Enrica Audero

Identifying Molecular Substrates in a Mouse Model of the Serotonin Transporter × Environment Risk Factor for Anxiety and Depression

BACKGROUND A polymorphism in the serotonin transporter (5-HTT) gene modulates the association between adverse early experiences and risk for major depression in adulthood. Although human imaging studies have begun to elucidate the neural circuits involved in the 5-HTT x environment risk factor, a molecular understanding of this phenomenon is lacking. Such an understanding might help to identify novel targets for the diagnosis and therapy of mood disorders. To address this need, we developed a gene-environment screening paradigm in the mouse. METHODS We established a mouse model in which a heterozygous null mutation in 5-HTT moderates the effects of poor maternal care on adult anxiety and depression-related behavior. Biochemical analysis of brains from these animals was performed to identify molecular substrates of the gene, environment, and gene x environment effects. RESULTS Mice experiencing low maternal care showed deficient gamma-aminobutyric acid-A receptor binding in the amygdala and 5-HTT heterozygous null mice showed decreased serotonin turnover in hippocampus and striatum. Strikingly, levels of brain-derived neurotrophic factor (BDNF) messenger RNA in hippocampus were elevated exclusively in 5-HTT heterozygous null mice experiencing poor maternal care, suggesting that developmental programming of hippocampal circuits might underlie the 5-HTT x environment risk factor. CONCLUSIONS These findings demonstrate that serotonin plays a similar role in modifying the long-term behavioral effects of rearing environment in diverse mammalian species and identifies BDNF as a molecular substrate of this risk factor.

Sporadic Autonomic Dysregulation and Death Associated with Excessive Serotonin Autoinhibition

Sudden infant death syndrome is the leading cause of death in the postneonatal period in developed countries. Postmortem studies show alterations in serotonin neurons in the brainstem of such infants. However, the mechanism by which altered serotonin homeostasis might cause sudden death is unknown. We investigated the consequences of altering the autoinhibitory capacity of serotonin neurons with the reversible overexpression of serotonin 1A autoreceptors in transgenic mice. Overexpressing mice exhibited sporadic bradycardia and hypothermia that occurred during a limited developmental period and frequently progressed to death. Moreover, overexpressing mice failed to activate autonomic target organs in response to environmental challenges. These findings show that excessive serotonin autoinhibition is a risk factor for catastrophic autonomic dysregulation and provide a mechanism for a role of altered serotonin homeostasis in sudden infant death syndrome.

Suppression of Serotonin Neuron Firing Increases Aggression in Mice

Numerous studies link decreased serotonin metabolites with increased impulsive and aggressive traits. However, although pharmacological depletion of serotonin is associated with increased aggression, interventions aimed at directly decreasing serotonin neuron activity have supported the opposite association. Furthermore, it is not clear if altered serotonin activity during development may contribute to some of the observed associations. Here, we used two pharmacogenetic approaches in transgenic mice to selectively and reversibly reduce the firing of serotonin neurons in behaving animals. Conditional overexpression of the serotonin 1A receptor (Htr1a) in serotonin neurons showed that a chronic reduction in serotonin neuron firing was associated with heightened aggression. Overexpression of Htr1a in adulthood, but not during development, was sufficient to increase aggression. Rapid suppression of serotonin neuron firing by agonist treatment of mice expressing Htr1a exclusively in serotonin neurons also led to increased aggression. These data confirm a role of serotonin activity in setting thresholds for aggressive behavior and support a direct association between low levels of serotonin homeostasis and increased aggression.

Stress-Induced Susceptibility to Sudden Cardiac Death in Mice with Altered Serotonin Homeostasis

In humans, chronic stressors have long been linked to cardiac morbidity. Altered serotonergic neurotransmission may represent a crucial pathophysiological mechanism mediating stress-induced cardiac disturbances. Here, we evaluated the physiological role of serotonin (5-HT) 1A receptors in the autonomic regulation of cardiac function under acute and chronic stress conditions, using 5-HT1A receptor knockout mice (KOs). When exposed to acute stressors, KO mice displayed a higher tachycardic stress response and a larger reduction of vagal modulation of heart rate than wild type counterparts (WTs). During a protocol of chronic psychosocial stress, 6 out of 22 (27%) KOs died from cardiac arrest. Close to death, they displayed a severe bradycardia, a lengthening of cardiac interval (P wave, PQ and QRS) duration, a notched QRS complex and a profound hypothermia. In the same period, the remaining knockouts exhibited higher values of heart rate than WTs during both light and dark phases of the diurnal rhythm. At sacrifice, KO mice showed a larger expression of cardiac muscarinic receptors (M2), whereas they did not differ for gross cardiac anatomy and the amount of myocardial fibrosis compared to WTs. This study demonstrates that chronic genetic loss of 5-HT1A receptors is detrimental for cardiovascular health, by intensifying acute, stress-induced heart rate rises and increasing the susceptibility to sudden cardiac death in mice undergoing chronic stress.

Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo.

Serotonin 1A auto-receptors are not sufficient to modulate anxiety in mice

The neurotransmitter serotonin plays an important role in modulating diverse behavioral traits. Mice lacking the serotonin 1A receptor (Htr1a) show elevated avoidance of novel open spaces, suggesting that it has a role in modulating anxiety behavior. Htr1a is a Gαi‐coupled G‐protein‐coupled receptor expressed on serotonin neurons (auto‐receptor), where it mediates negative feedback of serotonin neuron firing. Htr1a is also expressed on non‐serotonin neurons (hetero‐receptor) in diverse brain regions, where it mediates an inhibitory effect of serotonin on neuronal activity. Debate exists about which of these receptor populations is responsible for the modulatory effects of Htr1a on anxiety. Studies using tissue‐specific transgenic expression have suggested that forebrain Htr1a hetero‐receptors are sufficient to restore normal anxiety behavior to Htr1a knockout mice. At the same time, experiments using tissue‐specific transgenic suppression of Htr1a expression have demonstrated that Htr1a auto‐receptors, but not forebrain hetero‐receptors, are necessary for normal anxiety behavior. One interpretation of these data is that multiple Htr1a receptor populations are involved in modulating anxiety. Here, we aimed to test this hypothesis by determining whether Htr1a auto‐receptors are sufficient to restore normal anxiety to Htr1a knockout animals. Transgenic mice expressing Htr1a under the control of the tryptophan hydroxylase 2 (Tph2) promoter showed restored Htr1a‐mediated serotonin negative feedback and hypothermia, but anxiety behavior indistinguishable from that of knockout mice. These data show that, in the absence of Htr1a hetero‐receptors, auto‐receptors are unable to have an impact on anxiety. When combined with previous data, these findings support the hypothesis that Htr1a auto‐receptors are necessary, but not sufficient, to modulate anxiety.

Impaired Chemosensitivity of Mouse Dorsal Raphe Serotonergic Neurons Overexpressing Serotonin 1A (Htr1a) Receptors

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 Htr1aRO mice). Htr1aRO 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 Htr1aRO mice. Principal findings Loose-seal cell-attached recordings in brainstem slices revealed that serotonergic neurons in dorsal raphe nucleus of Htr1aRO mice have dramatically reduced responses to hypercapnic challenge as compared with control littermates. In control mice, application of 9% CO2 produced an increase in firing rate of serotonergic neurons (0.260±0.041 Hz, n = 20, p = 0.0001) and application of 3% CO2 decreased their firing rate (−0.142±0.025 Hz, n = 17, p = 0.0008). In contrast, in Htr1aRO mice, firing rate of serotonergic neurons was not significantly changed by 9% CO2 (0.021±0.034 Hz, n = 16, p = 0.49) and by 3% CO2 (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.

Modulation of anxiety by cortical serotonin 1A receptors

Serotonin (5-HT) plays an important role in the modulation of behavior across animal species. The serotonin 1A receptor (Htr1a) is an inhibitory G-protein coupled receptor that is expressed both on serotonin and non-serotonin neurons in mammals. Mice lacking Htr1a show increased anxiety behavior suggesting that its activation by serotonin has an anxiolytic effect. This outcome can be mediated by either Htr1a population present on serotonin (auto-receptor) or non-serotonin neurons (hetero-receptor), or both. In addition, both transgenic and pharmacological studies have shown that serotonin acts on Htr1a during development to modulate anxiety in adulthood, demonstrating a function for this receptor in the maturation of anxiety circuits in the brain. However, previous studies have been equivocal about which Htr1a population modulates anxiety behavior, with some studies showing a role of Htr1a hetero-receptor and others implicating the auto-receptor. In particular, cell-type specific rescue and suppression of Htr1a expression in either forebrain principal neurons or brainstem serotonin neurons reached opposite conclusions about the role of the two populations in the anxiety phenotype of the knockout. One interpretation of these apparently contradictory findings is that the modulating role of these two populations depends on each other. Here we use a novel Cre-dependent inducible allele of Htr1a in mice to show that expression of Htr1a in cortical principal neurons is sufficient to modulate anxiety. Together with previous findings, these results support a hetero/auto-receptor interaction model for Htr1a function in anxiety.

Dorsal raphe serotonin neurons mediate CO2- induced arousal from sleep

Arousal from sleep in response to CO2 is a critical protective phenomenon. Dysregulation of CO2-induced arousal contributes to morbidity and mortality from prevalent diseases, such as obstructive sleep apnea and sudden infant death syndrome. Despite the critical nature of this protective reflex, the precise mechanism for CO2-induced arousal is unknown. Because CO2 is a major regulator of breathing, prevailing theories suggest that activation of respiratory chemo- and mechano-sensors is required for CO2-induced arousal. However, populations of neurons that are not involved in the regulation of breathing are also chemosensitive. Among these are serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) that comprise a component of the ascending arousal system. We hypothesized that direct stimulation of these neurons with CO2 could cause arousal from sleep independently of enhancing breathing. Dialysis of CO2-rich acidified solution into DRN, but not medullary raphe responsible for modulating breathing, caused arousal from sleep. Arousal was lost in mice with a genetic absence of 5-HT neurons, and with acute pharmacological or optogenetic inactivation of DRN 5-HT neurons. Here we demonstrate that CO2 can cause arousal from sleep directly, without requiring enhancement of breathing, and that chemosensitive 5-HT neurons in the DRN critically mediate this arousal. Better understanding mechanisms underlying this protective reflex may lead to interventions to reduce disease-associated morbidity and mortality. SIGNIFICANCE STATEMENT Although CO2-induced arousal is critical to a number of diseases, the specific mechanism is not well understood. We previously demonstrated that serotonin (5-HT) neurons are important for CO2-induced arousal, as mice without 5-HT neurons do not arouse to CO2. Many have interpreted this to mean that medullary 5-HT neurons that regulate breathing are important in this arousal mechanism. Here we found that direct application of CO2-rich aCSF to the dorsal raphe nucleus, but not the medullary raphe, causes arousal from sleep, and that this arousal was lost with genetic ablation or acute inhibition of 5-HT neurons. We propose that 5-HT neurons in the dorsal raphe nucleus can be activated directly by CO2 to cause arousal independently of respiratory activation.