Impaired Respiratory and Body Temperature Control Upon Acute Serotonergic Neuron Inhibition

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Science  29 Jul 2011:
Vol. 333, Issue 6042, pp. 637-642
DOI: 10.1126/science.1205295


Physiological homeostasis is essential for organism survival. Highly responsive neuronal networks are involved, but their constituent neurons are just beginning to be resolved. To query brain serotonergic neurons in homeostasis, we used a neuronal silencing tool, mouse RC::FPDi (based on the synthetic G protein–coupled receptor Di), designed for cell type–specific, ligand-inducible, and reversible suppression of action potential firing. In mice harboring Di-expressing serotonergic neurons, administration of the ligand clozapine-N-oxide (CNO) by systemic injection attenuated the chemoreflex that normally increases respiration in response to tissue carbon dioxide (CO2) elevation and acidosis. At the cellular level, CNO suppressed firing rate increases evoked by CO2 acidosis. Body thermoregulation at room temperature was also disrupted after CNO triggering of Di; core temperatures plummeted, then recovered. This work establishes that serotonergic neurons regulate life-sustaining respiratory and thermoregulatory networks, and demonstrates a noninvasive tool for mapping neuron function.

Normal mammalian cell and organism function requires relative constancy around optimal internal physiological conditions. Maintaining this dynamic equilibrium involves neuronal networks affecting numerous brain and body systems. One such homeostatic reflex, the respiratory chemoreflex, controls ventilation in response to deviations in arterial and brainstem pH/Pco2 (partial pressure of CO2) (16). Various classes of brainstem cells (3, 711) have been implicated, with integration and redundancy among components likely essential. Serotonergic neurons of the lower brainstem have been proposed as one critical constituent (3, 1216). They also have been implicated in other homeostatic circuitry, such as the thermoregulatory network for body temperature maintenance (1719). Fatal or life-threatening clinical disorders of homeostatic dysfunction also point to serotonergic involvement, such as in sudden infant death syndrome (SIDS) (20, 21) and serotonin syndrome (22). Direct evidence demonstrating a requirement for serotonergic neurons in homeostasis, however, is only now emerging (15, 18) as tools with sufficient resolving power become available.

To test whether serotonergic neuron activity is critical to homeostatic control, we engineered a genetic tool, allele RC::FPDi (Fig. 1A), for suppressing neuron excitability in conscious mice inducibly, reversibly, with cell-subtype precision, and with minimal invasiveness. Conditional intersectional genetics (2325) were used to activate the expression of the synthetic receptor Di (DREADD, hM4D) (26), a Gi/o protein–coupled receptor (GPCR) with engineered selectivity for the biologically inert synthetic ligand clozapine-N-oxide (CNO) while being refractive to endogenous ligands. Like endogenous Gi/o GPCRs, the binding of CNO by Di can trigger cell-autonomous hyperpolarization and diminished cell excitability (2628). RC::FPDi, a knock-in ROSA26 allele using the CAG promoter (29), offers nonviral means for Di expression, exploiting the dual-recombinase methodology that endows Cre and Flpe transgenics with the potential to activate Di expression in combinatorially defined neuron subtypes, as previously demonstrated for various intersectional alleles (2325).

Fig. 1

Cell-selective Di expression via RC::FPDi. (A) This Gt(ROSA)26Sor knock-in allele consists of CAG regulatory elements, an FRT-flanked transcriptional Stop, a loxP-flanked mCherry-Stop, and HA-tagged Di-encoding sequence. A hypothetical example (left) illustrates intersectional restriction of Di to a serotonergic neuron subset (gray area, lower schema) after Flpe- and Cre-recombination; mCherry marks Flpe-only cells (red areas represent serotonergic nuclei); neither Di nor mCherry are expressed in Cre-only cells (yellow circumscribed area). (B) Derivative Di alleles. (C and D) In RC::PDi, RC::rePe, Slc6a4-cre brainstems, we observed concurrent reproducible expression of HA-tagged Di (far-red indirect HA immunofluorescence), eGFP, and Tph2 (indirect immunofluorescence). DAPI, nuclear stain 4′,6-diamidino-2-phenylindole.

To meet needs for single recombinase–mediated Di expression (versus the dual-recombinase strategy), we generated derivatives of RC::FPDi (Fig. 1B): RC::PDi requires only Cre to activate Di expression because the FRT-cassette was excised in the ancestral germ line; reciprocally, RC::FDi requires only Flpe to activate Di expression. By partnering RC::PDi with Slc6a4-cre, we expressed Di in virtually all serotonergic neurons (Fig. 1, C and D) and a small subset of thalamic neurons. We also incorporated a Cre-responsive green fluorescent protein (GFP) allele, RC::rePe, to visualize the Slc6a4-cre lineage (Fig. 1, C and D), thereby facilitating validation of lineage-specific Di expression (Fig. 1D) as well as electrophysiological study of Di-expressing neurons (Fig. 2).

Fig. 2

Inducible and reversible suppression of serotonergic neuron excitability using RC::PDi. (A and B) Recordings of cultured medullary serotonergic neurons from RC::PDi; RC::rePe; Slc6a4-cre mice showing CNO-induced abolishment (A) or moderate suppression (B) of action potential firing, with recovery after return to aCSF superfusate. (C) Firing rate during sequential applications of 8-OH-DPAT and CNO. (D) Average firing rates (normalized to baseline ± SEM) of Di/GFP-expressing versus control neurons. Inhibition by CNO was observed for Di-neurons compared to pre-CNO, post-CNO aCSF superfusate [aCSF washout (WO) of CNO], and CNO-exposed control neurons, **P < 0.0001 (Friedman test), *P < 0.005 (Mann-Whitney test). CNO-inhibition was comparable to that of 8-OH-DPAT (N = 9 RC::PDi; RC::rePe; Slc6a4-cre neurons; N = 8 control neurons). (E) Sample trace of BaCl2-mediated block of CNO-induced suppression. (F) Average ratio of firing rate for CNO versus pre-CNO, expressed as a percentage ± SEM. Neurons were assayed in aCSF [a and b in (E)] and upon subsequent application of BaCl2 (c and d) *P < 0.05 (Friedman test). (G) Voltage clamp recording showing the current-voltage relationship of a Di-expressing serotonergic neuron with or without CNO. (H) Average current elicited by CNO and 8-OH-DPAT at –60 mV, *P = 0.002 (Mann-Whitney test).

To test Di in modulating serotonergic neuron activity, as driven by RC::PDi, we made current-clamp membrane potential recordings (Fig. 2) from lower brainstem serotonergic neurons cultured from RC::PDi; Slc6a4-cre mice, again coupled with RC::rePe to fluorescently identify Cre-expressing (and thus Di-expressing) neurons. CNO reduced the firing rate of Di-expressing neurons by ~40% on average (Fig. 2D)—a reduction similar to that successively observed in the same neurons after application of 8-OH-DPAT (Fig. 2, C and D), an agonist for the endogenous inhibitory serotonin autoreceptor 5HT1A, which is a GPCR known to act through Gi/o and Kir3 channels to inhibit serotonergic neuron excitability (27, 30). The response onset to CNO occurred on average 1.3 ± 0.1 min (all errors are SEM) after switching the perfusion port from artificial cerebrospinal fluid (aCSF) to CNO, with bath superfusate exchange taking ~1 min; this finding suggested response onset within seconds. Peak responses to CNO occurred 2.8 ± 0.3 min after port switching, thus within ~2 min of CNO exposure. The CNO-induced suppression of action potential firing reversed on return to control superfusate (Fig. 2, A to D), reaching full recovery on average in 12.4 ± 2.9 min (although with notable variation from 0.9 to 25.4 min); these values are loosely correlated with duration of CNO exposure. The CNO-induced suppression could be blocked by the potassium channel inhibitor barium chloride (300 μM) (Fig. 2, E and F), consistent with a Kir3 mechanism of neuron inhibition. There was neuron-to-neuron variation in firing rate suppression not only in response to CNO (0.58 ± 0.07 normalized to baseline values pre-CNO, Fig. 2D) but also to 8-OH-DPAT (0.37 ± 0.09), likely reflecting endogenous heterogeneity among 5-HT neuron subtypes: Not all serotonergic neurons express 5HT1A autoreceptors (31), and they may also not all express Kir3-type channels.

The current-voltage relationships characterizing Di-expressing serotonergic neurons revealed activation, by CNO/Di, of an inwardly rectifying conductance reversing at −78 ± 1.9 mV, which is near the predicted reversal potential for potassium (EK = −81 mV) (Fig. 2G). Responsive neurons (8 of 17 in this assay) demonstrated an increase in slope conductance averaging 2.7 ± 1.1 pS between –110 and –90 mV, and a hyperpolarizing current of 16 ± 3.6 pA at –60 mV (Fig. 2H) ranging between 5 and 45 pA across potentials of –60 to –50 mV—a resting-potential range inclusive of most serotonergic neurons (16, 32). The response to 8-OH-DPAT in the same neurons was similar, with hyperpolarizing currents of 17.7 ± 4.7 pA at –60 mV, ranging from 5 to 85 pA across –50 to –60 mV, and an increase in slope conductance of 4.7 ± 1.3 pS between –110 and –90 mV. Control serotonergic neurons (Fig. 2, D and H) exhibited a similarly robust response to 8-OH-DPAT but not to CNO, indicating that CNO in the absence of Di does not measurably affect membrane conductance or action potential firing rate. Collectively, these findings point to CNO/Di, like 8-OH-DPAT and the 5HT1A autoreceptor, evoking hyperpolarizing outward potassium currents via Kir3-type channels.

Hypercapnic acidosis, a decrease in tissue pH caused by Pco2 elevation from ventilatory dysfunction or cellular metabolism, is a powerful respiratory stimulus. To assess a requirement for serotonergic neuron activity in the CO2-driven respiratory chemoreflex, we measured the ventilatory response of adult RC::PDi; Slc6a4-cre mice and sibling controls to an increase in inspired CO2 from 0% (room air) to 5% (a modest rise) before and after CNO injection (Fig. 3, A to C). The typical increase in ventilation for restoring normal arterial pH/PCO2 levels was reduced by ~50% in RC::PDi; Slc6a4-cre mice within minutes after CNO administration (Fig. 3B). Before CNO, CO2 exposure evoked a ventilatory response comparable to that seen in control siblings, indicating that Di expression alone does not affect the chemoreflex. Nor is CNO alone inhibitory, given the normal ventilatory response of CNO-treated control siblings (Fig. 3C). Similar results were obtained when the Slc6a4-cre driver was replaced with Pet1::Flpe (24), an alternative serotonergic neuron driver, and partnered with RC::FDi (fig. S1), thereby independently confirming attribution of function to serotonergic neurons.

Fig. 3

CNO/Di perturbation of serotonergic neurons disrupts the central respiratory CO2 chemoreflex. (A) Protocol for plethysmographic assessment at 34°C of respiratory responses to inspired CO2 in the awake animal before and after CNO administration; boxes (a, b, c, d) represent points analyzed from continuous recordings. (B) RC::PDi; Slc6a4-cre mice upon CNO administration showed reduced minute ventilation responses to inspired CO2 as compared to pre-CNO baselines, *P = 0.002 [repeated-measures analysis of variance (ANOVA) followed by Tukey post hoc]. (C) Pre-CNO and CNO responses to CO2 were indistinguishable for control mice and from the pre-CNO response of RC::PDi; Slc6a4-cre mice. (D) RC::PDi; Slc6a4-cre mice exhibited reduced CO2 upon CNO administration as compared to controls, *P < 0.05 (paired t test). (E) Firing rate and simultaneous bath pH recordings from cultured serotonergic neurons from controls (top) compared to RC::PDi; RC::rePe; Slc6a4-cre mice (bottom). Changing CO2 from 5% to 9% shifted pH from ~7.4 to ~7.16 and induced an increase in firing rate. In Di neurons (bottom), this response was inhibited by CNO and reversed on return to aCSF. (F) Peak firing rates (normalized to baseline) in 9% CO2-saturated aCSF were suppressed with CNO application in RC::PDi; RC::rePe; Slc6a4-cre neurons, *P < 0.05 (Mann-Whitney test).

Room air ventilation was unaffected after CNO/Di manipulation of Slc6a4-cre–expressing neurons (Fig. 3, A to D). This is reminiscent of phenotypes reported for adult mice in which embryonic deletion of the gene lmx1b in Pet1-expressing cells results in developmental loss of serotonergic neurons (18). Notably, after CNO injection, oxygen consumption in room air dropped from normal to below-normal range (0.053 ± 0.003 ml g–1 min–1 to 0.043 ± 0.003 ml g–1 min–1) in RC::PDi; Slc6a4-cre mice but not in controls (Fig. 3D). Thus, Di-mediated perturbation of Slc6a4-cre–expressing neurons led to a decrease in metabolic rate without proportionately matching effects on ventilation. An additional role for serotonergic neurons may thus be supported: influencing metabolic rate and the ability of ventilation to properly track with metabolic state.

Triggering the chemoreflex requires sensing and transducing milieu changes in pH/Pco2 into cellular activity changes, such as action potential firing rates capable of affecting respiratory circuit output and breathing. We found that cultured serotonergic neurons from the lower brainstem reproducibly increased their firing rate by a factor of 2 to 3 in response to acidosis (0.2 to 0.3 pH units, a magnitude of physiological relevance) caused by increasing the amount of CO2 bubbled in the superfusate from 5 to 9% (Fig. 3E and fig. S2); this is consistent with previous findings in serotonergic neurons from rats (13, 16). About 70% of medullary serotonergic neurons exhibited chemosensitivity, in line with the view that not all medullary serotonergic neurons project to brainstem respiratory centers and that this property likely distinguishes a specific functional subset of serotonergic neurons. Because recordings were performed under conditions of relative synaptic isolation (blockade of GABAA, NMDA, and AMPA/kainate receptors by picrotoxin, AP5,and CNQX, respectively), the observed chemosensitivity may be an intrinsic property of these serotonergic neurons. CNO administration inhibited this chemoresponsiveness [1 μM (fig. S2) or 30 μM (Fig. 3, E and F), concentrations less than or comparable to that delivered in vivo], which could be restored upon return to control superfusate; control, non–Di-expressing serotonergic neurons showed no response to CNO (Fig. 3E, upper panel). CNO was continuously applied over two to three pH cycles for 20 min on average (versus exposure time of ~5 min during firing rate assays) (Fig. 2, A to D); recovery times were longer (76.3 ± 60.7 min) after this extended CNO exposure.

These data support the parsimonious model in which a subset of serotonergic neurons in the lower brainstem act as central respiratory chemoreceptors capable of regulating the downstream respiratory network, and thus lung ventilation, in an attempt to restore normal arterial pH/Pco2 (3, 12, 14, 18, 33). Pco2 stabilization is also served by Phox2b-expressing glutamatergic neurons of the brainstem retrotrapezoid nucleus (34, 35). It will be important to determine how these and likely other (3, 79, 35) neural systems integrate to control breathing, and under what developmental stages, arousal states, and conditions they may be differentially used. Astrocytes along the brainstem surface have also been implicated as respiratory chemosensors, capable of influencing retrotrapezoid neuron activity (11, 36); it will be important to assess whether astrocytes also influence serotonergic neurons and whether their effects on chemoreception persist in conscious animals.

During the whole-body plethysmography (Fig. 3), chamber temperature was held at ~34°C to reduce effects of body temperature fluctuations on respiratory parameters and oxygen consumption; under these conditions, core body temperature remained within ±0.65°C of baseline for controls and Di mice. In the absence of this thermoneutral environment, double-transgenic RC::PDi; Slc6a4-cre mice exhibited a decrease in body temperature within minutes of CNO administration. While housed individually at room temperature (~23°C), RC::PDi; Slc6a4-cre mice exhibited a decrease in body temperature from 36.9° ± 0.2°C to 30.33° ± 0.2°C within 30 min of CNO injection, dipping to 27.1° ± 0.9°C by ~2.5 hours (Fig. 4A), after which recovery ensued with restoration to within normal range by 11.8 ± 1.3 hours. Sibling controls receiving CNO showed normal thermoregulation (37.2° ± 0.03°C). These findings establish that Slc6a4-cre–expressing neuron activity is required for adult thermal homeostasis, with most thermoregulatory capacity lost given the near equilibration of body temperature to ambient room temperature. The kinetics of body temperature change (Fig. 4A) reflect, to a large degree, body thermal inertia, in contrast to the more rapid response observed for individual neurons (Fig. 2). Similar body temperature dysregulation was observed upon replacing Slc6a4-cre; RC::PDi with Pet1::Flpe (24); RC::FDi (fig. S3), thereby independently confirming attribution of function to serotonergic neurons (albeit the extent of temperature dysregulation is less severe, likely because the Pet1::Flpe driver shows mosaicism in recombinase expression in medullary serotonergic neurons). Delineating the effectors of body temperature homeostasis acting downstream of serotonergic circuitry will be an important next step.

Fig. 4

CNO/Di inhibition of serotonergic neurons induced severe yet reversible and repeatable hypothermia. Trials consisted of body temperature assessments taken at room temperature just before a single CNO administration and then every 10 min for the first half hour, followed by every 30 min until recovery. Animals underwent four sequential trials. (A) Body temperature averages of RC::PDi; Slc6a4-cre mice versus controls before and after CNO injection, *P < 0.05 (unpaired t test). (B) Average lowest temperatures achieved per trial, *P < 0.05 (one-way repeated-measures ANOVA). (C) Average duration to return to 36°C after CNO injection, *P < 0.01 (one-way repeated-measures ANOVA).

By contrast to the response evoked upon acute serotonergic neuron inhibition, adult mice devoid of serotonin-producing neurons from mid-gestation onward can maintain a near wild-type body temperature (~36° to 38°C) while housed at room temperature (~24°C) (14, 18). This phenotypic difference indicates that compensatory circuitry arises in the face of developmental loss of serotonergic neurons and highlights how acute neuron perturbation avoids such confounds.

Repeated neuron perturbation in the same animal is possible. With each round of CNO administration, body temperature plummeted within minutes (Fig. 4A); the severity of the induced hypothermia, though, showed modest adaptation with each subsequent trial (Fig. 4B). The time required for return to baseline body temperature also showed adaptation (Fig. 4C). We observed no overt behavioral deficits (short- or long-term) after these bouts of CNO/Di-triggered hypothermia, perhaps not surprisingly given rodents’ capacity for daily torpor.

By using this RC::FPDi strategy, we have revealed impaired respiratory and body temperature control upon acute perturbation of serotonergic neuron activity, providing direct evidence that serotonergic neurons play key roles in the central chemoreflex and thermoregulation. These findings offer potential mechanistic explanation for fatal or life-threatening disorders of homeostasis that associate with serotonin abnormalities, such as in SIDS and the serotonin syndrome. In SIDS, the leading cause of death in children between 1 month and 1 year of age (21), multiple abnormalities in the brainstem serotonergic system have been identified, including serotonin insufficiency (20, 21). Our findings suggest that such insufficiency might compromise an infant’s respiratory response to hypercapnic acidosis that may occur upon rebreathing exhaled air as a result of sleeping prone in the face-down position (SIDS infants are often found prone), contributing to respiratory failure and death. By contrast, serotonin syndrome is a disorder of serotonin excess and extreme hyperthermia, shivering, seizures, coma, and in some cases death that can result acutely from serotonin drug interactions (22). This association between serotonin excess and hyperthermia is consistent with our reciprocal findings of serotonergic inhibition inducing hypothermia. We presume that in vivo CNO/Di signaling suppresses action potential firing, resulting in net inhibition of serotonergic neuron activity. Other scenarios are possible, such as net excitation, but seem improbable given the whole-animal phenotypes observed upon CNO/Di signaling in serotonergic neurons, the molecular mechanism by which Di appears to act, and the paucity of evidence that net excitation could occur. For example, such net excitation has not been observed in cultured serotonergic neurons, nor in extensive studies on 5HT1A receptor agonists in vivo or ex vivo, which act mechanistically like CNO/Di (37, 38). Assessing the precise in vivo electrophysiological effects of CNO/Di signaling on serotonergic neuron activity, including chemoresponsiveness, awaits the means to record from individual medullary serotonergic neurons over the course of hours (see Fig. 3E) in unanesthetized, conscious mice. By contrast, recordings made during either anesthesia (15, 3941) or decerebration (42, 43) suffer from the confound of perturbing serotonergic neuron activity and chemoreflex properties—the very processes under examination.

The present studies, as well as our analyses of broad constitutive Di expression and activity (fig. S4), establish RC::FPDi as a neuronal perturbation tool featuring in vivo ligand inducibility within seconds to minutes, inhibition for minutes to hours, reversibility within hours, intra-animal repeatability, and high cell-subtype selectivity (including anatomically dispersed populations) and thus resolution for functional mapping. This tool can be applied in the awake, freely behaving animal without confounding interference from anesthesia, surgical procedures such as cannulation, or compensatory changes in circuitry that can develop in response to constitutive genetic alterations. RC::FPDi can be applied to map other behaviors in which dysregulation of specific populations has been implicated.

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Figs. S1 to S4

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References and Notes

  1. Acknowledgments: Supported by NIH grants F32HD063257-01A1 (R.R.), 5R21DA023643-02 (R.B., S.D.), 5R21MH083613-02 (R.R., J.C.K., S.D.), 5P01HD036379-13 (R.R., A.C., R.B., J.C.K., E.N., G.R., S.D.), 5R01HL028066-30 (A.C., E.N.), and 5R01HD052772 (G.R.). Clozapine-N-oxide used in some preliminary experiments was provided through the NIH Rapid Access to Intervention Development Program. We thank B. Roth for providing the Di-encoding cDNA; B. Roth, W. Regehr, C. Hull, B. Sabatini, and members of the Dymecki lab for helpful discussions; and Y. Wu and J. J. Mai for technical assistance. The authors declare no conflicts of interest.
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