Spinal Inhibitory Interneuron Diversity Delineates Variant Motor Microcircuits.

Animals generate movement by engaging spinal circuits that direct precise sequences of muscle contraction, but the identity and organizational logic of local interneurons that lie at the core of these circuits remain unresolved. Here, we show that V1 interneurons, a major inhibitory population that controls motor output, fractionate into highly diverse subsets on the basis of the expression of 19 transcription factors. Transcriptionally defined V1 subsets exhibit distinct physiological signatures and highly structured spatial distributions with mediolateral and dorsoventral positional biases. These positional distinctions constrain patterns of input from sensory and motor neurons and, as such, suggest that interneuron position is a determinant of microcircuit organization. Moreover, V1 diversity indicates that different inhibitory microcircuits exist for motor pools controlling hip, ankle, and foot muscles, revealing a variable circuit architecture for interneurons that control limb movement.

Air-stepping in the neonatal mouse: a powerful tool for analyzing early stages of rhythmic limb movement development.

There is a lack of experimental methods in genetically tractable mouse models to analyze the developmental period at which newborns mature weight-bearing locomotion. To overcome this deficit, we introduce methods to study l-3,4-dihydroxyphenylalanine (l-DOPA)-induced air-stepping in mice at postnatal day (P)7 and P10. Air-stepping is a stereotypic rhythmic behavior that resembles mouse walking overground locomotion but without constraints imposed by weight bearing, postural adjustments, or sensory feedback. We propose that air-stepping represents the functional organization of early spinal circuits coordinating limb movements. After subcutaneous injection of l-DOPA (0.5 mg/g), we recorded air-stepping movements in all four limbs and electromyographic (EMG) activity from ankle flexor (tibialis anterior, TA) and extensor (lateral gastrocnemius, LG) muscles. Using DeepLabCut pose estimation, we analyzed rhythmicity and limb coordination. We demonstrate steady rhythmic stepping of similar duration from P7 to P10 but with some fine-tuning of interlimb coordination with age. Hindlimb joints undergo a greater range of flexion at older ages, indicating maturation of flexion-extension cycles as the animal starts to walk. EMG recordings of TA and LG show alternation but with more focused activation particularly in the LG from P7 to P10. We discuss similarities to neonatal rat l-DOPA-induced air-stepping and infant assisted walking. We conclude that limb coordination and muscle activations recorded with this method represent basic spinal cord circuitry for limb control in neonates and pave the way for future investigations on the development of rhythmic limb control in genetic or disease models with correctly or erroneously developing motor circuitry.NEW & NOTEWORTHY We present novel methods to study neonatal air-stepping in newborn mice. These methods allow analyses at the onset of limb coordination during the period in which altricial species like rats, mice, and humans "learn" to walk. The methods will be useful to test a large variety of mutations that serve as models of motor disease in newborns or that are used to probe for specific circuit mechanisms that generate coordinated limb motor output.

Endothelin Receptor Antagonists in Kidney Disease.

Endothelin (ET) is found to be increased in kidney disease secondary to hyperglycaemia, hypertension, acidosis, and the presence of insulin or proinflammatory cytokines. In this context, ET, via the endothelin receptor type A (ETA) activation, causes sustained vasoconstriction of the afferent arterioles that produces deleterious effects such as hyperfiltration, podocyte damage, proteinuria and, eventually, GFR decline. Therefore, endothelin receptor antagonists (ERAs) have been proposed as a therapeutic strategy to reduce proteinuria and slow the progression of kidney disease. Preclinical and clinical evidence has revealed that the administration of ERAs reduces kidney fibrosis, inflammation and proteinuria. Currently, the efficacy of many ERAs to treat kidney disease is being tested in randomized controlled trials; however, some of these, such as avosentan and atrasentan, were not commercialized due to the adverse events related to their use. Therefore, to take advantage of the protective properties of the ERAs, the use of ETA receptor-specific antagonists and/or combining them with sodium-glucose cotransporter 2 inhibitors (SGLT2i) has been proposed to prevent oedemas, the main ERAs-related deleterious effect. The use of a dual angiotensin-II type 1/endothelin receptor blocker (sparsentan) is also being evaluated to treat kidney disease. Here, we reviewed the main ERAs developed and the preclinical and clinical evidence of their kidney-protective effects. Additionally, we provided an overview of new strategies that have been proposed to integrate ERAs in kidney disease treatment.

Mechanism Insights into the Iridium(III)- and B(C6F5)3-Catalyzed Reduction of CO2 to the Formaldehyde Level with Tertiary Silanes.

The catalytic system [Ir(CF3CO2)(κ2-NSiMe)2] [1; NSiMe = (4-methylpyridin-2-yloxy)dimethylsilyl]/B(C6F5)3 promotes the selective reduction of CO2 with tertiary silanes to the corresponding bis(silyl)acetal. Stoichiometric and catalytic studies evidenced that species [Ir(CF3COO-B(C6F5)3)(κ2-NSiMe)2] (3), [Ir(κ2-NSiMe)2][HB(C6F5)3] (4), and [Ir(HCOO-B(C6F5)3)(κ2-NSiMe)2] (5) are intermediates of the catalytic process. The structure of 3 has been determined by X-ray diffraction methods. Theoretical calculations show that the rate-limiting step for the 1/B(C6F5)3-catalyzed hydrosilylation of CO2 to bis(silyl)acetal is a boron-promoted Si-H bond cleavage via an iridium silylacetal borane adduct.

Synthesis and Characterization of Ir-(κ2-NSi) Species Active toward the Solventless Hydrolysis of HSiMe(OSiMe3)2.

The reaction of [IrH(Cl)(κ2-NSitBu2)(coe)] (1) with 1 equiv of PCy3 (or PHtBu2) gives the species [IrH(Cl)(κ2-NSitBu2)(L)] (L = PCy3, 2a; PHtBu2, 2b), which reacts with 1 equiv of AgOTf to afford [IrH(OTf)(κ2-NSitBu2)(L)] (L = PCy3, 3a and PHtBu2, 3b). Complexes 2a, 2b, 3a, and 3b have been characterized by means of NMR spectroscopy and HR-MS. The solid-state structures of complexes 2a, 2b, and 3a have been determined by X-ray diffraction studies. The reversible coordination of water to 3a, 3b, and 4 to afford the corresponding adduct [IrH(OTf)(κ2-NSitBu2)(L)(H2O)] (L = PCy3, 3a-H2O; PHtBu2, 3b-H2O; coe, 4-H2O) has been demonstrated spectroscopically by NMR studies. The structure of complexes 3b-H2O and 4-H2O have been determined by X-ray diffraction studies. Computational analyses of the interaction between neutral [NSitBu2]• and [Ir(H)L(X)]• fragments in Ir-NSitBu2 species confirm the electron-sharing nature of the Ir-Si bond and the significant role of electrostatics in the interaction between the transition metal fragment and the κ2-NSitBu2 ligand. The activity of Ir-(κ2-NSitBu2) species as catalysts for the hydrolysis of HSiMe(OSiMe3)2 depends on the nature of the ancillary ligands. Thus, while the triflate derivatives are active, the related chloride species show no activity. The best catalytic performance has been obtained when using complexes 3a, with triflate and PCy3 ligands, as a catalyst precursor, which allows the selective obtention of the corresponding silanol.

Spinal Motor Circuit Synaptic Plasticity after Peripheral Nerve Injury Depends on Microglia Activation and a CCR2 Mechanism.

Peripheral nerve injury results in persistent motor deficits, even after the nerve regenerates and muscles are reinnervated. This lack of functional recovery is partly explained by brain and spinal cord circuit alterations triggered by the injury, but the mechanisms are generally unknown. One example of this plasticity is the die-back in the spinal cord ventral horn of the projections of proprioceptive axons mediating the stretch reflex (Ia afferents). Consequently, Ia information about muscle length and dynamics is lost from ventral spinal circuits, degrading motor performance after nerve regeneration. Simultaneously, there is activation of microglia around the central projections of peripherally injured Ia afferents, suggesting a possible causal relationship between neuroinflammation and Ia axon removal. Therefore, we used mice (both sexes) that allow visualization of microglia (CX3CR1-GFP) and infiltrating peripheral myeloid cells (CCR2-RFP) and related changes in these cells to Ia synaptic losses (identified by VGLUT1 content) on retrogradely labeled motoneurons. Microgliosis around axotomized motoneurons starts and peaks within 2 weeks after nerve transection. Thereafter, this region becomes infiltrated by CCR2 cells, and VGLUT1 synapses are lost in parallel. Immunohistochemistry, flow cytometry, and genetic lineage tracing showed that infiltrating CCR2 cells include T cells, dendritic cells, and monocytes, the latter differentiating into tissue macrophages. VGLUT1 synapses were rescued after attenuating the ventral microglial reaction by removal of colony stimulating factor 1 from motoneurons or in CCR2 global KOs. Thus, both activation of ventral microglia and a CCR2-dependent mechanism are necessary for removal of VGLUT1 synapses and alterations in Ia-circuit function following nerve injuries.SIGNIFICANCE STATEMENT Synaptic plasticity and reorganization of essential motor circuits after a peripheral nerve injury can result in permanent motor deficits due to the removal of sensory Ia afferent synapses from the spinal cord ventral horn. Our data link this major circuit change with the neuroinflammatory reaction that occurs inside the spinal cord following injury to peripheral nerves. We describe that both activation of microglia and recruitment into the spinal cord of blood-derived myeloid cells are necessary for motor circuit synaptic plasticity. This study sheds new light into mechanisms that trigger major network plasticity in CNS regions removed from injury sites and that might prevent full recovery of function, even after successful regeneration.

MERS-CoV 4b protein interferes with the NF-κB-dependent innate immune response during infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that emerged in 2012, causing severe pneumonia and acute respiratory distress syndrome (ARDS), with a case fatality rate of ~36%. When expressed in isolation, CoV accessory proteins have been shown to interfere with innate antiviral signaling pathways. However, there is limited information on the specific contribution of MERS-CoV accessory protein 4b to the repression of the innate antiviral response in the context of infection. We found that MERS-CoV 4b was required to prevent a robust NF-κB dependent response during infection. In wild-type virus infected cells, 4b localized to the nucleus, while NF-κB was retained in the cytoplasm. In contrast, in the absence of 4b or in the presence of cytoplasmic 4b mutants lacking a nuclear localization signal (NLS), NF-κB was translocated to the nucleus leading to the expression of pro-inflammatory cytokines. This indicates that NF-κB repression required the nuclear import of 4b mediated by a specific NLS. Interestingly, we also found that both in isolation and during infection, 4b interacted with α-karyopherin proteins in an NLS-dependent manner. In particular, 4b had a strong preference for binding karyopherin-α4 (KPNA4), which is known to translocate the NF-κB protein complex into the nucleus. Binding of 4b to KPNA4 during infection inhibited its interaction with NF-κB-p65 subunit. Thereby we propose a model where 4b outcompetes NF-κB for KPNA4 binding and translocation into the nucleus as a mechanism of interference with the NF-κB-mediated innate immune response.

Genetic and pharmacological inactivation of apical Na+-K+-2Cl- cotransporter 1 in choroid plexus epithelial cells reveals the physiological function of the cotransporter.

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF). They express Na+-K+-ATPase and Na+-K+-2Cl- cotransporter 1 (NKCC1) on their apical membrane, deviating from typical basolateral membrane location in secretory epithelia. Given this peculiarity, the direction of basal net ion fluxes mediated by NKCC1 in CPECs is controversial, and cotransporter function is unclear. Determining the direction of basal NKCC1-mediated fluxes is critical to understanding the function of apical NKCC1. If NKCC1 works in the net efflux mode, it may be directly involved in CSF secretion. Conversely, if NKCC1 works in the net influx mode, it would have an absorptive function, contributing to intracellular Cl- concentration ([Cl-]i) and cell water volume (CWV) maintenance needed for CSF secretion. We resolve this long-standing debate by electron microscopy (EM), live-cell-imaging microscopy (LCIM), and intracellular Na+ and Cl- measurements in single CPECs of NKCC1+/+ and NKCC1-/- mouse. NKCC1-mediated ion and associated water fluxes are tightly linked, thus their direction is inferred by measuring CWV changes. Genetic or pharmacological NKCC1 inactivation produces CPEC shrinkage. EM of NKCC1-/- CPECs in situ shows they are shrunken, forming large dilations of their basolateral extracellular spaces, yet remaining attached by tight junctions. Normarski LCIM shows in vitro CPECs from NKCC1-/- are ~17% smaller than NKCC1+/+. CWV measurements in calcein-loaded CPECs show that bumetanide (10 µM) produces ~16% decrease in CWV in NKCC1+/+ but not in NKCC1-/- CPECs. Our findings suggest that under basal conditions apical NKCC1 is continuously active and works in the net inward flux mode maintaining [Cl-]i and CWV needed for CSF secretion.

A Substantial Structural Conversion of the Native Monomer Leads to in-Register Parallel Amyloid Fibril Formation in Light-Chain Amyloidosis.

Amyloid light-chain (AL) amyloidosis is a rare disease in which plasma-cell-produced monoclonal immunoglobulin light chains misfold and become deposited as fibrils in the extracellular matrix. λ6 subgroup light chains are particularly fibrillogenic, and around 25 % of amyloid-associated λ6 light chains exist as the allotypic G24R variant that renders the protein less stable. The molecular details of this process, as well as the structures of the fibrils, are unknown. We have used solid-state NMR to investigate different fibril polymorphs. The secondary structures derived from NMR predominantly show ß-strands, including in former turn or helical regions, and provide a molecular basis for previously identified fibrillogenic hotspots. We have determined, by using differentially 15 N:13 C-labeled samples, that the ß-strands are stacked in-register parallel in the fibrils. This supramolecular arrangement shows that the native globular folds rearrange substantially upon fibrillization, and rules out the previously hypothesized fibril formation from native monomers.

Mechanistic Insights on the Reduction of CO2 to Silylformates Catalyzed by Ir-NSiN Species.

The hydrosilylation of CO2 with different silanes such as HSiEt3 , HSiMe2 Ph, HSiMePh2 , HSiMe(OSiMe3 )2 , and HSi(OSiMe3 )3 in the presence of catalytic ammounts of the iridium(III) complex [Ir(H)(CF3 CO2 )(NSiN*)(coe)] (1; NSiN*=fac-bis-(4-methylpyridine-2-yloxy); coe=cis-cyclooctene) has been comparatively studied. The activity of the hydrosilylation catalytic system based on 1 depends on the nature of the reducing agent, where HSiMe(OSiMe3 )2 has proven to be the most active. The aforementioned reactions were found to be highly selective toward the formation of the corresponding silylformate. It has been found that using 1 as catalyst precursor above 328 K decreases the activity through a thermally competitive mechanistic pathway. Indeed, the reduction of the ancillary trifluoroacetate ligand to give the corresponding silylether CF3 CH2 OSiR3 has been observed. Moreover, mechanistic studies for the 1-catalyzed CO2 -hydrosilylation reaction based on experimental and theoretical studies suggest that 1 prefers an inner-sphere mechanism for the CO2 reduction, whereas the closely related [Ir(H)(CF3 SO3 )(NSiN)(coe)] catalyst, bearing a triflate instead of trifluoroacetate ligand, follows an outer-sphere mechanism.

Rhodium-Catalyzed Dehydrogenative Silylation of Acetophenone Derivatives: Formation of Silyl Enol Ethers versus Silyl Ethers.

A series of rhodium-NSiN complexes (NSiN=bis (pyridine-2-yloxy)methylsilyl fac-coordinated) is reported, including the solid-state structures of [Rh(H)(Cl)(NSiN)(PCy3 )] (Cy=cyclohexane) and [Rh(H)(CF3 SO3 )(NSiN)(coe)] (coe=cis-cyclooctene). The [Rh(H)(CF3 SO3 )(NSiN)(coe)]-catalyzed reaction of acetophenone with silanes performed in an open system was studied. Interestingly, in most of the cases the formation of the corresponding silyl enol ether as major reaction product was observed. However, when the catalytic reactions were performed in closed systems, formation of the corresponding silyl ether was favored. Moreover, theoretical calculations on the reaction of [Rh(H)(CF3 SO3 )(NSiN)(coe)] with HSiMe3 and acetophenone showed that formation of the silyl enol ether is kinetically favored, while the silyl ether is the thermodynamic product. The dehydrogenative silylation entails heterolytic cleavage of the Si-H bond by a metal-ligand cooperative mechanism as the rate-determining step. Silyl transfer from a coordinated trimethylsilyltriflate molecule to the acetophenone followed by proton transfer from the activated acetophenone to the hydride ligand results in the formation of H2 and the corresponding silyl enol ether.

Somatostatin activates Ras and ERK1/2 via a G protein ßγ-subunit-initiated pathway in thyroid cells.

Somatostatin (SST) is one of the main regulators of thyroid function. It acts by binding to its receptors, which lead to the dissociation of G proteins into Gαi and Gßγ subunits. However, much less is known about the function of Gßγ in thyroid cells. Here, we studied the role of SST and Gßγ dimers released upon SST stimulation on the Ras-ERK1/2 pathway in FTRL-5 thyroid cells. We demonstrate that SST activates Ras through Gi proteins, since SST-induced Ras activation is inhibited by pertussis toxin. Moreover, the specific sequestration of Gßγ dimers decreases Ras-GTP and phosphorylated ERK1/2 levels, and overexpression of Gßγ increases ERK1/2 phosphorylation induced by SST, indicating that Gßγ dimers released after SST treatment mediate activation of Ras and ERK1/2. On the other hand, SST treatment does not modify the expression of the thyroid differentiation marker sodium/iodide symporter (NIS) through ERK1/2 activation. However, SST increases AKT activation and the inhibition of the Src/PI3K/AKT pathway increases NIS levels in SST-treated cells. Thus, we conclude that, in thyroid cells, signalling from SST receptors to ERK1/2 involves a Gßγ-mediated signal acting on a Ras-dependent pathway. Moreover, we demonstrate that SST might regulates NIS expression through a Src/PI3K/AKT-dependent mechanism, but not through ERK1/2 signalling, showing the main role of this hormone in thyroid function.

Normal distribution of VGLUT1 synapses on spinal motoneuron dendrites and their reorganization after nerve injury.

Peripheral nerve injury induces permanent alterations in spinal cord circuitries that are not reversed by regeneration. Nerve injury provokes the loss of many proprioceptive IA afferent synapses (VGLUT1-IR boutons) from motoneurons, the reduction of IA EPSPs in motoneurons, and the disappearance of stretch reflexes. After motor and sensory axons successfully reinnervate muscle, lost IA VGLUT1 synapses are not re-established and the stretch reflex does not recover; however, electrically evoked EPSPs do recover. The reasons why remaining IA synapses can evoke EPSPs on motoneurons, but fail to transmit useful stretch signals are unknown. To better understand changes in the organization of VGLUT1 IA synapses that might influence their input strength, we analyzed their distribution over the entire dendritic arbor of motoneurons before and after nerve injury. Adult rats underwent complete tibial nerve transection followed by microsurgical reattachment and 1 year later motoneurons were intracellularly recorded and filled with neurobiotin to map the distribution of VGLUT1 synapses along their dendrites. We found in control motoneurons an average of 911 VGLUT1 synapses; ~62% of them were lost after injury. In controls, VGLUT1 synapses were focused to proximal dendrites where they were grouped in tight clusters. After injury, most synaptic loses occurred in the proximal dendrites and remaining synapses were declustered, smaller, and uniformly distributed throughout the dendritic arbor. We conclude that this loss and reorganization renders IA afferent synapses incompetent for efficient motoneuron synaptic depolarization in response to natural stretch, while still capable of eliciting EPSPs when synchronously fired by electrical volleys.

Diverse nitrogen sources in seminal fluid act in synergy to induce filamentous growth of Candida albicans.

The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality-of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N-acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO2 and reduced temperatures (30 °C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.

The effect of chitin size, shape, source and purification method on immune recognition.

The animal immune response to chitin is not well understood and needs to be investigated further. However, this is a challenging topic to study because of the technical difficulties in purifying chitin, and because this material usually comes associated with contaminating components that can activate the immune system. In this study, improvements to previously described purification protocols were investigated for chitin obtained from different sources, including commercial shellfish, Candida albicans yeast and hyphal cell walls, as well as cell walls of the filamentous fungi Aspergillus fumigatus and Mucor circinelloides. The immune response to these different chitin preparations was tested using human peripheral blood mononuclear cells. In agreement with previous literature, small chitin particles of an average size of 0.2 µm were not immunogenic. On the other hand, bigger chitin particles induced in some cases a pro-inflammatory response. The results of this work suggest that not only the purity and size of the chitin particles, but also their shape can influence immune recognition.

Postoperative adjuvant combination therapy with doxorubicin and noncytotoxic suramin in dogs with appendicular osteosarcoma.

Although conventional treatment of dogs with osteosarcoma (OSA) by amputation and chemotherapy results in reported survival times (STs) of 262-413 days, no major improvements in STs have occurred in the past 2 decades. Suramin is a polysulfonated napthylurea, which at noncytotoxic concentrations in vitro, increases tumor sensitivity to chemotherapy, including doxorubicin. The study authors evaluated the combination of noncytotoxic suramin and doxorubicin after amputation in dogs with OSA. The hypothesis was that treatment of dogs with appendicular OSA with amputation, adjuvant doxorubicin, and noncytotoxic suramin would be well tolerated and result in STs at least comparable to those of doxorubicin alone. Forty-seven dogs received 6.75 mg/kg of suramin IV followed by 30 mg/m(2) of doxorubicin IV 4 hr later. Treatment was repeated q 2 wk for five doses. The median disease free time (DFI) was 203 days (range, 42-1,580+ days) and the median ST for all dogs was 369 days (range, 92-1,616+ days). There was no statistical difference in ST and DFI between greyhounds and nonngreyhounds. Adjuvant doxorubicin and noncytotoxic suramin was well tolerated in dogs with OSA following amputation. Additional studies are needed to determine if this combination treatment protocol provides additional clinical benefit compared with doxorubicin alone.

Modulation of central synapse remodeling after remote peripheral injuries by the CCL2-CCR2 axis and microglia.

Microglia-mediated synaptic plasticity after CNS injury varies depending on injury severity, but the mechanisms that adjust synaptic plasticity according to injury differences are largely unknown. This study investigates differential actions of microglia on essential spinal motor synaptic circuits following different kinds of nerve injuries. Following nerve transection, microglia and C-C chemokine receptor type 2 signaling permanently remove Ia axons and synapses from the ventral horn, degrading proprioceptive feedback during motor actions and abolishing stretch reflexes. However, Ia synapses and reflexes recover after milder injuries (nerve crush). These different outcomes are related to the length of microglia activation, being longer after nerve cuts, with slower motor-axon regeneration and extended expression of colony-stimulating factor type 1 in injured motoneurons. Prolonged microglia activation induces CCL2 expression, and Ia synapses recover after ccl2 is deleted from microglia. Thus, microglia Ia synapse removal requires the induction of specific microglia phenotypes modulated by nerve regeneration efficiencies. However, synapse preservation was not sufficient to restore the stretch-reflex function.

Renshaw cells and Ia inhibitory interneurons are generated at different times from p1 progenitors and differentiate shortly after exiting the cell cycle.

Spinal interneurons modulating motor output are highly diverse but surprisingly arise from just a few embryonic subgroups. The principles governing their development, diversification, and integration into spinal circuits are unknown. This study focuses on the differentiation of adult Renshaw cells (RCs) and Ia inhibitory interneurons (IaINs), two subclasses that respectively mediate recurrent and reciprocal inhibition of motoneurons from embryonic V1 interneurons (V1-INs). V1-INs originate from p1 progenitors and, after they become postmitotic, specifically express the transcription factor engrailed-1, a property that permits genetic labeling of V1 lineages from embryo to adult. RCs and IaINs are V1 derived, but differ in morphology, location, calcium-binding protein expression, synaptic connectivity, and function. These differences are already present in neonates, and in this study we show that their differentiation starts in the early embryo. Using 5'-bromodeoxyuridine birth dating we established that mouse V1-INs can be divided into early (E9.5-E10.5) and late (E11.5-E12.5) groups generated from the p1 domain (where E is embryonic day). The early group upregulates calbindin expression soon after becoming postmitotic and includes RCs, which express the transcription factor MafB during early differentiation and maintain calbindin expression throughout life. The late group includes IaINs, are calbindin-negative, and express FoxP2 at the start of differentiation. Moreover, developing RCs follow a characteristic circumferential migratory route that places them in unique relationship with motor axons with whom they later synaptically interact. We conclude that the fate of these V1-IN subclasses is determined before synaptogenesis and circuit formation by a process that includes differences in neurogenesis time, transcription factor expression, and migratory pathways.

An alternative mechanistic paradigm for the ß-Z hydrosilylation of terminal alkynes: the role of acetone as a silane shuttle.

The ß-Z selectivity in the hydrosilylation of terminal alkynes has been hitherto explained by introduction of isomerisation steps in classical mechanisms. DFT calculations and experimental observations on the system [M(I)2{κ-C,C,O,O-(bis-NHC)}]BF4 (M=Ir (3a), Rh (3b); bis-NHC=methylenebis(N-2-methoxyethyl)imidazole-2-ylidene) support a new mechanism, alternative to classical postulations, based on an outer-sphere model. Heterolytic splitting of the silane molecule by the metal centre and acetone (solvent) affords a metal hydride and the oxocarbenium ion [R3Si-O(CH3)2](+), which reacts with the corresponding alkyne in solution to give the silylation product [R3Si-CH=C-R](+). Thus, acetone acts as a silane shuttle by transferring the silyl moiety from the silane to the alkyne. Finally, nucleophilic attack of the hydrido ligand over [R3Si-CH=C-R](+) affords selectively the ß-(Z)-vinylsilane. The ß-Z selectivity is explained on the grounds of the steric interaction between the silyl moiety and the ligand system resulting from the geometry of the approach that leads to ß-(E)-vinylsilanes.