In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording

In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. Voltage-sensitive dye (VSD) imaging directly assays the spatiotemporal dynamics of neuronal activity, including the functional connectivity of the neurons involved. In spatially segregated structures, VSD imaging can define how physiology and connectivity interact, and can identify functional abnormalities in models of neurological and psychiatric disorders. In the following protocol, we describe the in vitro slice preparation, epifluorescence setup and analyses necessary for fast charge-coupled device (CCD)-based VSD imaging combined with simultaneous whole-cell patch recording. The addition of single-cell recordings validates imaging results, and can reveal the relationship between single-cell activity and the VSD-imaged population response; in synchronously activated neurons, this change in whole-cell recorded V(m) can accurately represent population V(m) changes driving the VSD responses. Thus, the combined VSD imaging and whole-cell patch approach provides experimental resolution spanning single-cell electrophysiology to complex local circuit responses.     

Adult neurogenesis and functional plasticity in neuronal circuits

The adult brain is a plastic place. To ensure that the mature nervous system's control of behaviour is flexible in the face of a varying environment, morphological and physiological changes are possible at many levels, including that of the entire cell. In two areas of the adult brain - the olfactory bulb and the dentate gyrus - new neurons are generated throughout life and form an integral part of the normal functional circuitry. This process is not fixed, but highly modulated, revealing a plastic mechanism by which the brain's performance can be optimized for a given environment. The functional benefits of this whole-cell plasticity, however, remain a matter for debate.     

Electrical activity and gene expression in the development of vertebrate neural circuits.

Over the past several decades, anatomical and electrophysiological analyses have demonstrated that the electrical activity of neurons is required for development of the precise patterns of synaptic connectivity found in the adult central nervous system. However, knowledge of the molecular cascades that underlie activity-dependent synaptic development remains rudimentary. As a result, many fundamental issues remain unresolved. Recent advances in differential cloning have begun to provide the tools and insight necessary to bring a molecular level of understanding to principles of activity-dependent synaptic development established via classic systems approaches.     

Ratiometry of transmembrane voltage-sensitive fluorescent dye emission in hearts

Transmembrane voltage-sensitive fluorescence measurements are limited by baseline drift that can obscure changes in resting membrane potential and by motion artifacts that can obscure repolarization. Voltage-dependent shift of emission wavelengths may allow reduction of drift and motion artifacts by emission ratiometry. We have tested this for action potentials and potassium-induced changes in resting membrane potential in rabbit hearts stained with di-4-ANEPPS [Pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt] using laser excitation (488 nm) and a two-photomultiplier tube system or spectrofluorometer (resolution of 500-1,000 Hz and <1 mm). Green and red emissions produced upright and inverted action potentials, respectively. Ratios of green emission to red emission followed action potential contours and exhibited larger fractional changes than either emission alone (P < 0.001). The largest changes and signal-to-noise ratio (signal/noise) were obtained with numerator wavelengths of 525-550 nm and denominator wavelengths of 650-700 nm. Ratiometry lessened drift 56-66% (P < 0.015) and indicated decreases in resting membrane potential. Ratiometry lessened motion artifacts and increased magnitudes of deflections representing phase-zero depolarizations relative to total deflections by 123-188% in intact hearts (P < 0.02). Durations of action potentials at different pacing rates, temperatures, and potassium concentrations were independent of whether they were measured ratiometrically or with microelectrodes (P > or = 0.65). The ratiometric calibration slope was 0.017/100 mV and decreased with time. Thus emission ratiometry lessens the effects of motion and drift and indicates resting membrane potential changes and repolarization.     

A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos

Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP)(s843) transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.     

Correction of motion artifact in transmembrane voltage-sensitive fluorescent dye emission in hearts

Fast voltage-sensitive dyes are widely used to image cardiac electrical activity. Typically, the emission spectrum of these fluorochromes is wavelength shifted with altered membrane potential, but the optical signals obtained also decay with time and are affected by contraction. Ratiometry reduces, but may not fully remove, these artifacts. An alternate approach has been developed in which the time decay in simultaneously acquired short- and long-wavelength signals is characterized nonparametrically and removed. Motion artifact is then identified as the time-varying signal component common to both decay-corrected signals and subtracted. Performance of this subtraction technique was compared with ratiometry for intramural optical signals acquired with a fiber-optic probe in an isolated, Langendorff-perfused pig heart preparation (n = 4) stained with di-4-ANEPPS. Perfusate concentration of 2,3-butanedione monoxime was adjusted (7.5-12.5 mM) to alter contractile activity. Short-wavelength (520-600 nm) and long-wavelength (>600 nm) signals were recorded over 8-16 cardiac cycles at 6 sites across the left ventricular free wall in sinus rhythm and during pacing. A total of 451 such data sets were acquired. Appreciable wall motion was observed in 225 cases, with motion artifact classed as moderate (less than modulation due to action potential) in 187 and substantial (more than modulation due to action potential) in 38. In all cases, subtraction performed as well as, or better than, ratiometry in removing motion artifact and decay. Action potential morphology was recovered more faithfully by subtraction than by ratiometry in 58 of 187 and 31 of 38 cases with moderate and substantial motion artifact, respectively. This novel subtraction approach may therefore provide a means of reducing the concentration of uncoupling agents used in cardiac optical mapping studies.     

Schistosoma mansoni: functional proteasomes are required for development in the vertebrate host

Proteasomes are multi-subunit proteases involved in several mechanisms and thought to contribute to the regulation of cellular homeostasis. Here, we report for the first time biochemical evidence for the existence of a ubiquitin-proteasome proteolytic pathway in this parasite. Proteasomes from both cercariae and adult worms exhibited a high preference for hydrolysis of the substrate Suc-LLVY-AMC, although in the cercariae extract the rate of hydrolysis was 50% lower when compared to adult worms extracts. The same difference in proteasome activities was observed when endogenous proteins were broken down in the presence of ATP and ubiquitin. Additionally, accumulation of high molecular weight conjugates was observed when cercariae were pre-incubated with proteasome inhibitors. Finally, we present evidence that during experimental schistosomiasis, proteasome inhibitors were able to reduce the number of lung stage schistosomula, reduce the worm burden and consequently decrease the egg output in infected mice.     

WNTs in the vertebrate nervous system: from patterning to neuronal connectivity

WNT signalling has a key role in early embryonic patterning through the regulation of cell fate decisions, tissue polarity and cell movements. In the nervous system, WNT signalling also regulates neuronal connectivity by controlling axon pathfinding, axon remodelling, dendrite morphogenesis and synapse formation. Studies, from invertebrates to mammals, have led to a considerable understanding of WNT signal transduction pathways. This knowledge provides a framework for the study of the mechanisms by which WNTs regulate diverse neuronal functions. Manipulation of the WNT pathways could provide new strategies for nerve regeneration and neuronal circuit modulation.     

Long-term recording on multi-electrode array reveals degraded inhibitory connection in neuronal network development

Spontaneous neuronal activity plays an important role in development. However, the mechanism that underlies the long-term spontaneous developmental change of cultured neuronal networks in vitro is not well understood. To investigate the contribution of inhibitory and excitatory connections to the development of neuronal networks, dissociated neurons from an embryonic rat hippocampal formation were cultured on a multi-electrode array plate and spontaneous activities were recorded by multi-channel system. These spontaneous activities were compared to bicuculline-induced firings, which were recorded by 60 electrodes simultaneously from 1 to 14 weeks in vitro (WIV). The phenomena showed that the spontaneous firing activities changed from an initial pattern of synchronized bursts to a later pattern of high frequency random spikes. The bicuculline-induced firing activities transformed from a pattern of synchronized bursts throughout all active sites in 3 WIV, to a pattern of local synchronized or random spikes appearing in the intervals of synchronized bursts after 11 WIV, while the firing rate hardly changed. Kynurenic acid, a broad-spectrum glutamate receptor antagonist, blocked all activities while CNQX inhibited only the local synchronized or random spikes. These suggest that the inhibitory connection was age-dependent degraded in vitro and the developmental spontaneous firing pattern was built by the homeostatic balance of the excitatory-inhibitory connection networks. Long-term cultures on MEA provided a useful tool to measure the relationship between spontaneous developmental change and pharmacological influence in vitro.     

脊椎动物胚胎骨骼肌的生成

肌肉发生的起始和生肌节的形成是胚胎肌肉发育中的两个关键事件。研究表明,肌肉发生的起始有赖于体节周围组织所产生的分泌因子的影响。这些组织包括体轴结构,侧板中胚层及体节正上方的外胚层,代表性的分子有Ｗｎｔｓ家族的一些成员以及Ｓｈｈ和ＢＭＰ－４;而生肌节的形成则首先依赖与分节化相关的基因,如ｄｅｌｔａ,ｈｅｒ１等的正常功能,分节之后也同样需在周围环境的作用之下形成生肌节。两栖类非洲爪蟾的肌肉发生有其特殊性。本文对这一领域中最近的研究进展作一综合介绍。     

MEA-based recording of neuronal activity in vitro

Based on the advantages of MEA-based recording, developmental changes of spontaneous activity and tetanus-induced modification of evoked activity were studied. Rat cortical neurons were cultured on MEAs and the spontaneous activity was continuously monitored for two months. The activity started a few days after plating. During the second week, the cultures generated periodic synchronized bursts, which were the characteristic properties of cortical neurons in vitro. In about one month, the cultured networks reached a steady state. Between these two, we found a critical period during which only weak activities were generated. This critical period might reflect the transition from immature networks to mature networks including precisely controlled excitatory and inhibitory synapses. We could elicit clear evoked responses with high reproducibility in mature cultures. A focal tetanic stimulation was applied to the mature cultures and how the tetanus affects 64 kinds of evoked activity was studied. The evoked responses showed bi-directional changes in their propagation patterns, potentiation and depression. These induced changes reflected the correlation properties with the tetanized activity pattern. The next step will be the combination of long-term recording and multi-site stimulation. How long does the induced change last, as well as how additional strong activity affects the previously induced changes, will be studied.     

Systems control of BMP morphogen flow in vertebrate embryos

Embryonic morphogenetic programs coordinate cell behavior to ensure robust pattern formation. Having identified components of those programs by molecular genetics, developmental biology is now borrowing concepts and tools from systems biology to decode their regulatory logic. Dorsal-ventral (D-V) patterning of the frog gastrula by Bone Morphogenetic Proteins (BMPs) is one of the best studied examples of a self-regulating embryonic patterning system. Embryological analyses and mathematical modeling are revealing that the BMP activity gradient is maintained by a directed flow of BMP ligands towards the ventral side. Pattern robustness is ensured through feedback control of the levels of extracellular BMP pathway modulators that adjust the flow to the dimensions of the embryonic field.