Impaired chemosensitivity of mouse dorsal raphe serotonergic neurons overexpressing serotonin 1A (Htr1a) receptors

Abstract

Background: Serotonergic system participates in a wide range of physiological processes and behaviors, but its role is generally considered as modulatory and noncrucial, especially concerning life-sustaining functions. We recently created a transgenic mouse line in which a functional deficit in serotonin homeostasis due to excessive serotonin autoinhibition was produced by inducing serotonin 1A receptor (Htr1a) overexpression selectively in serotonergic neurons (Htr1a raphe-overexpressing or Htr1a(RO) mice). Htr1a(RO) mice exhibit episodes of autonomic dysregulation, cardiovascular crises and death, resembling those of sudden infant death syndrome (SIDS) and revealing a life-supporting role of serotonergic system in autonomic control. Since midbrain serotonergic neurons are chemosensitive and are implicated in arousal we hypothesized that their chemosensitivity might be impaired in Htr1a(RO) mice.

Principal findings: Loose-seal cell-attached recordings in brainstem slices revealed that serotonergic neurons in dorsal raphe nucleus of Htr1a(RO) mice have dramatically reduced responses to hypercapnic challenge as compared with control littermates. In control mice, application of 9% CO(2) produced an increase in firing rate of serotonergic neurons (0.260 ± 0.041 Hz, n=20, p=0.0001) and application of 3% CO(2) decreased their firing rate (-0.142 ± 0.025 Hz, n=17, p=0.0008). In contrast, in Htr1a(RO) mice, firing rate of serotonergic neurons was not significantly changed by 9% CO(2) (0.021 ± 0.034 Hz, n=16, p=0.49) and by 3% CO(2) (0.012 ± 0.046 Hz, n=12, p=0.97).

Conclusions: Our findings support the hypothesis that chemosensitivity of midbrain serotonergic neurons provides a physiological mechanism for arousal responses to life-threatening episodes of hypercapnia and that functional impairment, such as excessive autoinhibition, of midbrain serotonergic neuron responses to hypercapnia may contribute to sudden death.

Figure 1. Intrinsic chemosensitive responses of serotonergic DRN neurons are greatly decreased in Htr1a^RO mice.

A, B, Representative loose-seal cell-attached voltage-clamp recordings performed in the presence of synaptic blockers (see results) showing time-courses of serotonergic neuron firing in response to bath application of 9% and 3% CO₂ in slices from control (A) and Htr1a^RO (B) mice. Each panel reports three time-courses from different neurons. In B, one neuron with basal firing rate higher than the average of Htr1a^RO group is shown to illustrate that the lack of responses to CO₂ changes did not depend on basal firing rate of the recorded neuron (see results). Lines show firing rate calculated over 10 s bins. Traces illustrate recorded action currents for each experiment. C, Bar graph of baseline firing rate in the two groups. D, Summary bar graph comparing the effects of 9% and 3% CO₂ in control and Htr1a^RO mice. * p<0.05; *** p<0.001 (Mann-Whitney test). Number of recorded neurons is indicated in parentheses.

Figure 2. In the absence of α₁-adrenoceptor stimulation, 9% CO₂ does not change firing rate of spontaneously active serotonergic neurons in control mice.

A, Time-course of a representative experiment. Phenylephrine was omitted from ACSF containing synaptic blockers. Inset shows the recorded action current. B, Distribution of responses to 9% CO₂ for all recorded neurons.

Figure 3. Decreased chemosensitive responses of serotonergic DRN neurons in Htr1a^RO mice in the absence of synaptic blockade.

A, B, Representative recordings performed in normal phenylephrine-supplemented ACSF showing time-courses of serotonergic neuron firing in response to bath application of 9% and 3% CO₂ in slices from control (A) and Htr1a^RO (B) mice. Lines show firing rate calculated over 10 s bins. Traces illustrate recorded action currents for each experiment. Arrows indicate the application of the Htr1a agonist R-8-OH-DPAT (30 nM) that silenced recorded neurons confirming that they are serotonergic. C, Bar graph of baseline firing rate in control and Htr1a^RO mice. D, Summary bar graph comparing the effects of 9% and 3% CO₂ in two groups. In Htr1a^RO mice the response to 9% CO₂ was significantly reduced when compared to control littermates. ** p<0.01 (Mann-Whitney test). Number of recorded neurons is indicated in parentheses.

Figure 4. Distribution of pooled responses to hypercapnic challenge.

A, Bar graph showing the distribution of responses in control and Htr1a^RO mice. Curves represent best fit of data to a double (Control, R² = 0.901) and single Gaussian function (Htr1a^RO, R² = 0.952). B, Schematic diagram of frontal sections at various rostrocaudal levels of the DRN mouse raphe in which positions of the recorded serotonergic neurons in control (open circles) and Htr1a^RO mice (filled circles) are reported. Numbers correspond to plates in . C, Individual responses to 9% CO₂ application in slices from control (open circles) and Htr1a^RO mice (filled circles) plotted against the rostrocaudal position of the corresponding recorded neuron indicated by the plate number. Continuous and broken lines are best linear regressions of data in control and Htr1a^RO, respectively. D, Individual responses to 9% CO₂ application in slices from control (open circles) and Htr1a^RO (filled circles) plotted against the postnatal age of the mouse at time of recording. Continuous and broken lines are best linear regressions of data in control and Htr1a^RO, respectively.