RESUMO
The organization of sensory maps in the cerebral cortex depends on experience, which drives homeostatic and long-term synaptic plasticity of cortico-cortical circuits. In the mouse primary somatosensory cortex (S1) afferents from the higher-order, posterior medial thalamic nucleus (POm) gate synaptic plasticity in layer (L) 2/3 pyramidal neurons via disinhibition and the production of dendritic plateau potentials. Here we address whether these thalamocortically mediated responses play a role in whisker map plasticity in S1. We find that trimming all but two whiskers causes a partial fusion of the representations of the two spared whiskers, concomitantly with an increase in the occurrence of POm-driven N-methyl-D-aspartate receptor-dependent plateau potentials. Blocking the plateau potentials restores the archetypical organization of the sensory map. Our results reveal a mechanism for experience-dependent cortical map plasticity in which higher-order thalamocortically mediated plateau potentials facilitate the fusion of normally segregated cortical representations.
Assuntos
Potenciais de Ação/fisiologia , Potenciais Somatossensoriais Evocados/fisiologia , Rede Nervosa/fisiologia , Córtex Somatossensorial/fisiologia , Tálamo/fisiologia , Vibrissas/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Mapeamento Encefálico/métodos , Maleato de Dizocilpina/farmacologia , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/anatomia & histologia , Plasticidade Neuronal/efeitos dos fármacos , Imagem Óptica , Técnicas de Patch-Clamp , Picrotoxina/farmacologia , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Córtex Somatossensorial/anatomia & histologia , Tálamo/anatomia & histologia , Vibrissas/lesõesRESUMO
Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).
Assuntos
Microscopia de Fluorescência/instrumentação , Animais , Embrião de Galinha , Microscopia de Fluorescência/métodos , SoftwareRESUMO
Long-term synaptic potentiation (LTP) is thought to be a key process in cortical synaptic network plasticity and memory formation. Hebbian forms of LTP depend on strong postsynaptic depolarization, which in many models is generated by action potentials that propagate back from the soma into dendrites. However, local dendritic depolarization has been shown to mediate these forms of LTP as well. As pyramidal cells in supragranular layers of the somatosensory cortex spike infrequently, it is unclear which of the two mechanisms prevails for those cells in vivo. Using whole-cell recordings in the mouse somatosensory cortex in vivo, we demonstrate that rhythmic sensory whisker stimulation efficiently induces synaptic LTP in layer 2/3 (L2/3) pyramidal cells in the absence of somatic spikes. The induction of LTP depended on the occurrence of NMDAR (N-methyl-d-aspartate receptor)-mediated long-lasting depolarizations, which bear similarities to dendritic plateau potentials. In addition, we show that whisker stimuli recruit synaptic networks that originate from the posteromedial complex of the thalamus (POm). Photostimulation of channelrhodopsin-2 expressing POm neurons generated NMDAR-mediated plateau potentials, whereas the inhibition of POm activity during rhythmic whisker stimulation suppressed the generation of those potentials and prevented whisker-evoked LTP. Taken together, our data provide evidence for sensory-driven synaptic LTP in vivo, in the absence of somatic spiking. Instead, LTP is mediated by plateau potentials that are generated through the cooperative activity of lemniscal and paralemniscal synaptic circuitry.
Assuntos
Dendritos/fisiologia , Potenciação de Longa Duração , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , Potenciais de Ação , Animais , Channelrhodopsins , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Física , Receptores de N-Metil-D-Aspartato/metabolismo , Tálamo/citologia , Tálamo/fisiologia , Vibrissas/fisiologiaRESUMO
In cultured vertebrate neurons, axons have a uniform arrangement of microtubules with plus-ends distal to the cell body (plus-end-out), whereas dendrites contain mixed polarity orientations with both plus-end-out and minus-end-out oriented microtubules. Rather than non-uniform microtubules, uniparallel minus-end-out microtubules are the signature of dendrites in Drosophila and Caenorhabditis elegans neurons. To determine whether mixed microtubule organization is a conserved feature of vertebrate dendrites, we used live-cell imaging to systematically analyze microtubule plus-end orientations in primary cultures of rat hippocampal and cortical neurons, dentate granule cells in mouse organotypic slices, and layer 2/3 pyramidal neurons in the somatosensory cortex of living mice. In vitro and in vivo, all microtubules had a plus-end-out orientation in axons, whereas microtubules in dendrites had mixed orientations. When dendritic microtubules were severed by laser-based microsurgery, we detected equal numbers of plus- and minus-end-out microtubule orientations throughout the dendritic processes. In dendrites, the minus-end-out microtubules were generally more stable and comparable with plus-end-out microtubules in axons. Interestingly, at early stages of neuronal development in nonpolarized cells, newly formed neurites already contained microtubules of opposite polarity, suggesting that the establishment of uniform plus-end-out microtubules occurs during axon formation. We propose a model in which the selective formation of uniform plus-end-out microtubules in the axon is a critical process underlying neuronal polarization. SIGNIFICANCE STATEMENT: Live-cell imaging was used to systematically analyze microtubule organization in primary cultures of rat hippocampal neurons, dentate granule cells in mouse organotypic slices, and layer 2/3 pyramidal neuron in somatosensory cortex of living mice. In vitro and in vivo, all microtubules have a plus-end-out orientation in axons, whereas microtubules in dendrites have mixed orientations. Interestingly, newly formed neurites of nonpolarized neurons already contain mixed microtubules, and the specific organization of uniform plus-end-out microtubules only occurs during axon formation. Based on these findings, the authors propose a model in which the selective formation of uniform plus-end-out microtubules in the axon is a critical process underlying neuronal polarization.
Assuntos
Axônios/fisiologia , Polaridade Celular/fisiologia , Microtúbulos/fisiologia , Neurônios/citologia , Animais , Células Cultivadas , Centríolos/fisiologia , Córtex Cerebral/citologia , Dendritos/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Fatores de Tempo , Tubulina (Proteína)/metabolismoRESUMO
We provide here a procedure enabling light sheet fluorescence microscopy (LSFM) of entire human eyes after iDISCO + -based clearing (ClearEye) and immunolabeling. Demonstrated here in four eyes, post-processing of LSFM stacks enables three-dimensional (3D) navigation and customized display, including en face viewing of the fundus similarly to clinical imaging, with resolution of retinal capillaries. This method overcomes several limitations of traditional histology of the eyes. Tracing of spatially complex structures such as anterior ciliary vessels and Schlemm's canal was achieved. We conclude that LSFM of immunolabeled human eyes after iDISCO + -based clearing is a powerful tool for 3D histology of large human ocular samples, including entire eyes, which will be useful in both anatomopathology and in research.
Assuntos
Imageamento Tridimensional , Humanos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodosRESUMO
BACKGROUND: The intrinsic cardiac nervous system (ICNS) refers to clusters of neurons, located within the heart, that participate in the neuronal regulation of cardiac functions and that are involved in the initiation of cardiac arrhythmias. Therefore, deciphering its role in cardiac physiology and physiopathology is mandatory. OBJECTIVE: The aim of this study was to provide a phenotypic, electrophysiological, and pharmacological characterization of the mouse ICNS, which is still poorly characterized. METHODS: Global cardiac innervation and phenotypic diversity were investigated using immunohistochemistry on cleared murine hearts and on tissue sections. The patch clamp technique was used for the electrophysiological and pharmacological characterization of isolated mouse intracardiac neurons. RESULTS: We have identified the expression of 7 distinct neuronal markers within the mouse ICNS, thus proving the neurochemical diversity of this network. Of note, it was the first time that the existence of neurons expressing the calcium-binding protein calbindin, neuropeptide Y, and cocaine and amphetamine regulated transcript peptide was described in the mouse. Electrophysiology studies also revealed the existence of 4 different neuronal populations on the basis of their electrical behavior. Finally, we showed that these neurons can be modulated by several neuromodulators. CONCLUSION: This study showed that the mouse ICNS presents a molecular and functional complexity similar to other species and is therefore a suitable model to decipher the role of individual neuronal subtypes regarding the modulation of cardiac function and the initiation of cardiac arrhythmias.
Assuntos
Arritmias Cardíacas , Coração , Animais , Coração/inervação , Camundongos , Sistema Nervoso , Neurônios/metabolismo , Técnicas de Patch-ClampRESUMO
The growth of data throughput in optical microscopy has triggered the extensive use of supervised learning (SL) models on compressed datasets for automated analysis. Investigating the effects of image compression on SL predictions is therefore pivotal to assess their reliability, especially for clinical use. We quantify the statistical distortions induced by compression through the comparison of predictions on compressed data to the raw predictive uncertainty, numerically estimated from the raw noise statistics measured via sensor calibration. Predictions on cell segmentation parameters are altered by up to 15% and more than 10 standard deviations after 16-to-8 bits pixel depth reduction and 10:1 JPEG compression. JPEG formats with higher compression ratios show significantly larger distortions. Interestingly, a recent metrologically accurate algorithm, offering up to 10:1 compression ratio, provides a prediction spread equivalent to that stemming from raw noise. The method described here allows to set a lower bound to the predictive uncertainty of a SL task and can be generalized to determine the statistical distortions originated from a variety of processing pipelines in AI-assisted fields.
Assuntos
Compressão de Dados , Algoritmos , Compressão de Dados/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia , Reprodutibilidade dos Testes , Aprendizado de Máquina SupervisionadoRESUMO
Projections from the lateral habenula (LHb) control ventral tegmental area (VTA) neuronal populations' activity and both nuclei shape the pathological behaviors emerging during cocaine withdrawal. However, it is unknown whether cocaine withdrawal modulates LHb neurotransmission onto subsets of VTA neurons that are part of distinct neuronal circuits. Here we show that, in mice, cocaine withdrawal, drives discrete and opposing synaptic adaptations at LHb inputs onto VTA neurons defined by their output synaptic connectivity. LHb axons innervate the medial aspect of VTA, release glutamate and synapse on to dopamine and non-dopamine neuronal populations. VTA neurons receiving LHb inputs project their axons to medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and lateral hypothalamus (LH). While cocaine withdrawal increases glutamate release from LHb onto VTA-mPFC projectors, it reduces presynaptic release onto VTA-NAc projectors, leaving LHb synapses onto VTA-to-LH unaffected. Altogether, cocaine withdrawal promotes distinct adaptations at identified LHb-to-VTA circuits, which provide a framework for understanding the circuit basis of the negative states emerging during abstinence of drug intake.
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Neurons in primary sensory cortex encode a variety of stimulus features upon perceptual learning. However, it is unclear whether the acquired stimulus selectivity remains stable when the same input is perceived in a different context. Here, we monitor the activity of individual neurons in the mouse primary somatosensory cortex during reward-based texture discrimination. We track their stimulus selectivity before and after changing reward contingencies, which allows us to identify various classes of neurons. We find neurons that stably represented a texture or the upcoming behavioral choice, but the majority is dynamic. Among those, a subpopulation of neurons regains texture selectivity contingent on the associated reward value. These value-sensitive neurons forecast the onset of learning by displaying a distinct and transient increase in activity, depending on past behavioral experience. Thus, stimulus selectivity of excitatory neurons during perceptual learning is dynamic and largely relies on behavioral contingencies, even in primary sensory cortex.
Assuntos
Percepção/fisiologia , Reversão de Aprendizagem/fisiologia , Córtex Somatossensorial/fisiologia , Animais , Comportamento Animal , Sinalização do Cálcio , Comportamento de Escolha , Discriminação Psicológica , Masculino , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Recompensa , Sensação , Fatores de TempoRESUMO
In rodents, the decrease of felid aversion induced by Toxoplasma gondii, a phenomenon termed fatal attraction, is interpreted as an adaptive manipulation by the neurotropic protozoan parasite. With the aim of understanding how the parasite induces such specific behavioral modifications, we performed a multiparametric analysis of T. gondii-induced changes on host behavior, physiology, and brain transcriptome as well as parasite cyst load and distribution. Using a set of complementary behavioral tests, we provide strong evidence that T. gondii lowers general anxiety in infected mice, increases explorative behaviors, and surprisingly alters predator aversion without selectivity toward felids. Furthermore, we show a positive correlation between the severity of the behavioral alterations and the cyst load, which indirectly reflects the level of inflammation during brain colonization. Taken together, these findings refute the myth of a selective loss of cat fear in T. gondii-infected mice and point toward widespread immune-related alterations of behaviors.
Assuntos
Comportamento Animal/fisiologia , Encéfalo/parasitologia , Comportamento Exploratório/fisiologia , Medo/psicologia , Interações Hospedeiro-Parasita/fisiologia , Toxoplasma/patogenicidade , Toxoplasmose/transmissão , Animais , Masculino , CamundongosRESUMO
Severe spinal cord contusions interrupt nearly all brain projections to lumbar circuits producing leg movement. Failure of these projections to reorganize leads to permanent paralysis. Here we modeled these injuries in rodents. A severe contusion abolished all motor cortex projections below injury. However, the motor cortex immediately regained adaptive control over the paralyzed legs during electrochemical neuromodulation of lumbar circuits. Glutamatergic reticulospinal neurons with residual projections below the injury relayed the cortical command downstream. Gravity-assisted rehabilitation enabled by the neuromodulation therapy reinforced these reticulospinal projections, rerouting cortical information through this pathway. This circuit reorganization mediated a motor cortex-dependent recovery of natural walking and swimming without requiring neuromodulation. Cortico-reticulo-spinal circuit reorganization may also improve recovery in humans.
Assuntos
Córtex Motor/fisiologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Medula Espinal/fisiologia , Núcleo Vestibular Lateral/fisiologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Encéfalo/anatomia & histologia , Encéfalo/efeitos dos fármacos , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Motor/efeitos dos fármacos , Desempenho Psicomotor/efeitos dos fármacos , Quipazina/farmacologia , Ratos , Ratos Endogâmicos Lew , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/genética , Agonistas do Receptor de Serotonina/farmacologia , Medula Espinal/efeitos dos fármacos , Traumatismos da Medula Espinal/diagnóstico por imagem , Traumatismos da Medula Espinal/tratamento farmacológico , Antígenos Thy-1/administração & dosagem , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Núcleo Vestibular Lateral/efeitos dos fármacosRESUMO
Longitudinal imaging studies of neuronal structures in vivo have revealed rich dynamics in dendritic spines and axonal boutons. Spines and boutons are considered to be proxies for synapses. This implies that synapses display similar dynamics. However, spines and boutons do not always bear synapses, some may contain more than one, and dendritic shaft synapses have no clear structural proxies. In addition, synaptic strength is not always accurately revealed by just the size of these structures. Structural and functional dynamics of synapses could be studied more reliably using fluorescent synaptic proteins as markers for size and function. These proteins are often large and possibly interfere with circuit development, which renders them less suitable for conventional transfection or transgenesis methods such as viral vectors, in utero electroporation, and germline transgenesis. Single cell electroporation (SCE) has been shown to be a potential alternative for transfection of recombinant fluorescent proteins in adult cortical neurons. Here we provide proof of principle for the use of SCE to express and subsequently image fluorescently tagged synaptic proteins over days to weeks in vivo.
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[This corrects the article on p. 36 in vol. 9, PMID: 25904849.].
RESUMO
Cell to cell communication in the central nervous system is encoded into transient and local membrane potential changes (ΔVm). Deciphering the rules that govern synaptic transmission and plasticity entails to be able to perform V(m) recordings throughout the entire neuronal arborization. Classical electrophysiology is, in most cases, not able to do so within small and fragile neuronal subcompartments. Thus, optical techniques based on the use of fluorescent voltage-sensitive dyes (VSDs) have been developed. However, reporting spontaneous or small ΔV(m) from neuronal ramifications has been challenging, in part due to the limited sensitivity and phototoxicity of VSD-based optical measurements. Here we demonstrate the use of water soluble VSD, ANNINE-6plus, with laser-scanning microscopy to optically record ΔV(m) in cultured neurons. We show that the sensitivity (>10% of fluorescence change for 100 mV depolarization) and time response (sub millisecond) of the dye allows the robust detection of action potentials (APs) even without averaging, allowing the measurement of spontaneous neuronal firing patterns. In addition, we show that back-propagating APs can be recorded, along distinct dendritic sites and within dendritic spines. Importantly, our approach does not induce any detectable phototoxic effect on cultured neurons. This optophysiological approach provides a simple, minimally invasive, and versatile optical method to measure electrical activity in cultured neurons with high temporal (ms) resolution and high spatial (µm) resolution.
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In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca(2+) imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements.
Assuntos
Diagnóstico por Imagem/métodos , Medula Espinal/metabolismo , Animais , Cálcio/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
The ultrafast ground state recovery (GSR) dynamics of the radical cation of perylene, Pe(*+), generated upon bimolecular photoinduced electron transfer in acetonitrile, has been investigated using pump-pump-probe spectroscopy. With 1,4-dicyanobenzene as electron acceptor, the free ion yield is substantial and the GSR dynamics of Pe(*+) was found to depend on the time delay between the first and second pump pulses, Deltat(12), i.e., on the "age" of the ion. At short Deltat(12), the GSR dynamics is biphasic, and at Deltat(12) larger than about 500 ps, it becomes exponential with a time constant around 3 ps. With trans-1,2-dicyanoethylene as acceptor, the free ion yield is essentially zero and the GSR dynamics of Pe(*+) remains biphasic independently of Deltat(12). The change of dynamics observed with 1,4-dicyanobenzene is ascribed to the transition from paired to free solvated ion, because in the pair, the excited ion has an additional decay channel to the ground state, i.e., charge recombination followed by charge separation. The rate constants deduced from the analysis of these GSR dynamics are all fully consistent with this hypothesis.
RESUMO
The effect of the excitation wavelength on the charge recombination (CR) dynamics of several donor-acceptor complexes (DACs) composed of benzene derivatives as donors and of tetracyanoethylene or pyromellitic dianhydride as acceptors has been investigated in polar solvents using ultrafast time-resolved spectroscopy. Three different wavelength effects have been observed. (1) With complexes exhibiting two well-separated charge-transfer bands, the CR dynamics was found to be slower by a factor of about 1.5 upon excitation in the high-energy band. This effect was measured in both fast and slow relaxing solvents and was discussed in terms of different DAC geometries. (2) When the CR is faster than diffusive solvation, a slowing down of the CR with increasing excitation wavelength accompanied by an increase of the nonexponential character of the dynamics was measured. This effect appears only when exciting on the red edge of the charge-transfer absorption band. (3) When the driving force for CR is small, both nonequilibrium (hot) and thermally activated CR pathways can be operative. The results obtained with such a complex indicate that the relative contribution of these two paths depends on the excitation wavelength.
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Visible pump-probe spectroscopy has been used to identify and characterize short-lived metal-to-metal charge transfer (MMCT) excited states in a group of cyano-bridged mixed-valence complexes of the formula [LCo(III)NCM(II)(CN)(5)](-), where L is a pentadentate macrocyclic pentaamine (L(14)) or triamine-dithiaether (L(14S)) and M is Fe or Ru. Nanosecond pump-probe spectroscopy on frozen solutions of [L(14)Co(III)NCFe(II)(CN)(5)](-) and [L(14S)Co(III)NCFe(II)(CN)(5)](-) at 11 K enabled the construction of difference transient absorption spectra that featured a rise in absorbance in the region of 350-400 nm consistent with the generation of the ferricyanide chromophore of the photoexcited complex. The MMCT excited state of the Ru analogue [L(14)Co(III)NCRu(II)(CN)(5)](-) was too short-lived to allow its detection. Femtosecond pump-probe spectroscopy on aqueous solutions of [L(14)Co(III)NCFe(II)(CN)(5)](-) and [L(14S)Co(III)NCFe(II)(CN)(5)](-) at room temperature enabled the lifetimes of their Co(II)-Fe(III) MMCT excited states to be determined as 0.8 and 1.3 ps, respectively.
Assuntos
Metais Pesados/química , Nitrilas/química , Compostos Organometálicos/química , Conformação Molecular , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Termodinâmica , Fatores de TempoRESUMO
The influence of the excitation pulse carrier frequency on the ultrafast charge recombination dynamics of excited donor-acceptor complexes has been explored both theoretically and experimentally. The theoretical description involves the explicit treatment of both the optical formation of the nuclear wave packet on the excited free energy surface and its ensuing dynamics. The wave packet motion and the electronic transition are described within the framework of the stochastic point-transition approach. It is shown that the variation of the pulse carrier frequency within the absorption band can significantly change the effective charge recombination dynamics. The mechanism of this phenomenon is analyzed and a semiquantitative interpretation is suggested. The role of the vibrational coherence in the recombination dynamics is discussed. An experimental investigation of the ultrafast charge recombination dynamics of two donor-acceptor complexes in valeronitrile also is presented. The decays of the excited state population were found to be highly nonexponential, the degree of non-exponentiality depending on the excitation frequency. For one complex, the charge recombination dynamics was found to slow down upon increasing the excitation frequency, while the opposite behavior was observed with the other complex. These experimental observations follow qualitatively the predictions of the simulations.