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

Errors in the measurement of voltage-activated ion channels in cell-attached patch-clamp recordings

Patch-clamp recording techniques have revolutionized understanding of the function and sub-cellular location of ion channels in excitable cells. The cell-attached patch-clamp configuration represents the method of choice to describe the endogenous properties of voltage-activated ion channels in the axonal, somatic and dendritic membrane of neurons, without disturbance of the intracellular milieu. Here, we directly examine the errors associated with cell-attached patch-clamp measurement of ensemble ion channel activity. We find for a number of classes of voltage-activated channels, recorded from the soma and dendrites of neurons in acute brain-slices and isolated cells, that the amplitude and kinetics of ensemble ion channel activity recorded in cell-attached patches is significantly distorted by transmembrane voltage changes generated by the flow of current through the activated ion channels. We outline simple error–correction procedures that allow a more accurate description of the density and properties of voltage-activated channels to be incorporated into computational models of neurons

The calcineurin inhibitor cyclosporin A cyclophilin A complex reduces desensitization of GABA(A)-mediated responses in acutely dissociated rat hippocampal neurons

The effects of the selective inhibitor of calcineurin, cyclosporin A-cyclophilin A (CC) complex on the desensitization kinetics of GABA(A) receptors was studied in acutely dissociated hippocampal neurons, using the patch clamp technique in the whole cell configuration. In control conditions, the decay of GABA-evoked current could be fitted by a biexponential function having time constants of 0.65 +/- 0.24 s and 3.75 +/- 2 s. The plateau to peak ratio was 0.087 +/- 0.034. Recovery from desensitization was obtained in more than 2 min. In cells dialyzed with the CC complex, the decay of the currents could be fitted with the sum of two exponentials having time constants similar to controls (0.81 +/- 0.47 s and 3.62 +/- 2.1 s), but the percentage of the fast component was smaller. The plateau to peak ratio was significantly larger than control (0.185 +/- 0.07). With CC complex, recovery from desensitization was completed in almost 30 s. The cyclosporin A derivative PSC 833, which does not inhibit calcineurin, did not affect desensitization kinetics. These results suggest that phosphatase 2B regulates desensitization of GABA(A) receptors

Long term potentiation affects intracellular metalloproteinases activity in the mossy fiber-CA3 pathway.

Matrix Metalloproteinases (MMPs) are a family of endopeptidases known to process extracellular proteins. In the last decade, studies carried out mainly on the Schaffer collateral-CA1 hippocampal projection have provided solid evidence that MMPs regulate synaptic plasticity and learning. Recently, our group has shown that MMP blockade disrupts LTP maintenance also in the mossy fiber-CA3 (mf-CA3) projection (Wojtowicz and Mozrzymas, 2010), where LTP mechanisms are profoundly different (NMDAR-independent and presynaptic expression site). However, how plasticity of this pathway correlates with activity and expression of MMPs remains unknown. Interestingly, several potential MMP substrates (especially of gelatinases) are localized intracellularly but little is known about MMP activity in this compartment. In the present study we have asked whether LTP is associated with the expression and activity of gelatinases in apparent intra- and extracellular compartments along mf-CA3 projection. In situ zymography showed that LTP induction was associated with increased gelatinases activity in the cytoplasm of the hilar and CA3 neurons. Using gelatin zymography, immunohistochemistry and immunofluorescent staining we found that this effect was due to de novo synthesis and activation of MMP-9 which, 2-3h after LTP induction was particularly evident in the cytoplasm. In contrast, MMP-2 was localized preferentially in the nuclei and was not affected by LTP induction. In conclusion, we demonstrate that LTP induction in the mf-CA3 pathway correlates with increased expression and activity of MMP-9 and provide the first evidence that this increase is particularly evident in the neuronal cytoplasm and nucleus