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  • Addiction, Drugs
  • Information from Lay-Language Summaries is Embargoed Until the Conclusion of the Scientific Presentation

    424—Transmitter Release: Docking, Fusion, and Calcium Dependence

    Monday, November 11, 2013, 1:00 pm - 5:00 pm

    424.20: Recording of whole-cell calcium currents from sub-micrometer sized synaptic boutons in hippocampal neuronal cultures using scanning ion conductance microscopy

    Location: Halls B-H

    U. VIVEKANANDA1, P. NOVAK2, K. VOLYNSKI1, Y. E. KORCHEV2, *D. M. KULLMANN1;
    1Inst. Neurology, UCL, London WC1N 3BG, United Kingdom; 2Dept. of Med., Imperial Col. London, London, United Kingdom

    Abstract Body: Current knowledge of neurotransmitter release mechanisms relies largely on studies of relatively large specialised synapses, such as the calyx of Held or hippocampal mossy fiber bouton, which can be patch-clamped to control the presynaptic membrane potential and to manipulate or measure Ca2+ concentrations. However, the majority of central synapses are too small (~ 1 µm scale), to permit similar approaches. Indeed, the conventional patch-clamp technique relies on diffraction-limited optical microscopy to navigate a glass pipette to the target structure (~ 0.25 μm lateral and ~ 0.5 - 1.0 μm axial resolution for transmitted light images). In practice this imposes a lower limit on the size of the subcellular compartment that can be targeted for patch-clamp recording.
    Here we describe a novel approach that allows precise targeted patch-clamp recordings in small hippocampal synaptic terminals in culture. The technique is based on imaging of live neurons with super-resolution scanning ion conductance microscopy (SICM, ~ 50 - 100 nm 3D-resolution), followed by patch-clamp recordings from the identified structures using the same scanning nanopipette. With this method we obtained single K+ and Cl- channels recordings from the exposed surface of small synaptic boutons in the cell-attached or inside-out patch-clamp configurations. In contrast we found no evidence for voltage-gated calcium channels (VGCCs) on exposed bouton surface. This result is consistent with the accumulating evidence that presynaptic VGCCs are almost exclusively located at the active zone (AZ), which is not accessible to the scanning nanopipette.
    Currents mediated by ion channels in and near the AZ could be recorded by rupturing the membrane patch of the terminal to enter the whole-cell patch clamp configuration. However, in practice, the extremely small tip diameter of the scanning nanopipette, necessary for the initial high-resolution scanning, preclude whole-bouton recordings. To overcome this limitation we optimized a method to widen the ultra-fine pipette tip by breaking it against the glass coverslip by utilizing programmable feedback control of the SICM controller. This allowed us to reliably increase the inner diameter of the sharp nanopipette from ~100 nm to ~300 nm (with a corresponding decrease of the pipette resistance from ~100 MΩ to 30 - 40 MΩ) and to obtain the first whole-cell patch-clamp recordings of VGCC activity in small presynaptic boutons.

    Lay Language Summary: We have developed a novel method termed Scanning Ion Conductance Microscopy (SICM), analogous to atomic force microscopy, enabling us to now image and directly record from synapses up to ten times smaller than those previously recorded from. Using SICM will provide us unprecedented information about how neuronal information is processed at the most elemental level.
    Electrically charged ions such as sodium, calcium and potassium flow through specialised channels through the membranes of neurons, and the precise way they are gated is fundamental to normal brain activity and nerve excitability. This is seen most evidently in synapses, the communication points between neurons, where the relative movement and gating of ions is responsible for the electrical information transferred through neurons. Thus far the synapses focussed on to obtain direct electrophysiological information, have been relatively large and specialised. This is because the method of obtaining recordings, termed patch-clamp, relies on light microscopy which limits the resolution at which synapses can be viewed.
    This method uses the patch pipette as a scanning probe that passes over cultured neurons, using the resistance at the tip of the pipette as an indicator of the distance to the neuronal membrane. This provides information about the topography of the neurons, which can then be used to recreate a high resolution image. Employing a combination of light microscopy, fluorescence imaging and SICM small active pre-synaptic terminals or boutons (of about one micrometer in diameter) can be imaged. The same pipette can then be used to target the desired bouton to obtain direct electrophysiological information. In this instance we have used this technique to record sodium, potassium and calcium currents in the synaptic bouton, giving us valuable information about the relative density and distribution of these ion channels in the synapse.