The dynamics of single spike-evoked adenosine release in the cerebellum

The purine adenosine is a potent neuromodulator in the brain, with roles in a number of diverse physiological and pathological processes. Modulators such as adenosine are difficult to study as once released they have a diffuse action (which can affect many neurones) and, unlike classical neurotransmitters, have no inotropic receptors. Thus rapid postsynaptic currents (PSCs) mediated by adenosine (equivalent to mPSCs) are not available for study. As a result the mechanisms and properties of adenosine release still remain relatively unclear. We have studied adenosine release evoked by stimulating the parallel fibres in the cerebellum. Using adenosine biosensors combined with deconvolution analysis and mathematical modelling, we have characterised the release dynamics and diffusion of adenosine in unprecedented detail. By partially blocking K+ channels, we were able to release adenosine in response to a single stimulus rather than a train of stimuli. This allowed reliable sub-second release of reproducible quantities of adenosine with stereotypic concentration waveforms that agreed well with predictions of a mathematical model of purine diffusion. We found no evidence for ATP release and thus suggest that adenosine is directly released in response to parallel fibre firing and does not arise from extracellular ATP metabolism. Adenosine release events showed novel short-term dynamics, including facilitated release with paired stimuli at millisecond stimulation intervals but depletion-recovery dynamics with paired stimuli delivered over minute time scales. These results demonstrate rich dynamics for adenosine release that are placed, for the first time, on a quantitative footing and show strong similarity with vesicular exocytosis

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

Plasticity of Astrocytic Coverage and Glutamate Transporter Expression in Adult Mouse Cortex

Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton–spine interface and an increase in glutamate transporter expression in astrocytic processes

Simulation of Postsynaptic Glutamate Receptors Reveals Critical Features of Glutamatergic Transmission

Activation of several subtypes of glutamate receptors contributes to changes in postsynaptic calcium concentration at hippocampal synapses, resulting in various types of changes in synaptic strength. Thus, while activation of NMDA receptors has been shown to be critical for long-term potentiation (LTP) and long term depression (LTD) of synaptic transmission, activation of metabotropic glutamate receptors (mGluRs) has been linked to either LTP or LTD. While it is generally admitted that dynamic changes in postsynaptic calcium concentration represent the critical elements to determine the direction and amplitude of the changes in synaptic strength, it has been difficult to quantitatively estimate the relative contribution of the different types of glutamate receptors to these changes under different experimental conditions. Here we present a detailed model of a postsynaptic glutamatergic synapse that incorporates ionotropic and mGluR type I receptors, and we use this model to determine the role of the different receptors to the dynamics of postsynaptic calcium with different patterns of presynaptic activation. Our modeling framework includes glutamate vesicular release and diffusion in the cleft and a glutamate transporter that modulates extracellular glutamate concentration. Our results indicate that the contribution of mGluRs to changes in postsynaptic calcium concentration is minimal under basal stimulation conditions and becomes apparent only at high frequency of stimulation. Furthermore, the location of mGluRs in the postsynaptic membrane is also a critical factor, as activation of distant receptors contributes significantly less to calcium dynamics than more centrally located ones. These results confirm the important role of glutamate transporters and of the localization of mGluRs in postsynaptic sites in their signaling properties, and further strengthen the notion that mGluR activation significantly contributes to postsynaptic calcium dynamics only following high-frequency stimulation. They also provide a new tool to analyze the interactions between metabotropic and ionotropic glutamate receptors

A Glial Variant of the Vesicular Monoamine Transporter Is Required To Store Histamine in the Drosophila Visual System

Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs) are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B) is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems

Isolation of glutamate transport-coupled charge flux and estimation of glutamate uptake at the climbing fiber–Purkinje cell synapse

Excitatory amino acid transporters (EAATs) located on neurons and glia are responsible for limiting extracellular glutamate concentrations, but specific contributions made by neuronal and glial EAATs have not been determined. At climbing fiber to Purkinje cell (PC) synapses in cerebellum, a fraction of released glutamate is rapidly bound and inactivated by neuronal EAATs located on postsynaptic PCs. Because transport involves a stoichiometric movement of ions and is electrogenic, postsynaptic currents mediated by EAATs should permit precise calculation of the amount of postsynaptic glutamate uptake. However, this is possible only if a stoichiometric EAAT current can be isolated from all other contaminating signals. We used synaptic stimulation and photolysis of caged glutamate to characterize the current in PCs that is resistant to high concentrations of glutamate receptor antagonists. Some of this response is inhibited by the high-affinity EAAT antagonist TBOA (dl-threo-β-benzyloxyaspartic acid), whereas the remaining current shows properties inconsistent with glutamate transport. By subtracting this residual non-EAAT current from the response recorded in glutamate receptor antagonists, we have obtained an estimate of postsynaptic uptake near physiological temperature. Analysis of such synaptic EAAT currents suggests that, on average, postsynaptic EAATs take up ≈1,300,000 glutamate molecules in response to a single climbing fiber action potential