Determining the neurotransmitter concentration profile at active synapses

Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission

The Effects of NMDA Subunit Composition on Calcium Influx and Spike Timing-Dependent Plasticity in Striatal Medium Spiny Neurons

Calcium through NMDA receptors (NMDARs) is necessary for the long-term potentiation (LTP) of synaptic strength; however, NMDARs differ in several properties that can influence the amount of calcium influx into the spine. These properties, such as sensitivity to magnesium block and conductance decay kinetics, change the receptor's response to spike timing dependent plasticity (STDP) protocols, and thereby shape synaptic integration and information processing. This study investigates the role of GluN2 subunit differences on spine calcium concentration during several STDP protocols in a model of a striatal medium spiny projection neuron (MSPN). The multi-compartment, multi-channel model exhibits firing frequency, spike width, and latency to first spike similar to current clamp data from mouse dorsal striatum MSPN. We find that NMDAR-mediated calcium is dependent on GluN2 subunit type, action potential timing, duration of somatic depolarization, and number of action potentials. Furthermore, the model demonstrates that in MSPNs, GluN2A and GluN2B control which STDP intervals allow for substantial calcium elevation in spines. The model predicts that blocking GluN2B subunits would modulate the range of intervals that cause long term potentiation. We confirmed this prediction experimentally, demonstrating that blocking GluN2B in the striatum, narrows the range of STDP intervals that cause long term potentiation. This ability of the GluN2 subunit to modulate the shape of the STDP curve could underlie the role that GluN2 subunits play in learning and development

What can whiskers tell us about mammalian evolution, behaviour, and ecology?

Most mammals have whiskers; however, nearly everything we know about whiskers derives from just a handful of species, including laboratory rats Rattus norvegicus and mice Mus musculus, as well as some species of pinniped and marsupial. We explore the extent to which the knowledge of the whisker system from a handful of species applies to mammals generally. This will help us understand whisker evolution and function, in order to gain more insights into mammalian behaviour and ecology. This review is structured around Tinbergen’s four questions, since this method is an established, comprehensive, and logical approach to studying behaviour. We ask: how do whiskers work, develop, and evolve? And what are they for? While whiskers are all slender, curved, tapered, keratinised hairs that transmit vibrotactile information, we show that there are marked differences between species with respect to whisker arrangement, numbers, length, musculature, development, and growth cycles. The conservation of form and a common muscle architecture in mammals suggests that early mammals had whiskers. Whiskers may have been functional even in therapsids. However, certain extant mammalian species are equipped with especially long and sensitive whiskers, in particular nocturnal, arboreal species, and aquatic species, which live in complex environments and hunt moving prey. Knowledge of whiskers and whisker use can guide us in developing conservation protocols and designing enriched enclosures for captive mammals. We suggest that further comparative studies, embracing a wider variety of mammalian species, are required before one can make large-scale predictions relating to evolution and function of whiskers. More research is needed to develop robust techniques to enhance the welfare and conservation of mammals

Monitoring Calcium Levels With Genetically Encoded Indicators

Calcium indicators are widely used to monitor activity in living neuronal tissue because of the tight relation between action potential firing and increases in the intracellular calcium concentration. Here, we describe the use of genetically encoded calcium indicators (GECIs) of the latest generation for monitoring calcium levels in the mammalian brain. We discuss how to choose the sensor for a given experiment, how to introduce the sensor into the cells of interest and how to estimate the sensitivity of the sensor in situ and in vivo. Finally, we illustrate the application of these sensors for high resolution in vivo imaging of sensory-driven neuronal activity

Biosensor Imaging in Brain Slice Preparations

International audienceCyclic-AMP dependent protein kinase (PKA) is present in most branches of the animal kingdom, and is an example in the nervous system where a kinase effector integrates the cellular effects of various neuromodulators. The recent development of FRET-based biosensors, such as AKAR, now allows the direct measurement of PKA activation in living cells by simply measuring the ratio between the fluorescence emission at the CFP and YFP wavelengths upon CFP excitation. This novel approach provides data with a temporal resolution of a few seconds at the cellular and even subcellular level, opening a new avenue of understanding the integration processes in space and time. Our protocol has been optimized to study morphologically intact mature neurons and we describe how simple and cheap wide-field imaging, as well as more elaborate two-photon imaging, allows real-time monitoring of PKA activation in pyramidal cortical neurons in neonate rodent brain slices. In addition, many practical details presented here also pertain to image analysis in other cellular preparations, such as cultured cells. Finally, this protocol can also be applied to the various other CFP-YFP-based FRET biosensors that are available for other kinases or other intracellular signals. It is likely that this kind of approach will be generally applicable to a broad range of assays in the near future