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 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

The Neuroprotective Effects of mGlu1 Receptor Antagonists Are Mediated by an Enhancement of GABAergic Synaptic Transmission via a Presynaptic CB1 Receptor Mechanism

In this study, we investigated the cross-talk between mGlu1 and CB1 receptors in modulating GABA hippocampal output in whole-cell voltage clamp recordings in rat hippocampal acute slices, in organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD) and in gerbils subjected to global ischemia. CB1 receptor expression was studied using immunohistochemistry and the CA1 contents of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by LC-MS/MS. Our results show that mGlu1 receptor antagonists enhance sIPSCs in CA1 pyramidal cells and the basal and ischemic hippocampal release of GABA in vivo in a manner that is mediated by CB1 receptor activation. In hippocampal slices exposed to OGD and in ischemic gerbils, mGlu1 receptor antagonists protected CA1 pyramidal cells against post-ischemic injury and this effect was reduced by CB1 receptor activation. OGD induced a transient increase in the hippocampal content of AEA and this effect is prevented by mGlu1 receptor antagonist. Finally, OGD induced a late disruption of CB1 receptors in the CA1 region and the effect was prevented when CA1 pyramidal cells were protected by mGlu1 antagonists. Altogether, these results suggest a cooperative interaction between mGlu1 receptors and the endocannabinoid system in the mechanisms that lead to post-ischemic neuronal death

Commentary on: Peng, Y et al., Structured recurrent inhibition in the presubiculum could improve information processing

HIF-2 represents a tissue-specific isoform of the hypoxia-inducible factors (HIFs) which regulate oxygen homeostasis in the cell. In acute oxygen deficiency, HIF transcription factors ensure the timely restoration of adequate oxygen supply. Particularly in medical conditions such as stroke, which have a high mortality risk due to ischaemic brain damage, rapid recovery of oxygen supply is of extraordinary importance. Nevertheless, the endogenous mechanisms are often not sufficient to respond to severe hypoxic stress with restoring oxygenation and fail to protect the tissue. Herein, we analysed murine neurospheres without functioning HIF-2α and found that special importance in the differentiation of neurons can be attributed to HIF-2 in the brain. Other processes, such as cell migration and signal transduction of different signalling pathways, appear to be mediated to some extent via HIF-2 and illustrate the function of HIF-2 in brain remodelling. Without hypoxic stress, HIF-2 in the brain presumably focuses on the fine-tuning of the neural network. However, a therapeutically increase of HIF-2 has the potential to regenerate or replace destroyed brain tissue and help minimize the consequences of an ischaemic stroke

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

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

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

<p><i>A, B,</i> 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 (<i>A</i>) and Htr1a^RO (<i>B</i>) 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. <i>C,</i> Bar graph of baseline firing rate in control and Htr1a^RO mice. <i>D,</i> 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. ** <i>p</i><0.01 (Mann-Whitney test). Number of recorded neurons is indicated in parentheses.</p

Impacts of Brain Serotonin Deficiency following Tph2 Inactivation on Development and Raphe Neuron Serotonergic Specification

Brain serotonin (5-HT) is implicated in a wide range of functions from basic physiological mechanisms to complex behaviors, including neuropsychiatric conditions, as well as in developmental processes. Increasing evidence links 5-HT signaling alterations during development to emotional dysregulation and psychopathology in adult age. To further analyze the importance of brain 5-HT in somatic and brain development and function, and more specifically differentiation and specification of the serotonergic system itself, we generated a mouse model with brain-specific 5-HT deficiency resulting from a genetically driven constitutive inactivation of neuronal tryptophan hydroxylase-2 (Tph2). Tph2 inactivation (Tph2-/-) resulted in brain 5-HT deficiency leading to growth retardation and persistent leanness, whereas a sex- and age-dependent increase in body weight was observed in Tph2+/- mice. The conserved expression pattern of the 5-HT neuron-specific markers (except Tph2 and 5-HT) demonstrates that brain 5-HT synthesis is not a prerequisite for the proliferation, differentiation and survival of raphe neurons subjected to the developmental program of serotonergic specification. Furthermore, although these neurons are unable to synthesize 5-HT from the precursor tryptophan, they still display electrophysiological properties characteristic of 5-HT neurons. Moreover, 5-HT deficiency induces an up-regulation of 5-HT$_{1A}$ and 5-HT$_{1B}$ receptors across brain regions as well as a reduction of norepinephrine concentrations accompanied by a reduced number of noradrenergic neurons. Together, our results characterize developmental, neurochemical, neurobiological and electrophysiological consequences of brain-specific 5-HT deficiency, reveal a dual dose-dependent role of 5-HT in body weight regulation and show that differentiation of serotonergic neuron phenotype is independent from endogenous 5-HT synthesis

Quantitative autoradiography of 5-HT_1A and 5-HT_1B receptors in various brain regions.

<p>(<b><i>A</i></b>) Representative photomicrographs of autoradiograms following the binding of [³H]WAY100635 to 5-HT_1A receptors on whole coronal sections. The signal was visibly increased in e.g. the dorsal raphe, CA1 of hippocampus, frontal cortex and septum of <i>Tph2</i>−/− mice. (<b><i>B</i></b>) Binding density of 5-HT_1A receptors labeled by the radioligand [³H]WAY100635 was up-regulated in most of the brain regions of <i>Tph2</i>−/− mice. (<b><i>C</i></b>) 5-HT_1A receptor-mediated increase in [³⁵S]GTP-γ-S binding after stimulation revealed enhanced 5-HT_1A coupling in the frontal cortex and septum of <i>Tph2</i>−/− mice. (<b><i>D</i></b>) Binding density of 5-HT_1B receptors labeled by the radioligand [¹²⁵I]ICYP was also increased in some brain regions of <i>Tph2</i>−/− mice. For (<b><i>B, C, D</i></b>) results are expressed as optical density (OD = specific OD – nonspecific OD) and presented as means ± sem (n = 5). * indicates ANOVA significant output for genotype effect with *p<0.05, **p<0.01, ***p<0.001. For detailed statistical results see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043157#pone.0043157.s001" target="_blank">Table S1</a>. c: cortex, Ss: somatosensory, Retrospl.: retrosplenial, CA1: cornu ammonis area 1 of hippocampus, g: gyrus, D: dorsal, Enth.: enthorinal, Caud. put.: caudate putamen, V pal.: ventral pallidus, Globus pal.: globus pallidus, Hypo. lat.: lateral hypothalamus, D subic.: dorsal subicullum, Subst. nigra: substantia nigra.</p