Generation and characterization of a tamoxifen-inducible Vsx1-CreERT2 line to target V2 interneurons in the mouse developing spinal cord.

In the spinal cord, ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities including locomotion. Interneurons arise during embryonic development from distinct progenitor domains orderly distributed along the dorso-ventral axis of the neural tube. The p2 progenitor domain generates at least five V2 interneuron populations. However, identification and characterization of all V2 populations remain currently incomplete and the mechanisms that control their development remain only partly understood. In this study, we report the generation of a Vsx1-CreERT2 BAC transgenic mouse line that drives CreERT2 recombinase expression mimicking endogenous Vsx1 expression pattern in the developing spinal cord. We showed that the Vsx1-CreERT2 transgene can mediate recombination in V2 precursors with a high efficacy and specificity. Lineage tracing demonstrated that all the V2 interneurons in the mouse developing spinal cord derive from cells expressing Vsx1. Finally, we confirmed that V2 precursors generate additional V2 populations that are not characterized yet. Thus, the Vsx1-CreERT2 line described here is a useful genetic tool for lineage tracing and for functional studies of the mouse spinal V2 interneurons.

Vsx1 and Chx10 paralogs sequentially secure V2 interneuron identity during spinal cord development.

Paralog factors are usually described as consolidating biological systems by displaying redundant functionality in the same cells. Here, we report that paralogs can also cooperate in distinct cell populations at successive stages of differentiation. In mouse embryonic spinal cord, motor neurons and V2 interneurons differentiate from adjacent progenitor domains that share identical developmental determinants. Therefore, additional strategies secure respective cell fate. In particular, Hb9 promotes motor neuron identity while inhibiting V2 differentiation, whereas Chx10 stimulates V2a differentiation while repressing motor neuron fate. However, Chx10 is not present at the onset of V2 differentiation and in other V2 populations. In the present study, we show that Vsx1, the single paralog of Chx10, which is produced earlier than Chx10 in V2 precursors, can inhibit motor neuron differentiation and promote V2 interneuron production. However, the single absence of Vsx1 does not impact on V2 fate consolidation, suggesting that lack of Vsx1 may be compensated by other factors. Nevertheless, Vsx1 cooperates with Chx10 to prevent motor neuron differentiation in early V2 precursors although these two paralog factors are not produced in the same cells. Hence, this study uncovers an original situation, namely labor division, wherein paralog genes cooperate at successive steps of neuronal development.

The Small GTP-Binding Protein Rhes Influences Nigrostriatal-Dependent Motor Behavior During Aging.

BACKGROUND: Here we aimed to evaluate: (1) Rhes mRNA expression in mouse midbrain, (2) the effect of Rhes deletion on the number of dopamine neurons, (3) nigrostriatal-sensitive behavior during aging in knockout mice. METHODS: Radioactive in situ hybridization was assessed in adult mice. The beam-walking test was executed in 3-, 6- and 12-month-old mice. Immunohistochemistry of midbrain tyrosine hydroxylase (TH)-positive neurons was performed in 6- and 12-month-old mice. RESULTS: Rhes mRNA is expressed in TH-positive neurons of SNpc and the ventral tegmental area. Moreover, lack of Rhes leads to roughly a 20% loss of nigral TH-positive neurons in both 6- and 12-month-old mutants, when compared with their age-matched controls. Finally, lack of Rhes triggers subtle alterations in motor performance and coordination during aging. CONCLUSIONS: Our findings indicate a fine-tuning role of Rhes in regulating the number of TH-positive neurons of the substantia nigra and nigrostriatal-sensitive motor behavior during aging.

Rhes regulates dopamine D2 receptor transmission in striatal cholinergic interneurons.

Ras homolog enriched in striatum (Rhes) is highly expressed in striatal medium spiny neurons (MSNs) of rodents. In the present study, we characterized the expression of Rhes mRNA across species, as well as its functional role in other striatal neuron subtypes. Double in situ hybridization analysis showed that Rhes transcript is selectively localized in striatal cholinergic interneurons (ChIs), but not in GABAergic parvalbumin- or in neuropeptide Y-positive cell populations. Rhes is closely linked to dopamine-dependent signaling. Therefore, we recorded ChIs activity in basal condition and following dopamine receptor activation. Surprisingly, instead of an expected dopamine D2 receptor (D2R)-mediated inhibition, we observed an aberrant excitatory response in ChIs from Rhes knockout mice. Conversely, the effect of D1R agonist on ChIs was less robust in Rhes mutants than in controls. Although Rhes deletion in mutants occurs throughout the striatum, we demonstrate that the D2R response is altered specifically in ChIs, since it was recorded in pharmacological isolation, and prevented either by intrapipette BAPTA or by GDP-ß-S. Moreover, we show that blockade of Cav2.2 calcium channels prevented the abnormal D2R response. Finally, we found that the abnormal D2R activation in ChIs was rescued by selective PI3K inhibition thus suggesting that Rhes functionally modulates PI3K/Akt signaling pathway in these neurons. Our findings reveal that, besides its expression in MSNs, Rhes is localized also in striatal ChIs and, most importantly, lack of this G-protein, significantly alters D2R modulation of striatal cholinergic excitability.

An epilepsy-related ARX polyalanine expansion modifies glutamatergic neurons excitability and morphology without affecting GABAergic neurons development.

Epileptic encephalopathies comprise a heterogeneous group of severe infantile disorders for which the pathophysiological basis of epilepsy is inaccurately clarified by genotype-phenotype analysis. Because a deficit of GABA neurons has been found in some of these syndromes, notably in patients with X-linked lissencephaly with abnormal genitalia, epilepsy was suggested to result from an imbalance in GABAergic inhibition, and the notion of "interneuronopathy" was proposed. Here, we studied the impact of a polyalanine expansion of aristaless-related homeobox (ARX) gene, a mutation notably found in West and Ohtahara syndromes. Analysis of Arx((GCG)7/Y) knock-in mice revealed that GABA neuron development is not affected. Moreover, pyramidal cell migration and cortical layering are unaltered in these mice. Interestingly, electrophysiological recordings show that hippocampal pyramidal neurons displayed a frequency of inhibitory postsynaptic currents similar to wild-type (WT) mice. However, these neurons show a dramatic increase in the frequency of excitatory inputs associated with a remodeling of their axonal arborization, suggesting that epilepsy in Arx((GCG)7/Y)mice would result from a glutamate network remodeling. We therefore propose that secondary alterations are instrumental for the development of disease-specific phenotypes and should be considered to explain the phenotypic diversity associated with epileptogenic mutations.

Perturbation of Serotonin Homeostasis during Adulthood Affects Serotonergic Neuronal Circuitry.

Growing evidence shows that the neurotransmitter serotonin (5-HT) modulates the fine-tuning of neuron development and the establishment of wiring patterns in the brain. However, whether serotonin is involved in the maintenance of neuronal circuitry in the adult brain remains elusive. Here, we use a Tph2fl°x conditional knockout (cKO) mouse line to assess the impact of serotonin depletion during adulthood on serotonergic system organization. Data show that the density of serotonergic fibers is increased in the hippocampus and decreased in the thalamic paraventricular nucleus (PVN) as a consequence of brain serotonin depletion. Strikingly, these defects are rescued following reestablishment of brain 5-HT signaling via administration of the serotonin precursor 5-hydroxytryptophan (5-HTP). Finally, 3D reconstruction of serotonergic fibers reveals that changes in serotonin homeostasis affect axonal branching complexity. These data demonstrate that maintaining proper serotonin homeostasis in the adult brain is crucial to preserve the correct serotonergic axonal wiring.

A Tph2GFP Reporter Stem Cell Line To Model in Vitro and in Vivo Serotonergic Neuron Development and Function.

Modeling biological systems in vitro has contributed to clarification of complex mechanisms in simplified and controlled experimental conditions. Mouse embryonic stem (mES) cells can be successfully differentiated toward specific neuronal cell fates, thus representing an attractive tool to dissect, in vitro, mechanisms that underlie complex neuronal features. In this study, we generated and characterized a reporter mES cell line, called Tph2GFP, in which the vital reporter GFP replaces the tryptophan hydroxylase 2 (Tph2) gene. Tph2GFP mES cells selectively express GFP upon in vitro differentiation toward the serotonergic fate, they synthesize serotonin, possess excitable membranes, and show the typical morphological, morphometrical, and molecular features of in vivo serotonergic neurons. Thanks to the vital reporter GFP, we highlighted by time-lapse video microscopy several dynamic processes such as cell migration and axonal outgrowth in living cultures. Finally, we demonstrated that predifferentiated Tph2GFP cells are able to terminally differentiate, integrate, and innervate the host brain when grafted in vivo. On the whole, the present study introduces the Tph2GFP mES cell line as a useful tool allowing accurate developmental and dynamic studies and representing a reliable platform for the study of serotonergic neurons in health and disease.

Vsx1 Transiently Defines an Early Intermediate V2 Interneuron Precursor Compartment in the Mouse Developing Spinal Cord.

Spinal ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities. Interneurons arise during embryonic development from distinct progenitor domains distributed orderly along the dorso-ventral axis of the neural tube. A single ventral progenitor population named p2 generates at least five V2 interneuron subsets. Whether the diversification of V2 precursors into multiple subsets occurs within the p2 progenitor domain or involves a later compartment of early-born V2 interneurons remains unsolved. Here, we provide evidence that the p2 domain produces an intermediate V2 precursor compartment characterized by the transient expression of the transcriptional repressor Vsx1. These cells display an original repertoire of cellular markers distinct from that of any V2 interneuron population. They have exited the cell cycle but have not initiated neuronal differentiation. They coexpress Vsx1 and Foxn4, suggesting that they can generate the known V2 interneuron populations as well as possible additional V2 subsets. Unlike V2 interneurons, the generation of Vsx1-positive precursors does not depend on the Notch signaling pathway but expression of Vsx1 in these cells requires Pax6. Hence, the p2 progenitor domain generates an intermediate V2 precursor compartment, characterized by the presence of the transcriptional repressor Vsx1, that contributes to V2 interneuron development.

Rasd2 Modulates Prefronto-Striatal Phenotypes in Humans and 'Schizophrenia-Like Behaviors' in Mice.

Rasd2 is a thyroid hormone target gene, which encodes for a GTP-binding protein enriched in the striatum where, among other functions, it modulates dopaminergic neurotransmission. Here we report that human RASD2 mRNA is abundant in putamen, but it also occurs in the cerebral cortex, with a distinctive expression pattern that differs from that present in rodents. Consistent with its localization, we found that a genetic variation in RASD2 (rs6518956) affects postmortem prefrontal mRNA expression in healthy humans and is associated with phenotypes of relevance to schizophrenia, including prefrontal and striatal grey matter volume and physiology during working memory, as measured with magnetic resonance imaging. Interestingly, quantitative real-time PCR analysis indicated that RASD2 mRNA is slightly reduced in postmortem prefrontal cortex of patients with schizophrenia. In the attempt to uncover the neurobiological substrates associated with Rasd2 activity, we used knockout mice to analyze the in vivo influence of this G-protein on the prepulse inhibition of the startle response and psychotomimetic drug-related behavioral response. Data showed that Rasd2 mutants display deficits in basal prepulse inhibition that, in turn, exacerbate gating disruption under psychotomimetic drug challenge. Furthermore, we documented that lack of Rasd2 strikingly enhances the behavioral sensitivity to motor stimulation elicited by amphetamine and phencyclidine. Based on animal model data, along with the finding that RASD2 influences prefronto-striatal phenotypes in healthy humans, we suggest that genetic mutation or reduced levels of this G-protein might have a role in cerebral circuitry dysfunction underpinning exaggerated psychotomimetic drugs responses and development of specific biological phenotypes linked to schizophrenia.

Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin.

Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84-178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2null/null mice. Finally, we set up a tamoxifen administration protocol that allows an efficient, time-specific inactivation of brain serotonin synthesis. On the whole, we generated a suitable genetic tool to investigate how serotonin depletion impacts on time-specific events during central nervous system development and adulthood life.

Rhes influences striatal cAMP/PKA-dependent signaling and synaptic plasticity in a gender-sensitive fashion.

Mechanisms of gender-specific synaptic plasticity in the striatum, a brain region that controls motor, cognitive and psychiatric functions, remain unclear. Here we report that Rhes, a GTPase enriched in medium spiny neurons (MSNs) of striatum, alters the striatal cAMP/PKA signaling cascade in a gender-specific manner. While Rhes knockout (KO) male mice, compared to wild-type (WT) mice, had a significant basal increase of cAMP/PKA signaling pathway, the Rhes KO females exhibited a much stronger response of this pathway, selectively under the conditions of dopamine/adenosine-related drug challenge. Corticostriatal LTP defects are exclusively found in A2AR/D2R-expressing MSNs of KO females, compared to KO males, an effect that is abolished by PKA inhibitors but not by the removal of circulating estrogens. This suggests that the synaptic alterations found in KO females could be triggered by an aberrant A2AR/cAMP/PKA activity, but not due to estrogen-mediated effect. Consistent with increased cAMP signaling, D1R-mediated motor stimulation, haloperidol-induced catalepsy and caffeine-evoked hyper-activity are robustly enhanced in Rhes KO females compared to mutant males. Thus Rhes, a thyroid hormone-target gene, plays a relevant role in gender-specific synaptic and behavioral responses.

Generation of Pet1210-Cre transgenic mouse line reveals non-serotonergic expression domains of Pet1 both in CNS and periphery.

Neurons producing serotonin (5-hydroxytryptamine, 5-HT) constitute one of the most widely distributed neuronal networks in the mammalian central nervous system (CNS) and exhibit a profuse innervation throughout the CNS already at early stages of development. Serotonergic neuron specification is controlled by a combination of secreted molecules and transcription factors such as Shh, Fgf4/8, Nkx2.2, Lmx1b and Pet1. In the mouse, Pet1 mRNA expression appears between 10 and 11 days post coitum (dpc) in serotonergic post-mitotic precursors and persists in serotonergic neurons up to adulthood, where it promotes the expression of genes defining the mature serotonergic phenotype such as tryptophan hydroxylase 2 (Tph2) and serotonin transporter (SERT). Hence, the generation of genetic tools based on Pet1 specific expression represents a valuable approach to study the development and function of the serotonergic system. Here, we report the generation of a Pet1(210)-Cre transgenic mouse line in which the Cre recombinase is expressed under the control of a 210 kb fragment from the Pet1 genetic locus to ensure a reliable and faithful control of somatic recombination in Pet1 cell lineage. Besides Cre-mediated recombination accurately occurred in the serotonergic system as expected and according to previous studies, Pet1(210)-Cre transgenic mouse line allowed us to identify novel, so far uncharacterized, Pet1 expression domains. Indeed, we showed that in the raphe Pet1 is expressed also in a non-serotonergic neuronal population intermingled with Tph2-expressing cells and mostly localized in the B8 and B9 nuclei. Moreover, we detected Cre-mediated recombination also in the developing pancreas and in the ureteric bud derivatives of the kidney, where it reflected a specific Pet1 expression. Thus, Pet1(210)-Cre transgenic mouse line faithfully drives Cre-mediated recombination in all Pet1 expression domains representing a valuable tool to genetically manipulate serotonergic and non-serotonergic Pet1 cell lineages.