of 5-HT neuron firing rate changes in response to increasing concentrations of 5-HTP in brain slices obtained from wt controls (A,B), Tph2-/- (C,D), and Sert-/- mice (E–H). Traces show action current of corresponding neurons recorded. (I) Dots represent log EC₅₀ of concentration–responses from individual experiments. Red lines report mean ± SD of values. One-way ANOVA followed by Tukey’s multiple comparison test showed statistically significant differences [F _(4,58) = 6.723, p = 0.0002] Asterisks indicate level of statistical significance between the indicated genotypes: ***p < 0.001, **p < 0.01, *p < 0.05. (J) Average concentration–response curves obtained from all the experiments. Each data point corresponds to the mean from several neurons (numbers shown in parentheses). For the sake of clarity, error bars are omitted. Data are normalized on average baseline firing rates recorded before 5-HTP application.