The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.Cholinergic neurotransmission has key functions in life, as it regulates several central and peripheral nervous system outputs. Acetylcholine (ACh) is synthesized in the cytoplasm by the enzyme choline acetyltransferase (ChAT) (16). Choline supplied by the high-affinity choline transporter (CHT1) is required to maintain ACh synthesis (52). A lack of ChAT (4, 35) or the high-affinity choline transporter (21) in genetically modified mice is incompatible with life. ACh plays an important role in wiring the neuromuscular junction (NMJ) during development (38, 43). Embryonic synthesis of ACh is fundamental for the development of proper nerve-muscle patterning at the mammalian NMJ, as ChAT-null mice present aberrant nicotinic ACh receptor (nAChR) localization and increased motoneuron (MN) survival, axonal sprouting, and branching (4, 35).The vesicular ACh transporter (VAChT) exchanges cytoplasmic ACh for two vesicular protons (37, 41). Previously reported electrophysiological studies showed that quantal size is decreased by vesamicol, an inhibitor of VAChT, but only in nerve terminals that have been electrically stimulated (19, 59, 60, 63). VAChT overexpression in developing Xenopus MNs increases both the size and frequency of miniature-end-plate currents (54). In Caenorhabditis elegans, mutations in VAChT affect behavior (65). Moreover, a decrease in VAChT expression has functional consequences for mammals, as mutant mice with a 70% reduction in the expression levels of this transporter (VAChT knockdown [KD^HOM] mice) are myasthenic and have cognitive deficits (47). Hence, vesicular transport activity is rate limiting for neurotransmission “in vivo” (18, 47).Exocytosis of synaptic vesicle contents is the predominant mechanism for the regulated secretion of neurotransmitters (55). However, alternative mechanisms of secretion have been proposed (20, 56, 61). Quantal ACh release, comparable to that seen in developing nerve terminals, has been detected in myocytes and fibroblasts in culture, which presumably do not express VAChT (14, 24). More recently, it was found that the correct targeting of Drosophila photoreceptor axons is disrupted in flies with null mutations in ChAT (64). Remarkably, the inactivation of VAChT did not produce the same result (64). The result suggests that the release of ACh during development is not dependent on VAChT, perhaps because it is nonvesicular or because vesicular storage can occur without VAChT.To test if the VAChT-independent secretion of ACh has any physiological role in the mammalian nervous system, we generated a mouse line in which the VAChT gene is deleted. These mice lack the stimulated release of ACh from synaptosomes, die after birth, and show several alterations in neuromuscular wiring consistent with a severe decrease in the cholinergic input to muscles during development. These experiments indicate that VAChT has an important role in maintaining activity-dependent ACh release that supports life and the correct patterning of innervation at the NMJ.     

Two-Photon Analysis of Lead Accumulation in Rat Cerebellar Granule Neurons

Lead (Pb²⁺) is a common pollutant and potent central neurotoxin. We have studied its pathways of permeation by two-photon fluorescence microscopy in rat cerebellar granule neurons loaded with the fluorescent dye indo-1. Pb²⁺ binds indo-1 with high affinity acting as a quencher. Its permeation through the neuronal membrane was indicated by a decrease of the fluorescence emission, which occurred even in resting condition. In the presence of 20 μM Pb²⁺, uptake reached a plateau level (≈45% of initial fluorescence) in 4 min and was partially antagonized by 25 μM lanthanum. Subsequent addition of a membrane permeant ionophore caused a further (>70%) quenching of the dye, suggesting that previous saturation was due to inactivation of the transport system. Intracellular Pb²⁺ concentrations were evaluated from the fluorescence intensity and this estimate indicated that the concentration of free Pb²⁺ sufficient to inactivate the transport system is close to 50 pM.     

Diiron and diruthenium aminocarbyne complexes containing pseudohalides: stereochemistry and reactivity

Acetonitrile is easily displaced from [Fe₂{μ-CN(Me)(R)}(μ-CO)(CO)(MeCN)(Cp)₂][SO₃CF₃] (R = 2,6-Me₂C₆H₃ (Xyl) (1a); Me (1b)) upon stirring in THF at room temperature in the presence of [NBu₄][SCN]. The resulting complexes trans-[Fe₂{μ-CN(Me)(R)}(μ-CO)(CO)(NCS)(Cp)₂] (R = Xyl (trans-2a); Me (trans-2b)) are completely isomerised to cis-[Fe₂{μ-CN(Me)(R)}(μ-CO)(CO)(NCS)(Cp)₂] (R = Xyl (cis-2a); Me (cis-2b)) when heated at reflux temperature. Similarly, the complexes cis-[M₂{μ-CN(Me)(R)}(μ-CO)(CO)(NCO)(Cp)₂] (M = Fe, R = Me (4a); M = Ru, R = Xyl (4b); M = Ru, R = Me (4c)) and cis-[M₂{μ-CN(Me)(R)}(μ-CO)(CO)(N₃)(Cp)₂] (M = Fe, R = Xyl (5a); M = Fe, R = Me (5b); M = Ru, R = Xyl (5c)) can be obtained by heating at reflux temperature a THF solution of [M₂{μ-CN(Me)(R)}(μ-CO)(CO)(MeCN)(Cp)₂][SO₃CF₃] (M = Fe, R = Xyl (1a); M = Fe, Me (1b); M = Ru, R = Xyl (1c); M = Ru, R = Me (1d)) in the presence of NaNCO and NaN₃, respectively. The reactions of 5 with MeO₂CCCCO₂Me, HCCCO₂Me and (NC)(H)CC(H)(CN) afford the triazolato complexes [M₂{μ-CN(Me)(R)}(μ-CO)(CO){N₃C₂(CO₂Me)₂}(Cp)₂] (M = Fe, R = Xyl (6a); M = Fe, R = Me (6b); M = Ru, R = Xyl (6c)), [M₂{μ-CN(Me)(R)}(μ- CO)(CO){N₃C₂(H)(CO₂Me)}(Cp)₂] (M = Fe, R = Me (7a); M = Ru, R = Xyl (7b)) and [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){N₃C₂(H)(CN)}(Cp)₂] (8), respectively. The asymmetrically substituted triazolato complexes 7-8 are obtained as mixtures of N(1) and N(2) bonded isomers, whereas 6 exists only in the N(2) form. Methylation of 6-8 results in the formation of the triazole complexes [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){N₃(Me)C₂(CO₂Me)₂}(Cp)₂][CF₃SO₃] (9), [M₂{μ-CN(Me)(R)}(μ-CO)(CO){N₃(Me)C₂(H)(CO₂Me)}(Cp)₂][CF₃SO₃] (M = Fe, R = Me (10a); M = Ru, R = Xyl (10b)) and [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){N₃(Me)C₂(H)(CN)}(Cp)₂][CF₃SO₃], 11. The crystal structures of trans-2b, 4b · CH₂Cl₂, 5a, 6b · 0.5CH₂Cl₂ and 8 · CH₂Cl₂ have been determined.     

Synthesis and reactivity with amines of new diiron alkynyl methoxy carbene complexes

The new diiron alkynyl methoxy carbene complexes [Fe₂{μ-CN(Me)(R)}(μ-CO)(CO){C(OMe)CCR′}(Cp)₂]⁺ (R = 2,6-Me₂C₆H₃ (Xyl), R′ = Tol, 3a; R = Xyl, R′ = Ph, 3b; R = Xyl, R′=Buⁿ, 3c; R = Xyl, R′=SiMe₃, 3d; R = Me, R′ = Tol, 3e; R = Me, R′ = Ph, 3f) are obtained in two steps by addition of R′CCLi (R′ = Tol, Ph, Buⁿ, SiMe₃) to the carbonyl aminocarbyne complexes [Fe₂{μ-CN(Me)(R)}(μ-CO)(CO)₂(Cp)₂]⁺ (R = Xyl, 1a; Me, 1b), followed by methylation of the resulting alkynyl acyl compounds [Fe₂{μ-CN(Me)(R)}(μ-CO)(CO){C(O)CCR′}(Cp)₂] (R = Xyl, R′ = Tol, 2a; R = Xyl, R′ = Ph, 2b; R = Xyl, R′ = Buⁿ, 2c; R = Xyl, R′ = SiMe₃, 2d; R = Me, R′ = Tol, 2e; R = Me, R′ = Ph, 2f). Complexes 3 react with secondary amines (i.e., Me₂NH, C₅H₁₀NH) to give the 4-amino-1-metalla-1,3-dienes [Fe₂{μ-CN(Me)(R)}(μ-CO)(CO){C(OMe)CHC(R′)(NMe₂)}(Cp)₂]⁺ (R = Xyl, R′ = Tol, 4a; R = Xyl, R′ = Ph, 4b; R = Me, R′ = Ph, 4c) and [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C(OMe)CHC(Tol)(NC₅H₁₀)}(Cp)₂]⁺, 5. The addition occurs stereo-selectively affording only the E-configured products. Analogously, addition of primary amines R′NH₂ (R′ = Ph, Et, Prⁱ) affords the 4-(NH-amino)-1-metalla-1,3-diene complexes [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C(OMe)CHC(R)(NHR′)}(Cp)₂]⁺ (R = Ph, 6a; Et, 6b; Prⁱ, 6c). In the case of 6a, only the E isomer is formed, whereas a mixture of the E and Z isomers is present in the case of 6b,c, with prevalence of the latter. Moreover, the two isomeric forms exist under dynamic equilibrium conditions, as shown by VT NMR studies. Complexes 6 are deprotonated by strong bases (e.g., NaH) resulting in the formation of the neutral vinyl imine complexes [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C(OMe)CHC(NR)(Tol)}(Cp)₂] (R = Ph, 7a; Et, 7b; Prⁱ, 7c); the reaction can be reverted by addition of strong acids. X-ray crystal structures have been determined for 3a[CF₃SO₃] · Et₂O, 4c[CF₃SO₃], 6a[BF₄] · CH₂Cl₂, 6c[CF₃SO₃] · 0.5Et₂O and 7a · CH₂Cl₂.     

Respiratory heat loss of Holstein cows in a tropical environment

In order to develop statistical models to predict respiratory heat loss in dairy cattle using simple physiological and environmental measurements, 15 Holstein cows were observed under field conditions in a tropical environment, in which the air temperature reached up to 40°C. The measurements of latent and sensible heat loss from the respiratory tract of the animals were made by using a respiratory mask. The results showed that under air temperatures between 10 and 35°C sensible heat loss by convection decreased from 8.24 to 1.09 W m^–2, while the latent heat loss by evaporation increased from 1.03 to 56.51 W m^–2. The evaporation increased together with the air temperature in almost a linear fashion until 20°C, but it became increasingly high as the air temperature rose above 25°C. Convection was a mechanism of minor importance for respiratory heat transfer. In contrast, respiratory evaporation was an effective means of thermoregulation for Holsteins in a hot environment. Mathematical models were developed to predict both the sensible and latent heat loss from the respiratory tract in Holstein cows under field conditions, based on measurements of the ambient temperature, and other models were developed to predict respiration rate, tidal volume, mass flow rate and expired air temperature as functions of the ambient temperature and other variables.This paper forms part of A. S. Campos Maias doctoral thesis.     

Paternally transmitted chromosomal aberrations in mouse zygotes determine their embryonic fate

The developmental consequences of chromosomal aberrations in embryos include spontaneous abortions, morphological defects, inborn abnormalities, and genetic/chromosomal diseases. Six germ-cell mutagens with different modes of action and spermatogenic stage sensitivities were used to investigate the relationship between the types of cytogenetic damage in zygotes with their subsequent risk of postimplantation death and of birth as a translocation carrier. Independent of the mutagen used, over 98% of paternally transmitted aberrations were chromosome type, rather than chromatid type, indicating that they were formed during the period between exposure of male germ cells and initiation of the first S phase after fertilization. There were consistent one-to-one agreements between the proportions of a) zygotes with unstable aberrations and the frequencies of dead embryos after implantation (slope = 0.87, confidence interval [CI]: 0.74, 1.16) and b) zygotes with reciprocal translocations and the frequency of translocation carriers at birth (slope = 0.74, CI: 0.48, 2.11). These findings suggest that chromosomal aberrations in zygotes are highly predictive of subsequent abnormal embryonic development and that development appears to proceed to implantation regardless of the presence of chromosomal abnormalities. Our findings support the hypothesis that, for paternally transmitted chromosomal aberrations, the fate of the embryo is already set by the end of G1 of the first cell cycle of development.     

Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha

Variations in calcium concentration within the endoplasmic reticulum ([Ca(2+)](ER)) may play a role in cell growth. This study evaluates the regulation of calcium pools by growth modulators of prostate cancer (PC) cells, the insulin growth factor (IGF), and the tumor necrosis growth factor-alpha (TNFalpha) as well as evaluating the possible role of [Ca(2+)](ER) variations as signals for growth modulation. We show that IGF (5 ng/ml), which increases cell growth, induces an increase in [Ca(2+)](ER) whereas TNFalpha (1 ng/ml) which reduces cell proliferation and induces apoptosis, reduces [Ca(2+)](ER). IGF-induced [Ca(2+)](ER) increase is correlated to an overexpression of the sarcoendoplasmic calcium-ATPase 2B (SERCA2b), whereas TNFalpha-induced [Ca(2+)](ER) decrease is associated to a reduction in SERCA2b expression. Pretreatment with epidermal growth factors (EGF) or IGF does not prevent TNFalpha from affecting the induction of apoptosis, [Ca(2+)](ER) reduction and SERCA2b downregulation. Reduction in [Ca(2+)](ER) induced by thapsigargin (TG) (from 1 pM to 1 microM, 48 h) reduces LNCaP growth in a dose dependent manner and induces apoptosis when cells are treated with 1 microM TG. We also show that a transient TG application (1 pM, 1 nM, 1 microM 15 min) is insufficient to induce a long lasting decrease in [Ca(2+)](ER), since [Ca(2+)](ER) remains identical to the control for 48 h following TG application. These treatments (1 pM and 1 nM, 15 min) do not modify cell growth. However, TG (1 microM, 15 min) induces apoptosis. We thus identify [Ca(2+)](ER) and SERCA2b as a central targets for causing LNCaP PC cell life or death induced by growth modulators. Furthermore our results indicate that calcium pool contents can regulate cell growth.